KARINA INACIO LADISLAU DE CARVALHO SALMAZI
ASPECTOS IMUNOLÓGICOS DA CO-INFECÇÃO PELO
Mycobacterium leprae E O VÍRUS DA
IMUNODEFICIÊNCIA HUMANA.
Tese apresentada à Universidade
Federal de São Paulo – Escola
Paulista de Medicina, para obtenção
do título de Doutor em Ciências.
São Paulo
2008
KARINA INACIO LADISLAU DE CARVALHO SALMAZI
ASPECTOS IMUNOLÓGICOS DA CO-INFECÇÃO PELO
Mycobacterium leprae E O VÍRUS DA
IMUNODEFICIÊNCIA HUMANA.
Orientador: Prof. Dr. Esper Georges Kállas
Tese apresentada à Universidade
Federal de São Paulo – Escola
Paulista de Medicina, para obtenção
do título de Doutor em Ciências.
São Paulo
2008
Ficha Catalográfica
Carvalho, KI
Aspectos Imunológicos da co-infecção do Mycobacterium leprae e o vírus da
imunodeficiência humana. / Karina Incaio Ladislau de Carvalho Salmazi. São
Paulo, 2008.123p.
Tese (Doutorado) – Universidade Federal de São Paulo – Escola Paulista de
Medicina. Programa de Pós-graduação em Infectologia.
Título em inglês: Immune aspects in coinfection of Mycobacteirum lepare and
human immunodeficiency virus.
1. Imunidade inarta. 2. Células NKT. 3. Citometria de fluxo 4.Mycobacterium leprae.
Trabalho realizado na Disciplina de Infectologia do
Departamento de Medicina e no Centro Interdisciplinar
de Terapia Gênica (Cintergen) da Universidade Federal
de São Paulo - Escola Paulista de Medicina, com o
auxílio financeiro concedido pela Conselho Nacional de
Pesquisa e Desenvolvimento (CNPQ).
Aos meus pais pelo amor incondicional, incentivo e
apoio, indispensável para a realização deste projeto e de
muitos outros de minha vida.
Ao meu querido Cristiano, que com muito amor,
compreensão esteve sempre presente. À minha filha
Julia, que é a luz de minha vida.
À vocês, minha eterna gratidão.
AGRADECIMENTOS
Ao meu orientador Prof. Dr. Esper George Kállas, que me ensina a cada
minuto, pela sua amizade, compreensão e por sempre procurar meu limite para
tornar os trabalhos sempre melhores e mais completos.
Ao Prof. Dr. Douglas Nixon, pelo apoio e confiança, pelas oportunidades
de interação e colaboração junto a sua equipe e principalmente pelas
sugestões.
Ao Prof. Dr. Patrick Hasllett, pelo incentivo de trabalhar com hanseníase,
e por suas sugestões.
À querida amiga Daniela Santoro Rosa pelo seu apoio, por muitas
discussões científicas e pela amizade maravilhosa.
À querida amiga Ana Lucia Girello sua fidelidade, incentivo e apoio
durantes todos estes anos.
À minha querida amiga Maria Regina, que me iniciou neste processo de
pesquisa, meu eterno agradecimento.
À minha querida amiga Jennifer Snyder-Cappione, pelo seu incentivo e
ajuda.
À todos que fizeram e fazem parte do grupo Kallas Lab, pelo apoio e
incentivo. Em especial a Helena Tomiyama, por sua paciência e ensinamento
em todos estes anos.
À minha borbes Fernanda Romano Bruno pela cumplicidade e carinho
desses últimos tempos.
À minha querida amiga Mariana Melillo Sauer pelo carinho, apoio e
incentivo.
À minha querida amiga Candida, que sua experiência é um brilho
constante.
Ao grupo do Prof. Dr. Mauricio Rodrigues e Prof.Dr. Edécio Cunha-Neto,
pelo apoio e incentivo, em especial a Susan Ribeiro pelo carinho e apoio.
Ao Issler Silva pelas informações importantes e por tantos favores que
me prestou.
À Profª.Drª. Marília Xavier Brasil, pela colaboração entre dois laboratório
distantes, que proporcionou enriquecer este trabalho.
À minha querida amiga Solange Maeda, pelo seu ensinamento, ajuda e
alegria, que foram importantes para a realização deste doutorado.
À todos os voluntários, pela compreensão, paciência e dedicação.
Aos amigos que me apoiam e me alegram sempre.
Ao CNPq, pelo auxílio financeiro que proporcionou a realização deste
trabalho.
SUMÁRIO
LISTA DE ABREVIATURAS…..…………………………………………….…….
v
RESUMO…………………………………………………………………………….
vi
ABSTRACT………………………………………………………………………….
viii
INTRODUÇÃO………………………………………………………………………
1
OBJETIVOS…………………………………………………………………………
20
ARTIGOS PUBLICADOS OU EM PREPARAÇÃO:
RESUMO 1………………………………………………………………………….
21
ARTIGO
22
“Immune cellular parameters of leprosy and human immunodeficiency virus1 co-infected subjects.”
RESUMO 2…………………………………………………………………………..
31
ARTIGO……………………………………………………………………………… 32
“Lower Th1 cytokine secretion ex vivo by CD1d-restricted NKT cells in HIV1-infected individuals is associated with high CD161 expression.”
RESUMO 3…………………………………………………………………………..
46
ARTIGO……………………………………………………………………………… 47
“NKT cells profile in HIV and leprosy coinfected patients.”
CONCLUSÕES……………………………………………………………………..
67
REFERÊNCIA
68
BIBLIOGRÁFICA…………………………………………………………………...
ANEXOS……………………………………………………………………………..
80
LISTA DE ABREVIATURAS
AIDS
-
Sindrome da imunodeficiência adiquirida
APC
-
Células Apresentadoras de antígeno
CCR
-
Co-receptores de quimiocinas
DNA
-
Ácido desoxiboribonucléico
Gp
-
Glicoproteína
HIV
-
Vírus da imunodeficiência humana
HLA
-
Antígeno leucocitário humano
IFN
-
Interferon
IL
-
Interleucina
MB
-
Multibacilar
MHC
-
Complexo major de histocompatibilidade
NK
-
Células natural killer
NKT
-
Células natural killer T
NRAMP1 - natural resistance-associated macrophage protein one
OMS
-
Organização Mundial da Saúde
PB
-
Paucibacilar
PACRG
-
Parkin co-regulated gen
PQT
-
Polioquimioterapia
TLR
-
Receptores do tipo toll
TCR
-
Receptor de células T
TNF
-
Fator de necrose tumoral
TR
-
Transcriptase reversa
v
Carvalho, KI
Abstract
RESUMO
As características da resposta imunológica em pacientes infectados pelo
Mycobacterium leprae e pelo virus da imunodeficiência humana (HIV) não estão
bem elucidadas. O objetivo geral desta tese foi avaliar diferentes parâmetros
imunológicos na interação destas doenças.
Inicialmente avaliamos a imunidade celular em quatro grupos de
pacientes
dividos
em:
indivíduos
saudáveis,
monoinfectados
pelo
Mycobacterium leprae, monoinfectados pelo virus da imunodeficiência humana e
coinfectados pelo M. leprae e HIV. Observamos que o grupo coinfectado
apresentou diminuição da razão de células CD4:CD8, aumento de níveis de
ativação em células T CD8+, aumento da razão de células T Vδ1:Vδ2 e
diminuição da porcentagem de células dendríticas plasmocitóides, comparadas
com o grupo de indivíduos monoinfectados pelo HIV-1. A produção de IL-4 por
linfócitos T CD4+ foi correlacionado positivamente com a porcentagem de
subpopulações de memória efetora de células T CD4+, sugerindo diferenciação
antigênica da população de células T em ambas as infecções de HIV-1 e M.
leprae. A coinfecção por M. leprae pode exacerbar a imunopatologia da doença
induzida pelo HIV-1. Houve uma tendência na expressão de citocinas Th2 na
resposta de células T CD4+ em ambas infecções de M. leprae e HIV-1, mas não
obtivemos efeitos aparentes nos pacientes coinfectados.
No trabalho subsequente, avaliamos de forma quantitativa e qualitativa as
células NKT da imunidade inata nos indivíduos saudáveis e infectados pelo HIV.
A frequência de células NKT que secretam IFN-γ e TNF-α estava
significantemente diminuída em pacientes HIV-1 quando comparados com os
indivíduos saudáveis. A magnitude da resposta de IFN-γ teve correlação inversa
com o número de anos da infecção, sugerindo que a função das células NKT
está diminuída progressivamente ao longo do tempo. Não houve alteração na
resposta das células NKT dos indivíduos infectados pelo HIV após tratamento
com Forbol12-Miristato13-Acetato (PMA) e ionomicina, sugerindo um defeito no
sinal do TCR prejudicando a produção de citocina. Foi observado uma
vi
Carvalho, KI
Abstract
diminuição na magnitude da resposta com produção de citocinas Th1 pelas
células NKT quando correlacionado com a expressão de CD161, sugerindo um
mecanismo inibitório deste receptor na regulação da resposta de células NKT.
Por último, nós demonstramos que pacientes coinfectados tem redução
da frequência de células NKT no sangue periférico, quando comparados com
indivíduos saudáveis e pacientes monoinfectados pelo M. lepare. Por outro lado,
as células NKT de pacientes coinfectados secretam mais IFN-γ quando
comparadas com pacientes monoinfectados pelo M. lepare. Estes resultados
sugerem que as células NKT têm atividade aumentada em pacientes
coinfectados, contudo em frequência diminuída no sangue periférico.
vii
Carvalho, KI
Abstract
ABSTRACT
The
immune
response
characteristics
in
patients
Mycobacterium leprae and human immunodeficiency
infected
with
virus (HIV) is not
elucidated. The aim of this study was to evaluate different immune parameters in
the overlapping of both diseases.
In the first paper we observaded The co-infected group exhibited lower
CD4:CD8 ratio, higher levels of CD8 T-cell activation, increased Vδ1 : Vδ2 T cell
ratio and lower percentage of plasmacytoid dendritic cells, compared to HIV-1
infected subjects. Across infected groups, IL-4 production by CD4 T lymphocytes
was positively correlated with the percentage of effector memory CD4 T cells,
suggesting antigenically-driven differentiation of such T cell population in both
HIV-1 and M. leprae infections. Co-infection with M. leprae may exacerbate the
immunopathology of HIV-1 induced disease. A T helper 2 (Th2) bias in the CD4
T-cell response was evident in both HIV-1-infection and leprosy, but no additive
effect was apparent in co-infected patients.
Subsequently, we evaluated quantitatively and qualitatively the NKT cells
from innate immune response in HIV-infected subjects and healthy controls. The
frequencies of NKT cells secreting IFN-γ and TNF-α were significantly lower in
HIV-1-infected subjects and the magnitude of the IFN-γ production was
negatively correlated with the number of years of infection, suggesting that NKT
cell function is progressively lost over time. NKT cell responses in HIV-1 infected
subjects were essentially normal after treatment with Phorbol12-Myristate13Acetato (PMA) and ionomycin, suggesting that defective TCR-signaling was the
underlying defect in the cytokine production. The lower levels of the NKT Th1
response correlated with higher CD161 expression, suggesting a role for this
inhibitory receptor in regulating NKT cell responsiveness.
Finally, we have investigated the NKT cells in the context of HIV and M.
leprae coinfection. The volunteers were enrolled into four groups: twenty-seven
healthy controls, seventeen HIV seropositive patients, seventeen patients with
leprosy, and twenty-three co-infected patients with leprosy and HIV-1 infection.
viii
Carvalho, KI
Abstract
Flow cytometric and ELISPOT assays were performed in stored PBMC. We
demonstrated that coinfected patients have reduced NKT cells in the peripheral
blood when compared to healthy subjects and leprosy monoinfected patients.
On the other hand, NKT cells from coinfected patients secrete more IFN-γ when
compared to leprosy monoinfected patients. These results suggest that NKT
cells are highly active in coinfected patients, although occurring in lower
frequency in the peripheral blood.
ix
Carvalho KI
Introdução
1. HANSEN
Há muitos séculos a hanseníase tem passado uma imagem de horror e
fascinação. Até os dias de hoje vários aspectos da doença permanecem
obscuros e abertos à investigação científica.
A primeira descrição do bacilo da hanseníase foi realizada pelo médico
norueguês Gerhard Henrik Hansen. Ele descobriu o bacilo em 1873 em nódulos
lepromatosos e foi sua primeira idéia da etiologia da doença. Tentou demonstrar
que a hanseníase era transmissível e acabou com uma ação criminosa. Em 3 de
novembro de 1879, Hansen inoculou material proveniente de um nódulo
hansênico em uma mulher. O experimento não alcançou o objetivo proposto,
que era comprovar que a hanseníase era uma doença infecto-contagiosa. No
entanto, Hansen estava convencido que era uma doença transmissível e
colocou a primeira lei (1877 “Norweigian Leprosy Act”) de isolamento dos
pacientes baseados em teorias não comprovadas de transmissão (Alter, Alcais
et al. 2008)
A hanseníase é causada pelo Mycobacterium leprae (M. leprae) e
continua sendo um dos maiores problemas de saúde pública mundial (Cole,
Eiglmeier et al. 2001). É um bacilo álcool-ácido-resistente, parasita intracelular
obrigatório, possui tropismo por macrófagos e células de Schwann e cresce de
preferência em regiões frias do organismo (Britton and Lockwood 2004). Além
disso, é relativamente resistente e pode viver fora do corpo humano por cerca de
45 dias. O M. leprae é encontrado em abundância na mucosa do septo-nasal de
pacientes sem tratamento e provavelmente sobrevive no ambiente antes de
infectar um novo indivíduo (Lockwood and Suneetha 2005). O único animal
conhecido por hospedar o M. leprae é o Dasypus novemcinctus, conhecido
como tatu, comum nas regiões do Texas e da Lousiana, nos Estados Unidos, e
não há evidências de outros animais que possam atuar como vetores do agente
(Kirchheimer and Storrs 1971; Lockwood and Suneetha 2005; Alter, Alcais et al.
2008).
1
Carvalho KI
Introdução
A hanseníase consiste em uma infecção crônica que afeta nervos
periféricos e pele. As lesões nos nervos resultam em deformidades e/ou
desabilidades sensoriais e motoras. Os bacilos da hanseníase parecem
depender de produtos metabolizados pelo hospedeiro, fato este que poderia
explicar a cronicidade da doença e a incapacidade de seu crescimento em
culturas (Cole, Eiglmeier et al. 2001). O fato de ser um microorganismo que não
é cultivado em cultura e ser uma doença de curso lento faz com que os estudos
in vitro e ensaios clínicos se tornem complexos (Britton and Lockwood 2004).
Nas últimas décadas, vários avanços foram obtidos no estudo da patogênese da
hanseníase, enquanto mais de 11 milhões de pacientes foram tratados com o
esquema de polioquimioterapia (PQT).
Nos dias atuais, é díficil obter-se classificação das formas clínicas
universalmente aceitas, devido a discordância na valorização dos critérios
habitualmente utilizados. Entre as classificações existentes, é importante fazer
referência a Ridley e Jopling, que utilizam as características imunológicas dos
indivíduos afetados e os classifica em um espectro que varia entre o pólo
tuberculóide, onde há grande resposta imunológica celular ao bacilo, e o pólo
lepromatoso, onde há uma anergia relativa ao M. leprae (Ridley and Jopling
1966). O Ministério da Saúde do Brasil adotou a recomendação da Organização
Mundial da Saúde (OMS), que propõe o agrupamento dos pacientes em formas
paucibacilares (PB), multibacilares (MB). Os pacientes classificados como PB
têm lesões eritêmato-hipocrômicas, eritematosas e com bordas discretamente
elevadas. O centro da lesão é habitualmente poupado, mostrando a evolução
centrífuga do processo. É frequente o comprometimento de nervos de forma
assimétrica, podendo ser a única manifestação clínica em alguns casos. Já nos
pacientes com a forma MB, as lesões são eritematosas, eritemato-violáceas,
nodulares e infiltradas. As lesões das formas disformes apresentam contorno
interno bem definido e externo mal definido, conhecidas como lesões foveolares.
A presença de nódulos e infiltrações na face são comuns, juntamente com o
comprometimento
neurológico.
A
hanseníase
indeterminada
é
uma
manifestação inicial da doença, caracterizada por manchas hipocrômicas,
2
Carvalho KI
Introdução
planas, únicas ou múltiplas, e com alteração na sensibilidade (Organization
2007).
O exame baciloscópico auxilia na classificação do paciente. A
baciloscopia deve ser realizada em todos com a suspeita clínica de hanseníase,
embora nem sempre seja possível evidenciar o M. leprae nas lesões hansênicas
ou em outros sítios de coleta. O M. leprae apresenta-se sob a forma de
bastonete pela microscopia, na maioria das vezes reto ou ligeiramente
encurvado ou, mais raramente, formando ângulos. Mede 1,5 a 8 micra de
comprimento por 0,2 a 0,5 micron de largura. A hanseníase apresenta um amplo
aspecto nas manifestações clínicas e histopatológicas. Biópsias realizadas em
lesões
de
pacientes
hansênicos
do
pólo
PB,
apresentam
infiltração
granulomatosa e raros bacilos. Aparentemente estes pacientes apresentam
resistência ao M. leprae. Contudo, no pólo MB as lesões são nodulares, com
presença de muitos bacilos, com infiltrado de macrófagos espumosos (células
de virchow) (Scollard, Adams et al. 2006).
Esta doença é tratada com o uso combinado de dapsona, rifampicina e
clofazimina, que tem efeito direto na bactéria e minimiza o desenvolvimento de
cepas resistentes a drogas (Alter, Alcais et al. 2008). A OMS recomenda tratar
por 6 a 12 meses os pacientes com a forma paucibacilar e multibacilar,
respectivamente (Tabela 1) (Organizaton 1982).
Tabela 1. Esquema de poliquimioterapia (PQT) recomendado pela Organização
Mundial da Sáude.
Classificação da Esquema de PQT
Duração do
Hanseníase
tratamento
Mensal
Diário
(meses)
Paucibacilar
Rifampicina 600 mg
Dapsona 100 mg
6
Multibacilar
Rifampicina 600 mg
Clofazimina 50 mg
12
Clofazimina 300 mg
Dapsona 100 mg
3
Carvalho KI
Introdução
Em alguns paises endêmicos, tais como Indonésia e Etiópia, mais de 5%
da população apresenta o ácido desoxicoribonucleíco (DNA) do M. leprae nos
septos-nasais, mas essas pessoas não apresentam os sintomas clínicos da
doença (Klatser, van Beers et al. 1993). Nas últimas décadas foi descrito que até
90% dos indivíduos expostos ao M. leprae permanecem assintomáticos (Convit,
Sampson et al. 1992; Chaudhury, Hazra et al. 1994; Gupte, Vallishayee et al.
1998), sugerindo que a progressão para a doença necessita de fatores
adicionais.
A hanseníase, assim como outras doenças infecciosas, apresenta um
fator causal (ex.: agente infeccioso) que é necessário, mas às vezes insuficiente
para a manifestação clínica. Esta doença aparentemente necessita de fatores de
risco adicionais, como meio ambiente e/ou genéticos (Alter, Alcais et al. 2008).
Em 1973, pela primeira vez foram descritas alterações genéticas, marco para
uma série de estudos que correlacionaram o desenvolvimento da hanseníase e
a suceptibilidade genética (Shields, Russell et al. 1987; Abel and Demenais
1988; Abel, Vu et al. 1995).
O fator genético de susceptibilidade ao bacilo pode estar presente em
dois níveis: imunidade inata ou adquirida. Um dos maiores avanços de
associação da suceptibilidade genética à doenças infecciosas foi realizado em
2003 (Scollard, Adams et al. 2006). Com relação a imunidade inata, foi
identificado que o locus específico PARK2, co-regulatório de PACRG (Mira,
Alcais et al. 2004) ou alterações na proteína NRAMP1 estão associados a
susceptibilidade na doença de Hansen. A proteína NRAMP1 possivelmente
interfere na expressão de complexo principal de histocompatibilidade classe II
(MHCII) e na regulação da expressão de fator de necrose tumoral alfa (TNF-α)
(Abel and Demenais 1988). Já em relação à imunidade adquirida, vários fatores
podem conferir maior susceptibilidade à doença. Diversos estudos sugerem que
o antígeno leucocitário humano (HLA) é determinante para a resposta ao M.
leprae e alguns estudos associam os alelos HLA-D2 e DR3 com a forma PB
(Ottenhoff, Converse et al. 1987; Ottenhoff and de Vries 1987). Outra molécula
4
Carvalho KI
Introdução
associada à suceptibilidade o TNF-α, relacionado à resistência ao M. leprae. Os
níveis séricos desta citocina em pacientes com a forma PB estão elevados,
como também a expressão da mesma nas células retiradas de lesões
hansências (Roy, McGuire et al. 1997; Moraes, Sarno et al. 1999; Ferreira,
Goulart et al. 2004). Os receptores semelhantes ao toll (toll-like receptors ou
TLRs), são moléculas de superfície que têm um papel importante no
reconhecimento de patógenos. Estudos recentes em pacientes hansênicos
indicaram que o TLR2 controla a produção de citocinas, sinais celulares e outros
aspectos da resistência ao M. leprae (Kang, Lee et al. 2002; Bochud, Hawn et al.
2003; Heine and Lien 2003; Krutzik, Ochoa et al. 2003; Kang, Yeum et al. 2004).
Evidências epidemiológicas e biológicas sugerem que a hanseníase não
pode ser eliminada apenas por esquema de PQT. Esta teoria resultou de um
estudo matemático recente, que sugere um declínio lento da doença (Meima,
Smith et al. 2004). Esse declínio, porém, se mantém incerto e comprova a
necessidade de uma política de controle mais eficaz da doença. Segundo a
OMS, a hanseníase foi eliminada como um problema de saúde pública mundial
no final do ano de 2000. A eliminação é definida por uma prevalência menor que
um paciente registrado para tratamento por 10.000 habitantes. A prevalência
mundial era de 5.35 milhões de pessoas (12 para 10.000) em 1985 e 597.035
mil pessoas (1 para 10.000) no final de 2000. No último boletim de casos de
hanseníase, observou-se que quatro países necessitam eliminar esta doença:
Brasil, República do Congo, Moçambique e Nepal (Smith and Richardus 2008).
O Brasil é o segundo país no mundo em número de casos novos registrados.
Mais de 64.000 indivíduos infectados estão em tratamento e cerca de 47.000
novos casos são detectados ao ano. Aproximadamente 8% dos novos casos são
em jovens abaixo de 15 anos e 6% apresentam algum grau de alteração motora.
Dos novos casos registrados pelo Ministério da Saúde do Brasil, 53%
apresentam a forma multibacilar (Gráfico 1) (Organization 2007).
5
Carvalho KI
Introdução
Gráfico 1. Incidência de casos de hanseníase no Brasil. Fonte: Ministério
da Saúde
60,000
50,000
40,000
30,000
20,000
10,000
0
Casos
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
14,515 17,133 16,994 18,798 18,854 19,303 18,497 19,728 26,615 27,844 28,765 30,874 33,396 34,251 33,190 36,263 40,505 45,125 42,444 42,389 41,305 44,609 47,506 49,026 49,366 38,410
A hanseníase era descrita como uma doença com características bem
definidas que dependia da resposta imunológica do hospedeiro (mediada por
células) contra o M. leprae
(Ridley and Jopling 1966; Bloom 1986).
Aparentemente a resposta imunológica ocorre em resposta a estimulação
antigênica de linfócitos T (timo-dependente), podendo ser desencadeada
diretamente pelo patógeno ou após o mecanismo de fagocitose por macrófagos.
Isso leva a uma proliferação linfocitária e secreção de citocinas, que aumenta a
capacidade antimicrobiana dos macrófagos. Pacientes que apresentam a forma
PB têm a capacidade de restringir o crescimento do patógeno e produzem uma
resposta de células T contra o M. leprae com especificidade de citocinas, como
o interferon gama (IFN-γ). Entretanto, pacientes que apresentam a forma MB
têm manifestação disseminada do M. leprae, sendo que a resposta de células T
é diminuída e as células T presentes nas lesões expressam interleucina 4 (IL-4)
e IL-10, associadas à resposta imunológica humoral, e suprimem a resposta
imunológica mediada por células (Quiroga, Martinez et al. 2004).
Estudos imunológicos nos extremos do espectro da hanseníase são raros
e geralmente realizados nas lesões de pele. Há evidências, no entanto, que o M.
leprae não induz supressão de maturação/ativação das células dendríticas in
vitro; por outro lado o Mycobacterium tuberculosis e o Mycobacterium bovis,
cepa Calmètte-Guérin, estimulam estas células (Murray, Siddiqui et al. 2007).
6
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Introdução
Um trabalho recente demonstrou que em pacientes com ambas formas de
apresentação clínica nos pólos da doença e com a forma indeterminada
apresentam contagem de células T CD4+ aumentadas, sendo que estas células
produzem uma quantidade maior de IFN-γ
enquanto as células T CD8+
apresentam-se diminuídas na forma multibacilar (Sridevi, Neena et al. 2004).
Embora a imunidade adaptativa tenha um papel essencial na resistência à
infecção pelo M. leprae, alguns indivíduos que apresentam a forma multibacilar
da doença têm danos permanentes em nervos periféricos (Scollard, Adams et al.
2006). A combinação de uma resposta imunológica efetiva e a baixa virulência
do bacilo pode levar o indivíduo a não desenvolver manifestações clínicas da
doença.
2. HIV
O primeiro caso da síndrome da imunodeficiência adquirida (AIDS) foi
relatado no início da década de 80 (Prevention 1983). É causada pelo vírus da
imunodeficiência humana (HIV) e é conhecida por uma infecção persistente que
pode ter um período latência clínica entre a infecção primária e os sintomas
associados a imunodeficiência grave (Barre-Sinoussi 1996). Uma das primeiras
características da infecção pelo HIV é a diminuição da resposta imunológica
celular, com alterações quantitativas nas subpopulações de linfócitos T CD4+ e
CD8+ (Barre-Sinoussi 1996). Esta diminuição pode resultar em infecções
oportunistas recorrentes, que podem ser causadas por fungos, bactérias, vírus e
micobactérias. Uma das infecções oportunistas mais frequentes em pacientes
infectados pelo HIV é a causada pelo M. tuberculosis. Não existem
esclarecimentos suficientes do comprometimento imunológico que as coinfecções exercem em indivíduos infectados pelo HIV.
Os cientistas têm respondido aos desafios desta pandemia: já foram
identificadas a etiologia, a rota de transmissão, os aspectos centrais na
patogênese da imunodeficiência, e foram desenvolvidos testes diagnósticos e
tratamento específico. No entanto, isto não resultou no controle global da
7
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Introdução
pandemia causada pelo HIV. Estima-se que ocorreram mais de 25 milhões de
mortes e 33 milhões de pessoas vivem com o vírus, ao final de 2007, com
consequências socio-econômicas, culturais, e políticas bastante significativas. O
alvo principal dessa epidemia são pessoas em idade economicamente produtiva,
os adultos jovens. A rota de transmissão pode ser sexual, vertical e parenteral,
essa última principalmente envolvendo o uso drogas ilícitas por via intravenosa
e, raramente hoje, transfusão de sangue e hemoderivados. A pandemia está
fora de controle, mesmo com tratamento anti-retroviral existente. No último
boletim epidemiológico do Ministério da Saúde do Brasil, aproximadamente
480.000 casos de AIDS foram notificados até junho de 2007, sendo 60%
localizados na região sudoeste do país (Saúde 2007).
Desde a descoberta do HIV do tipo 1 (HIV-1) em 1983, tem sido a doença
infecciosa mais estudada na história (Barre-Sinoussi, Chermann et al. 1983). O
HIV-1 é um retrovírus e o seu conteúdo genético está disposto em duas fitas
duplas de RNA. A extrema variabilidade dos retrovírus decorre do fato da
transcriptase reversa (TR) não possuir a propriedade de correção durante o
processo de replicação viral, característica comum à DNA-polimerase de outros
organismos. O genoma do HIV é pequeno e contém poucos genes, mas tem a
capacidade de neutralizar e escapar de diferentes componentes da defesa
(Barre-Sinoussi 1996; Emerman and Malim 1998). Atualmente, a classificação
adotada se baseia na análise do genoma completo de amostras de HIV-1
colhidas em diferentes regiões geográficas: grupo M (major), composto por nove
subtipos nomeados A – D, F – H, J e K (as variantes A e F são ainda
segregadas como sub subtipos A1 ou A2 e F1 e F2, respectivamente), e o grupo
O (out-group) e o N (new) (Simon, Ho et al. 2006). Em termos de diversidade
viral, o subtipo C continua dominante e é responsável por 55 a 60% de todas as
novas infecções pelo HIV-1 no mundo (Simon, Ho et al. 2006). Além destes,
várias formas recombinantes circulantes (circulating recombinant forms ou
CRFs) contribuem com o avanço da epidemia (Korber, Gaschen et al. 2001;
Thomson and Najera 2005).
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Introdução
O ciclo de vida do HIV é complexo e a duração e seu desenvolvimento
dependem do tipo e estado de ativação celulares. No início da infecção, o HIV
não causa danos letais às células do sistema imunológico, mas o processo pode
estimular sinais intracelulares que facilitam a replicação viral (Cicala, Arthos et
al. 2002; Balabanian, Harriague et al. 2004). Duas moléculas do envelope viral, a
glicoproteína externa (gp120) e a proteína transmembrana (gp41), formam a
ligação inicial do vírus com a célula alvo (Ray and Doms 2006). Durante este
processo, a gp120 se liga ao receptor de membrana das células T CD4+. Logo,
ocorre a interação entre o vírus e os co-receptores de quimiocinas
(principalmente CCR5 e CXCR4), formando uma ligação irreversível (Eckert and
Kim 2001; Ray and Doms 2006). Esta fusão leva minutos para formar um poro
na membrana celular (Eckert and Kim 2001; Platt, Shea et al. 2005) e liberar o
vírus para o interior citoplasmático. A distribuição destes receptores permitem
que a infecção não seja apenas direcionada às células T CD4+, mas também às
células apresentadoras de antígeno (APCs), incluindo macrófagos e células
dendríticas. O vírus HIV-1 parece ter características biológicas únicas para a
predileção destas células (Stevenson 2003). A destruição gradual de linfócitos T
CD4+ naïve e de memória é a marca da infecção pelo HIV-1, que leva ao
desenvolvimento da AIDS (Douek, Picker et al. 2003). Apesar da ausência de
sintomas durante a fase aguda e crônica na maioria dos casos, a replicação do
HIV é dinâmica. A meia vida do vírus é tão pequena, que metade da população
viral plasmática pode se replicar em menos de 30 minutos (Ramratnam,
Bonhoeffer et al. 1999), sendo que o total de partículas virais produzidas na
infecção crônica de um indivíduo infectado pode chegar à 1010 particulas por dia
(Ramratnam, Bonhoeffer et al. 1999; Simon and Ho 2003). A ativação celular é
um marcador de progressão (Giorgi, Hultin et al. 1999), e parece ser o foco
central da patogenia do HIV.
O diagnóstico da infecção pelo HIV pode ser realizado através da
detecção de anticorpos específicos, antígenos, ou ambos, e vários ensaios
sorológicos estão disponíveis nos dias de hoje. Testes imunoenzimáticos são
geralmente utilizados para triagem. Para acompanhamento da infecção é
9
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necessário realizar a contagem de células T CD4+ e a quantificação de cópias
de RNA do HIV no plasma (carga viral). A carga viral é utilizada principalmente
para
monitoramento
do
tratamento
anti-retroviral.
Vários
testes
para
quantificação carga viral também estão disponíveis nos dias atuais. A carga
viral, contudo, não determina o estágio destrutivo do sistema imunológico. Por
outro lado, o número de células T CD4+ revela o grau de imunodeficiência e é
utilizado como medidor do avanço para doença. O critério para classificação da
infecção do HIV é medido através da contagem de células T CD4+ e
manifestções clínicas (infecções oportunistas). A citometria de fluxo é o método
padrão para a quantificação de células T CD4+ no sangue periférico.
O tratamento anti-retroviral é a melhor opção para a supressão viral e,
subsequente, para a redução da morbidade e mortalidade. Nos dias de hoje, há
aproximadamente 20 drogas anti-retrovirais disponíveis para tratamento. Definir
o melhor momento para iniciar o tratamento anti-retroviral é uma das decisões
mais importantes no acompanhamento do indivíduo infectado pelo HIV. O
Ministério da Saúde do Brasil determina que pacientes com doença sintomática
e os que, apesar de assintomáticos, apresentam imunodeficiência avançada
(contagem de linfócitos T CD4+ abaixo de 200/mm3) devem iniciar tratamento; já
aqueles com contagem entre 200 e 350 céls./mm3 devem iniciar o tratamento na
presença de manifestações clínicas associadas a imunodeficiência ou a critério
do médico que está assistindo ao indivíduo (Saúde 2006).
3. Coinfecção HIV e Hanseníase
A infecção pelo HIV tem um efeito profundo na incidência e na patologia
clínica da tuberculose. No entanto, houve uma preocupação no início da
epidemia quanto a possibilidade de existir uma interação similar entre a infecção
pelo HIV e a hanseníase, que não foi confirmada até hoje.
A prevalência da infecção pelo HIV tem aumentado em países endêmicos
para a hanseníase. O número de pacientes com a co-infecção, no entanto, não
10
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Introdução
foi estimado e acredita-se que a sobreposição geográfica dessas duas doenças
poderá resultar no aumento do número de indivíduos com ambas infecções
(Figura 1) (Ustianowski, Lawn et al. 2006). O tempo prolongado de incubação da
infecção pelo M. leprae e sua baixa incidência tornam complicada a realização
de um estudo prospectivo de incidência da co-infecção ou um estudo casocontrole de pacientes infectados ou não pelo HIV.
Figura 1. Distribuição mundial do número de adultos e adolescente que
vivem com HIV/AIDS até o final de 2002 e seis paises com o maior número de
novos casos com hanseníase no mesmo ano (adaptado de Ustianowski, Lawn et
al. 2006).
Alguns estudos demonstraram que a prevalência de casos de pacientes
co-infectados pelo HIV/M. tuberculosis ou infecções pelo M. avium tende a ser
maior que os casos de HIV/M. leprae (Meeran 1989; Borgdorff, van den Broek et
al. 1993; Moses, Adelowo et al. 2003). Lucas B e cols. constataram que a
prevalência de pacientes infectados pelo HIV entre os casos de hanseníase não
é elevada quando comparada com o grupo de pacientes não infectados pelo
HIV, e a co-infecção não afetou o espectro clínico de pacientes que
apresentaram a forma multibacilar da hanseníase (Lucas, Fine et al. 1995).
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Um estudo realizado na Tanzânia, em 1990, identificou que 83 (12,2%)
estavam infectadas pelo HIV entre 679 pessoas infectadas pelo M. leprae. Este
resultado deve ser visto com cautela, já que o diagnóstico da infecção pelo HIV
contava com apenas um tipo de ensaio imunoenzimático (van den Broek, Chum
et al. 1997). De fato, já havia sido descrito que a hanseníase podia afetar a
especificidade e sensibilidade de alguns testes para o diagnóstico da infecção
pelo HIV (ShivRaj, Patil et al. 1988; Andrade, Avelleira et al. 1991), porque
pacientes com hanseníase multibacilar podem apresentar hipergamaglobulemia
que eventualmente resultava em testes falso-positivos em sorologias para sífilis
e ensaios de fator reumatóide (Murray 1982; Harboe 1988). Também foi descrito
que pacientes com forma multibacilar da hanseníase produzem anticorpos
contra antígenos micobacterianos, que podem ocasionar reações cruzadas em
ensaios sorológicos com proteínas Pol e Gag do HIV-1 (Kashala, Marlink et al.
1994; Milanga, Kashala et al. 1999), embora alguns trabalhos tenham falhado
em detectar estes efeitos (Lucas, Fine et al. 1995; Sterne, Turner et al. 1995).
O espectro clínico da hanseníase depende do sistema imunológico do
hospedeiro. O HIV afeta a resposta imunológica celular, sugerindo, portanto, que
pacientes co-infectados apresentariam a forma multibacilar da hanseníase com
maior freqüência (Turk and Rees 1988). Cinco grandes estudos descreveram
que a razão entre as formas paucibacilar e multibacilar não muda
significantemente com a co-infecção pelo HIV (Munyao, Bwayo et al. 1994; van
den Broek, Chum et al. 1997; Gebre, Saunderson et al. 2000).
Alguns estudos sugeriram que pacientes co-infectados podem ter uma
hanseníase mais severa, com neurite e aumento de reações reversas. Também
sugerem que o uso de terapia anti-retroviral pode potencializar efeitos adversos,
aumentando os episódios inflamatórios. Não há evidências, todavia, que a
neurite esteja aumentada na co-infecção, apesar do HIV poder ter efeito
neuropático concomitante (Pereira, Stefani et al. 2004; Ustianowski, Lawn et al.
2006). Um estudo histopatológico realizado em pacientes co-infectados
demonstrou que alguns genes produtores de citocinas, como, IL-4, IL-10, IFN-γ e
12
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Introdução
TNF-α tendem a ter sua expressão aumentada, mesmo com as células T CD4+
diminuídas no organismo (Sampaio, Caneshi et al. 1995).
A grande modificação clínica observada na co-infecção entre hanseníase
e o HIV ocorreu após o advento do tratamento anti-retroviral, que possibilitou a
caracterização de reações reversas como fenômenos de reconstituição imune.
Há atualmente poucos casos documentados desta associação, na qual há
aparecimento de manifestações clínicas da hanseníase previamente latente,
após a introdução do tratamento anti-retroviral (Couppie, Abel et al. 2004;
Hirsch, Kaufmann et al. 2004; Goebel 2005; Batista, SM. et al. 2007; Kharkar,
Bhor et al. 2007; Murdoch, Venter et al. 2007).
As diversas conclusões destes estudos refletem a dificuldade de estudar
a interação de ambas doenças, sendo que a maioria dos estudos realizados
possui amostragens pequenas de co-infectados.
4. Células T natural killer (NKT)
As células T natural killer (NKT) são linfócitos T especializados e com
características funcionais únicas. Originalmente descritos em camundongos,
essas células expressam o receptor de célula T (TCR) e o receptor C-lectina de
células natural killer (NK), o NK 1.1 (CD161 nos humanos) (Bendelac 1995;
Bendelac, Rivera et al. 1997; Godfrey, Hammond et al. 2000; Kronenberg and
Gapin 2002). Aproximadamente, 5 a 10% das células T do sangue periférico
expressam CD161 (Unutmaz 2003; Kronenberg 2005). A maioria das células
NKT humanas expressam TCRs V 24-J 18/V 11(Bendelac, Savage et al.
2007). Estas células têm um papel importante em várias respostas imunológicas,
incluindo ação anti-tumoral, em doenças auto-imunes e em infecções virais e
bacterianas (Godfrey, Hammond et al. 2000).
As subpopulações de células NKT diferem na expressão de moléculas de
integrina envolvidas na interação de célula-célula e na matriz extracelular (Rolf,
Berntman et al. 2008). Existem no mínimo duas subpopulações distintas, CD4+
e CD4-, nos humanos e nos macacos, que também podem expressar fenótipos
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CD8+ e CD4-CD8- (Prussin and Foster 1997; Kawano, Nakayama et al. 1999;
Nicol, Nieda et al. 2000; Metelitsa, Naidenko et al. 2001). As subpopulações
CD4+ tendem a produzir citocinas tanto Th1 (IFN-γ) e Th2 (IL-4, IL-10), embora
células NKT CD4- produzam apenas citocinas do tipo Th1 (Chen, Wang et al.
2007). Grumperz e cols., demonstraram que células NKT CD4+ e CD4- têm
diferentes expressões de perforina e células CD4- expressam o receptor
coestimulatório de células NK (NKG2D). Já a expressão de FASL é
aparentemente limitada à subpopulação CD4+ (Gumperz, Miyake et al. 2002).
Eger e cols., no entanto, descreveram que as células NKT dos neonatos são
diferentes na função e no fenótipo quando comparadas às células de adultos.
Apresentam alta expressão de marcadores de diferenciação, como CCR7 e
CD62L, e baixa expressão de marcadores de células NK (CD94) além de serem
mais efetoras (Eger, Sundrud et al. 2006).
A expressão de CD4 distingue as subpopulações de NKT fenotipicamente
e funcionalmente. Não é claro que a molécula CD4 é simplesmente um
marcador da linhagem das células NKT ou as subpopulações CD4 contribuam
diretamente para as propriedades funcionais das células NKT (Chen, Wang et al.
2007). A molécula CD4 tem a capacidade de ativar células T restritas ao MHC II,
via domínio terminal D1, e essa ligação parece estabilizar a interação TCR/MHC
classe II (Wang, Meijers et al. 2001; Wang and Reinherz 2002) . Chen e cols.,
demonstraram que a molécula CD4 pode contribuir com as vias de sinalização
das células NKT, com sinais de coestimulação na molécula MHC classe II nas
células T. Os resultados apresentados mostraram que a molécula CD4 pode
afetar a ativação das células NKT, independente do ligante na célula
apresentadora de antígeno (APC)
(Chen, Wang et al.
2007).
Estas
subpopulações podem ser distribuídas com diferentes frequências e em diversos
tecidos (Eberl, Lees et al. 1999; Hammond, Pelikan et al. 1999). No início da
gestação são encontrados os progenitores das células NKT e estes são mais
frequentes no timo, mas o número relativo declina com o tempo e são raros ou
ausentes no timo após o nascimento. No sangue periférico de adultos, 50% das
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Introdução
células NKT são CD4- com baixa ou nenhuma expressão de CD8 (Sandberg,
Stoddart et al. 2004).
Há muito interesse em descobrir os sinais necessários para o
desenvolvimento e funções das células NKT. Células T convencionais e NKT,
expressam receptores αβ. Um número considerável de estudos recentes, por
outro lado, sugerem que os sinais necessários para o desenvolveimento e
função destas células são diferentes dos necessários para as células T
convencionais (Au-Yeung and Fowell 2007). Alguns estudos têm sugerido que
uma função importante das células NKT pode ser a de proteger tecidos
(particularmente órgãos vitais) de danos inflamatórios ocasionados pela resposta
imunológica (Godfrey, Hammond et al. 2000). Estas células têm efeitos múltiplos
na resposta imunológica, incluindo ativação, regulação e atração de células do
sistema imunológico inato e regulação do sistema imunológico adaptativo (Rolf,
Berntman et al. 2008).
As células NKT têm capacidade especial de reconhecer antígenos
associados a molécula de CD1. Existem evidências que membros da família
CD1 nos humanos podem reconhecer algumas células T γδ e αβ que se ligam
ou são expressos em células T CD4+ e CD8+ (Bendelac, Lantz et al. 1995). A
familia CD1 consiste em dois grupos de lipídeos, incluindo CD1a, CD1b e CD1c
no Grupo I e CD1d no Grupo II. Após estímulo, as células NKT restritas a CD1d
rapidamente produzem várias citocinas do tipo Th1 e Th2 (Kronenberg and
Gapin 2002; Taniguchi, Harada et al. 2003; Godfrey and Kronenberg 2004;
Mercer, Ragin et al. 2005; Seino, Motohashi et al. 2006).
Nos camungongos, as células NKT foram detectadas em diversos tecidos
e as proporções destas células maduras nos tecidos são consideravelmente
diferentes: fígado 30 a 50%, medula óssea 20 a 30%, e no timo 10 a 20% e 0,3
a 0,5% dos timócitos totais. Há presença destas células em menor quantidade
no baço (3%), linfonodos (0,3%), sangue periférico (4%) e pulmão (7%)
(Godfrey, Hammond et al. 2000). Nos humanos a distribuição de células NKT
nos tecidos não esta bem definida, mas está claro que são raras no fígado
(<1%) quando comparadas ao modelo de camundongo (Ishihara, Nieda et al.
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Introdução
1999; Exley, He et al. 2002; Kenna, Golden-Mason et al. 2003) e tendem, nos
humanos, a ser aproximandamente dez vezes menos freqüêntes em todas as
outras localizações (Bendelac, Savage et al. 2007). A freqüência de células NKT
Vα24 no sangue periférico em indivíduos normais varia de 0.01 a 1%, fazendo
com que seja difícil de avaliar as diferenças entre indivíduos normais e aqueles
que apresentam algum tipo de patologia (Ohteki and MacDonald 1994; Nuti,
Rosa et al. 1998; Emoto, Emoto et al. 1999).
Um dos componentes mais eficientes para ativar as células NKT é um
glicolípedo sintético (originalmente derivado das esponjas marinhas) (Hong,
Scherer et al. 1999; Hayakawa, Godfrey et al. 2004) conhecido como
-
galctosilceramida ( -GalCer), que se liga efetivamente à molécula CD1d. O
complexo CD1d e o glicolípideo então se liga ao TCR das células NKT (Sidobre,
Naidenko et al. 2002). As células NKT podem ser identificadas por um
fluorocromo conjugado, a um complexo de tetrâmero CD1d com α-GalCer
(Benlagha, Weiss et al. 2000; Matsuda, Naidenko et al. 2000; Hammond, Pellicci
et al. 2001). Vários estudos têm utilizado o α-GalCer, um potente agonista das
células NKT (Hayakawa, Godfrey et al. 2004). Várias outras moléculas de
glicolipídeos foram testadas como estimuladores destas células e de suas
subpopulações, incluindo glangliosideo GD3 (Wu, Segal et al. 2003),
glicofosfatidilinositol (Schofield, McConville et al. 1999; Hansen, Siomos et al.
2003), fosfoetanolamina (Rauch, Gumperz et al. 2003), e algumas formas de βGalCer (Ortaldo, Young et al. 2004).
Quando ativadas, as células NKT respondem vigorosamente com
produção de citocinas após uma a duas horas (Godfrey, Hammond et al. 2000;
Kronenberg and Gapin 2002). Essas células têm capcidade de secretar IFN-γ,
TNF-α e também citocinas do tipo Th2, incluindo IL-4 e IL-13 (Smyth and
Godfrey 2000; Wilson, Johansson et al. 2003). Além disso, são capazes de
secretar simultaneamente citocinas tipo Th1 e Th2. Curiosamente, o estímulo de
sangue total ex vivo faz com que não secretem muita quantidade de citocinas
Th2 (Gadue and Stein 2002). O estímulo α-GalCer atua através da molécula de
CD4+ e CD40L, que, conseqüentemente, se liga ao CD40+ da APC, induzindo a
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Introdução
produção de IL-12 (Tomura, Yu et al. 1999). O mecanismo exato através do qual
as células NKT secretam as citocinas não está totalmente elucidado, o que é um
desafio para especialistas que atuam nessa área (Figura 2A e B).
APC
CD1d + glicolip’deo
(ex. α-GalCer, OCH, C-glicos’deo)
TCR
IL-7
IL-12
Diminui‹ o do sinal de TCR?
Aumento do sinal TCR?
Tipo de APC?
NKT
Tipo APC?
Outros fatores?
Outros fatores?
IL-4
IFN-γγ
IL-10
CD40L
IL-13
DC
Gr-1+
Tempo?
CTL
IL-12
CD8+
Treg
NK
Prote‹ o imunol—gica
e Th1
IFN-γγ
CD8DC
TGF-β
β
Supress‹ o imunol—gica
e Th2
Figura 2 A e B: Esquema de como as células NKT influenciam a resposta
imunológica. (A) O lado em verde demosntra fatores que podem gerar uma
resposta Th1 pelas células NKT, enquanto o lado vermelho demonstra fatores
que podem gerar uma resposta Th2 pelas células NKT. (B) Subpopulações de
células NKT humanas CD4- e CD4+ são programadas para produzir diferentes
razões de citocinas Th1/Th2 (adaptado de Godfrey & Kronenberg, 2004).
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Introdução
CD1d + glicolip’deo
CD1d + glicolip’deo
TCR
TCR
IFN-γγ
CD40L
IL-12
IL-4
IL-10
IL-13
TGF-β
β
Prote‹ o imunol—gica
e Th1
IFN-γγ
Supress‹ o imunol—gica
e Th2
As células NKT têm um papel importante no desenvolvimento de outras
respostas, incluindo a prevenção e o desenvolvimento de certas doenças autoimunes, inibição do desenvolvimento de tumores e crescimento e a eliminação
de algumas infecções. A depleção destas células pode ter um papel importante
durante a infecção pelo HIV (Sandberg, Fast et al. 2002; van der Vliet, von
Blomberg et al. 2002). É possível que a diminuição de células NKT vista em
pacientes infectados esteja relacionada à suceptibilidade destes indivíduos em
desenvolverem doenças como o sarcoma de Kaposi (Crowe, Godfrey et al.
2003).
As células NKT CD4+ expressam o receptor de quimiocina 5 (CCR5),
também co-receptor da ligação do HIV à célula alvo (impresso de unutmaz03).
Alguns trabalhos observaram que as células NKT (Vα24+Vβ11+) no sangue
18
Carvalho KI
Introdução
periférco de indivíduos infectados pelo HIV ocorrem em percentagens menores
quando comparados com indivíduos saudáveis. Também foi descrito que existe
uma correlação positiva entre a carga viral do HIV e o número de células NKT
CD4+ . Estas células são mais suceptíveis ao tropismo R5 na infecção do HIV
quando comparadas com o tropismo X4 (Motsinger, Haas et al. 2002; Sandberg,
Fast et al. 2002; van der Vliet, von Blomberg et al. 2002). A suceptibilidade das
células NKT para com o vírus HIV R5, é dependente do nível de moléculas
CCR5 expressas nestas células. O número reduzido de células NKT,
especialmente a subpopulação CD4+, em pacientes infectados pelo HIV deve ter
consequências significativas devido às suas importantes funções, descritas
anteriormente (Unutmaz 2003).
Alguns autores descreveram que as células NKT são protetoras durante
infecções bacterianas (Gumperz and Brenner 2001) e virais (Nuti, Rosa et al.
1998; Asselin-Paturel, Boonstra et al. 2001). Com isso, podem ter papel
importante durante as infecções oportunistas, freqüêntes nos pacientes
imunodeficientes (Unutmaz 2003). Umas das co-infecções mais comuns em
portadores do HIV são as causadas por micobactérias (Barnes, Bloch et al.
1991). As células NKT são conhecidas por reconhecer e responder às infecções
micobacterianas (Apostolou, Takahama et al. 1999; Emoto, Emoto et al. 1999).
Existem algumas evidências que antígenos glicolipídicos de micobactérias
apresentados pela molécula CD1d podem ativar funções efetoras das células
NKT, tais como secreção de citocinas e citotoxicidade contra células infectadas
por micobacterias (Apostolou, Takahama et al. 1999). Todavia, estas células são
capazes de agir diretamente com a secreção de granulosina na ação antimicobacteriana (Gansert, Kiessler et al. 2003). Portanto, a diminuição das
células NKT em indivíduos infectados pelo HIV podem contribuir para infecções
micobacterianas.
No presente trabalho, é apresentada uma série de experimentos para investigar
diferentes aspectos da resposta imunológica do tipo celular em pacientes coinfectados pelo M. leprae e pelo HIV-1. Entre os diversos aspectos analisados,
foi possível demonstrar várias alterações vistas nessa co-infecção, que podem
19
Carvalho KI
Introdução
auxiliar a compreensão de fenômenos envolvidos na fisiopatogênese de ambas
doenças e contribuir com o entendimento do impacto das co-infecções em
pessoas que vivem com HIV.
20
Carvalho KI
Objetivos
Objetivo Principal
Caracterizar parâmetros imunológicos em pacientes que apresentam a
coinfecção pelo HIV-1 e Mycobacterium leprae.
Objetivo 1
Caracterizar ativação celular, contagem de células T e distribuição de
células dendríticas.
Objetivo 2
Avaliar a distribuição do estado de maturação dos linfócitos T CD4+.
Objetivo 3
Identificar a resposta produtiva de IL-4, INF-γ e TNF-α após estímulo
inespecífico.
Objetivo 4
Identificar a distribuição e a função de células NKT na infecção pelo
HIV-1 comparadas com controles.
Objetivo 5
Identificar a distribuição e função da população de células NKT nos
grupos de indivíduos coinfectados e respectivos controles.
20
Carvalho KI
Trabalho 1
Hanseníase e o vírus da imunodeficiência humana do tipo 1 (HIV) são
exemplos de infecções humanas cuja interação entre o patógeno e a
imunidade celular do hospedeiro determina a manifestação clínica da doença.
No entanto, é esperada uma interação significantiva dos aspectos
imunopatológicos entre o HIV-1 e a hanseníase. Neste estudo, avaliamos
vários aspectos da imunidade celular em pacientes coinfectados com HIV-1 e
a Mycobacterium leprae. Vinte oito indivíduos foram estudados, divididos em
quarto grupos: controles saudáveis, coinfectados pelo HIV-1 e M. leprae,
monoinfectados pelo HIV-1, e monoinfectados pelo M.lepare. Os indivíduos
dos grupos monoinfectados e coinfectados foram pareados tanto quanto
possível pela carga bacilar e parâmetros imunológicos relacionados ao HIV.
Células mononucleares do sangue periférico (CMSP) foram analisadas no
citômetro de fluxo de seis e sete cores para avaliar subpopulações e níveis
de ativação cellular, distribuição de fenótipos de células dendríticas (DC) e
expressão de IL-4 por células T. O grupo coinfectado apresentou diminuição
da razão de células CD4:CD8, aumento de níveis de ativação de células T
CD8+, aumento da razão de células T Vδ1:Vδ2 e diminuição da porcentagem
de células dendríticas plasmocitóides, comparadas com o grupo de
indivíduos monoinfectados pelo HIV-1. A produção de IL-4 por linfócitos T
CD4+ foi correlacionado positivamente com a porcentagem da subpopulação
de memória efetora de células T CD4+, sugerindo diferenciação antigênica
da população de células T em ambas as infecções de HIV-1 e M. leprae. A
coinfecção com o M. lepare pode exacerbar a imunopatologia da doença do
HIV-1. Houve uma tendência na expressão de citocinas Th2 na resposta de
células T CD4+ em ambas infecções, mas não obtivemos efeitos aditivos
aparentes nos pacientes coinfectados.
21
IMMUNOLOGY
ORIGINAL ARTICLE
Immune cellular parameters of leprosy and human
immunodeficiency virus-1 co-infected subjects
Karina I. Carvalho,1 Solange
Maeda,1 Luciana Marti,2 Jane
Yamashita,1 Patrick A. J. Haslett3
and Esper G. Kallas1
1
Federal University of São Paulo, São Paulo,
Brazil, 2Albert Einstein Research Institute, São
Paulo, Brazil, and 3University of Miami, FL,
USA
doi:10.1111/j.1365-2567.2007.02756.x
Received 1 June 2007; revised 15 October
2007; accepted 16 October 2007.
Correspondence: E. G. Kallas, MD, PhD,
Laboratório de Imunologia, Disciplina de
Doenças Infecciosas e Parasitárias, Escola
Paulista de Medicina/UNIFESP, Rua Mirassol
207, 04044-010 - São Paulo – SP, Brazil.
Email: [email protected]
Senior author: Esper Kallas
Abstract
Leprosy and human immunodeficiency virus-1 (HIV-1) are examples of
human infections where interactions between the pathogen and the host
cellular immunity determine the clinical manifestations of disease. Hence,
a significant immunopathological interaction between HIV-1 and leprosy
might be expected. In the present study we explored several aspects of cellular immunity in patients co-infected with HIV-1 and Mycobacterium
leprae. Twenty-eight individuals were studied, comprising four groups:
healthy controls, HIV-1 and M. leprae co-infection, HIV-1 mono-infection, and M. leprae mono-infection. Subjects in the mono-infection and
co-infection groups were matched as far as possible for bacillary load and
HIV disease status, as appropriate. Peripheral blood mononuclear cells
(PBMC) were analysed using six- and seven-colour flow cytometry to
evaluate T-cell subpopulations and their activation status, dendritic cell
(DC) distribution phenotypes and expression of IL-4 by T cells. The
co-infected group exhibited lower CD4 : CD8 ratios, higher levels of
CD8+ T-cell activation, increased Vd1 : Vd2 T cell ratios and decreased
percentages of plasmacytoid DC, compared with HIV-1 mono-infected
subjects. Across infected groups, IL-4 production by CD4+ T lymphocytes
was positively correlated with the percentage of effector memory CD4+ T
cells, suggesting antigenically driven differentiation of this population of
T cells in both HIV-1 and M. leprae infections. Co-infection with M. leprae may exacerbate the immunopathology of HIV-1 disease. A T helper
2 (Th2) bias in the CD4+ T-cell response was evident in both HIV-1
infection and leprosy, but no additive effect was apparent in co-infected
patients.
Keywords: HIV; leprosy; co-infection; lymphocytes; IL-4
Introduction
Leprosy is a chronic infectious disease, affecting the skin
and peripheral nerves, caused by the intracellular bacillus
Mycobacterium leprae.1 The incidence of new cases of leprosy remains constant at 286 000 per year, and Brazil is
one of the countries worst affected, accounting for the
majority of new cases reported in the Americas.2 As the
prevalence rates of human immunodeficiency virus-1
(HIV-1) infection are escalating in some countries where
leprosy is endemic, one might expect that the geographic
overlap of the two epidemics may lead to increased
numbers of co-infected patients. The current situation
206
concerning leprosy endemicity and HIV-1 prevalence in
Brazil and other countries emphasizes the importance of
monitoring for co-infections.3 In addition to the public
health aspect of this co-infection, these pathogens may
have a potentially interesting immunologic interaction in
the human host.
It has been previously suggested that leprosy is a
human infection model in which to study the T helper
1/T helper 2 (Th1/Th2) paradigm,4 permitting the delineation of polarized human T helper responses in response
to a single pathogen. The spectrum of M. leprae-specific
immune responses between these poles correlates with the
range of clinical manifestations of the infection.5 At the
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
Immunity in M. leprae and HIV-1 co-infection
Th1 pole, tuberculoid, or paucibacillary, leprosy is
characterized by high levels of specific cell-mediated
immunity that effectively limits bacillary replication and
is associated with limited disease, although often with
concomitant immunological damage to the nerves. At the
other pole, lepromatous, or multibacillary, leprosy is
characterized by a selective unresponsiveness to M. leprae
antigens, diffuse cutaneous disease and the uncontrolled
multiplication of organisms in the skin, often to extraordinary numbers. Much of the morbidity of leprosy
results from episodic inflammatory exacerbations of leprosy lesions in the skin and nerves, called ‘lepra’ reactions, thought to be caused by spontaneous shifts in host
immunity.6
Because HIV-1 infection has a profound effect on the
incidence and clinico–pathological features of other mycobacterial diseases, such as tuberculosis, one might expect a
significant interaction also to exist between HIV-1 and
leprosy.7 In HIV-1/M. tuberculosis co-infections, immune
suppression secondary to HIV-1 infection accelerates the
progress of tuberculosis, and, conversely, the cellular
immune activation associated with tuberculosis is associated with more rapid progression of HIV-1 disease.8,9 In
the setting of HIV-1/M. leprae co-infections, there has
been a general expectation that immune deficiency caused
by HIV-1 infection would shift the spectrum of leprosy
towards the lepromatous (Th2) pole, although epidemiological data are sparse and conflicting.10 Paradoxically, the
most detailed description of leprosy immunopathology in
HIV-1 co-infected patients revealed no change in immune
cell infiltrates across the leprosy spectrum, despite
advanced HIV-1-associated immune deficiency.7,11 On the
other hand, there has been little or no attempt to evaluate
the impact of M. leprae infection on HIV-1 pathogenesis.
This interaction has been studied in the macaque/simian
immunodeficiency virus (SIV) model, however, where
M. leprae infection was observed to exert an unexpected
and unexplained ameliorating effect on SIV disease, prolonging survival of the animals, despite equal or increased
viral burdens.12
In light of these various reported interactions of mycobacterial infections with HIV and SIV pathogenesis, we
were interested in investigating whether human M. leprae
co-infection might exacerbate or attenuate HIV-1 pathogenesis. As an initial exploration of these questions, we
performed a cross-sectional analysis of immune cellular parameters in blood cells from relatively rare HIV-1
and M. leprae co-infected subjects, in comparison with
HIV-1 and M. leprae mono-infected subjects, and healthy
volunteers.
Our investigation focused on peripheral blood immune
cells that are known to be altered in HIV-1 disease and
that are implicated in the immunity and/or pathogenesis
of mycobacterial infections, including leprosy. Thus, in
addition to CD4 and CD8 T-cell subsets, we examined
the two main subsets of cd T cells: Vd2 cells and
Vd1 cells. Vd2 cells are stimulated by isoprenoid
phosphoantigens that are present in bacteria, including
mycobacteria.13 This population of cells plays a role in
antimycobacterial defense,14,15 but the cell population
shrinks dramatically during acute HIV-1 infection, with
variable recovery following antiviral chemotherapy.16,17 In
contrast, the Vd1 population, of unknown function,
expands during HIV-1 infection, so that the ratios of Vd1
to Vd2 T cells are increased with progressive HIV-1 infection.16,17 We also examined the two main subsets of
peripheral blood dendritic cells (DC), called plasmacytoid
and myeloid DC. DC are key components of the innate
immune system, acting as antigen-presenting cells that are
essential for the priming and regulation of T-cell immunity. Hence, the responses and interactions of these populations of DC are thought to determine whether T cells
differentiate into Th1 or Th2 cells,18 spanning the range
of phenotypes observed in leprosy. Mycobacteria are
known to stimulate DC via toll-like receptors (TLR) present on both myeloid (TLR2) and plasmacytoid (TLR9)
subsets.19–21 Both subsets of DC can be infected by HIV-1,
but a differential and striking loss of peripheral blood
plasmacytoid DC characterizes progressive HIV-1 disease.22 In light of the complex and contrasting effects of
HIV-1 and mycobacterial infections on cd T-cell and DC
populations, we were interested in examining these
immune cells in patients with HIV-1 and M. leprae
co-infections.
Materials and methods
Subjects and sample collection
This study was reviewed and approved by the local institutional review board (IRB, Comitê de Ética em Pesquisa
Humana da Universidade Federal de São Paulo/UNIFESP),
and IRB-approved informed consent was obtained from
all participants. Leprosy patients were treated according to
World Health Organization guidelines.23 Acquired immunodeficiency syndrome (AIDS) was defined using modified
criteria adopted by the Brazilian Ministry of Health that
includes patients with a CD4 cell count of < 200 cells/ll
or clinical conditions related to AIDS.24
Seven healthy controls and seven HIV-seropositive
patients, most of whom had CD4+ T-cell counts of < 400
cells/ll, were identified at UNIFESP. Seven patients with
leprosy were enrolled at the Leprosy Clinic at the State
Health Department (Sao Paulo, Brazil) and were classified
according to their bacillary load.25 Seven patients
co-infected with leprosy and HIV-1 infection were
recruited at UNIFESP, using local identification and referral from other services in Sao Paulo. Leprosy patients
were matched for bacillary load with the patients in the
co-infected group. In this study, the major presentations
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
207
K. I. Carvalho et al.
of leprosy were the paucibacillary form rather than the
multibacillary form.
The HIV mono-infected and co-infected patients were
receiving highly active antiretroviral therapy (HAART)
and multidrug therapy (MDT). Patients with immune
reconstitution inflammatory syndrome were not included
in the present study to avoid potential interference in the
immune parameters, as described in different mycobacterial diseases.26–28
Peripheral blood mononuclear cells (PBMC) were isolated from the study subjects by density-gradient sedimentation over Ficoll–Paque (Pharmacia Biotech,
Uppsala, Sweden). The isolated PBMC were then washed
twice in Hank’s balanced salt solution (Gibco, Grand
Island, NY). Cells were cryopreserved in RPMI 1640
(Gibco), supplemented with 20% heat-inactivated fetal
bovine serum (FBS; HyClone Laboratories, Logan, UT),
50 U/ml of penicillin (Gibco), 50 lg/ml of streptomycin
(Gibco), 10 mM glutamine (Gibco) and 75% dimethyl
sulphoxide (DMSO; Sigma, St Louis, MO). Cryopreserved cells were stored in liquid nitrogen until used in
the assays. At the time of the assay, PBMC were rapidly
thawed in a 37° water bath and washed in RPMI 1640
supplemented with 10% fetal calf serum, 100 U/ml of
penicillin, 100 lg/ml of streptomycin and 20 mM
glutamine (R10). Cells were counted, checked for viability and resuspended in R10 at a concentration of 106
cells/ml.
Plasma HIV-1 RNA detection
The plasma HIV RNA detection load was assessed using
the ultrasensitive AMPLICOR HIV-1 MONITOR test version 1.5 (Roche Diagnostics, Indianapolis, IN), according
to the manufacturer’s instructions.
Flow cytometry
The following monoclonal antibodies were used for
surface staining: CD3–allophycocyanin (APC) (clone
UCHT1), CD8–allophycocyanin carbocyanin 7 (APCCy7) (clone SK1), Vd2–phycoerythrin (PE) (cloneB6),
CD45RA–peridin chlorophyll protein (PerCP) (clone HI
100), CCR7–phycoerithrin carbocyanin 7 (PeCY7) (clone
3D12) and CD69–fluorescein isothiocyanate (FITC)
(clone FN50), from BD PharMingen (San Jose, CA);
CD4–Alexa 610 (clone S3.5) from Caltag Laboratories
(Burlingame, CA); Vd1–FITC (clone T58.2) from Endogen (Rockford, IL); Lineage Cocktail 1 (Lin 1: CD3,
CD14, CD16, CD19, CD20 and CD56) FITC, human
leucocyte antigen (HLA)-DR–PerCP (clone L243),
CD11c–APC (clone S-HCL3), CD123–PE [anti-interleukin
(IL)-3 receptor], CD38–PE (clone HB7), CD4–FITC (clone
L120), from BD Biosciences (San Jose, CA); and CD25–
PE–CY7 (clone BC96), from e-Bioscience, (San Diego,
208
CA). Intracellular staining for cytokines was performed
using mouse anti-human IL-4–PE (clone 3010.211), mouse
anti-human interferon (IFN)-c–PE–CY7 (clone B27) and
mouse anti-human tumour necrosis factor (TNF)-a–APC
(clone Mab11), all from BD PharMingen. Fluoresce minus
one (FMO) was used for gate strategy.29
In some experiments, thawed PBMC were incubated in
24-well plates (1 ml/well) (Becton Dickinson, San Jose,
CA) in the presence of 1 lM ionomycin (Sigma) and
20 ng/ml of phorbol 12-myristate 13-acetate (PMA;
Sigma), for 16 hr. After stimulation, cells were centrifuged
at 1500 g for 5 min and transferred into V-bottom
96-well plates (Nunc, Roskilde, Denmark) in 100 ll of
staining buffer [phosphate-buffered saline (PBS) supplemented with 01% sodium azide (Sigma) and 1% FBS,
pH 74–76] with the panel of surface monoclonal antibodies. Cells were incubated at 4° in darkness for 30 min,
washed twice and then resuspended in 100 ll of fixation
buffer [1% paraformaldehyde (Polysciences, Warrington,
PA) in PBS, pH 74–76].
Intracellular staining was performed after surface staining with CD4–FITC, CD3–PerCP and CD8–APC–CY7.
Cells were incubated with 100 ll of 4% fixation buffer
and washed with permeabilization buffer (PBS supplemented with 01% sodium azide, 1% FBS and 01% saponin; Sigma). Each sample was resuspended in 100 ll of
permeabilization buffer, incubated for 15 min at room
temperature in the dark, washed with 100 ll of staining
buffer and incubated for 30 min at 4° in the dark with
either no antibody (unstained tube) or anti-IL-4–PE, antiIFN-c–PE–CY7 and anti-TNF-a–APC in 50 ll of staining
buffer.30 Cells were washed with 200 ll of staining buffer
and resuspended in 100 ll of 1% paraformaldehyde (PFA)
for flow cytometry analysis. Samples were acquired on a
FACSCanto or FACSAria, using FACSDIVA software (BD
Biosciences), and the analysed with FLOWJO software (Tree
Star, San Carlo, CA). Fluorescence voltages were determined using matched unstained cells. Compensation was
carried out using CompBeads (BD Biosciences) singlestained with CD3–PerCP, CD4–FITC, CD8–APC–CY7,
CD4–PE–CY7, CD3–PE or CD3–APC. Samples were
acquired until at least 200 000 events in a live lymphocyte
gate or at least 500 000 events in a live DC gate were
obtained.
Statistical analyses
Groups were compared using non-parametric models;
data are reported as median and interquartile range.
Comparisons among groups were carried out using the
Kruskall–Wallis non-parametric test, followed by intergroup comparisons by the Dunnet test. Correlations were
performed using the Spearman non-parametric test.
P-values were considered significant if <005. Results are
expressed in medians and interquartile ranges (IQR).
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
Immunity in M. leprae and HIV-1 co-infection
Table 1. Demographic, clinical and laboratory characteristics of participants
Case
numbers1
Groups
Gender
Age
(years)
Leprosy
clinical
presentation
102
103
104
105
106
110
113
128
131
142
1001
1004
1020
1039
1050
2008
2011
1
2
3
4
5
6
7
10
11
12
13
14
15
16
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
HIV
HIV
HIV
HIV
HIV
HIV
HIV
HIV-Leprosy
HIV-Leprosy
HIV-Leprosy
HIV-Leprosy
HIV-Leprosy
HIV-Leprosy
HIV-Leprosy
Leprosy
Leprosy
Leprosy
Leprosy
Leprosy
Leprosy
Leprosy
Male
Female
Male
Male
Male
Male
Female
Male
Male
Male
Male
Male
Male
Male
Male
Male
Male
Male
Male
Male
Female
Male
Male
Male
Male
Male
Male
Male
Male
Female
Male
29
47
40
34
49
54
51
37
38
49
37
34
33
35
51
38
38
38
38
31
51
35
53
47
38
43
37
33
48
31
39
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
BL
BT
BT
BL
BT
BT
BT
LL
TT
BT
BT
LL
BL
LL
Bacillary
index
Leprosy
therapy
(months
of MDT)
Viral load
(HIV-RNA
copies/ml)
CD4+
T cells/
mm3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2+
Negative
1+
1+
Negative
Negative
1+
3+
Negative
Negative
Negative
3+
1+
3+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10
12
8
12
12
2
5
20
4
7
5
23
14
22
–
–
–
–
–
–
–
–
–
–
<399
925
<399
200
<399
<399
762
<399
<399
<399
<399
7220
<399
<399
–
–
–
–
–
–
–
1358
1695
742
774
1084
661
949
1571
713
980
405
503
410
170
265
275
297
161
269
235
390
127
236
481
ND
ND
ND
ND
ND
ND
ND
HIV
therapy
–
–
–
–
–
–
–
–
–
–
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
HAART
BL, borderline-lepromatous; BT, borderline-tuberculoid; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; LL,
lepromatous-lepromatous; MDT, multidrug therapy; ND, not done; TT, tuberculoid.
1
Case numbers reflect the enrollment sequences only within each individual group.
Results
Characteristics of the HIV-1-M. leprae co-infected
patients
Demographic, clinical, microbiological and laboratory
characteristics are detailed in Table 1. The median ages
of all participants was 38 years (IQR: 35–48) and most
were male (839%). No difference in gender distribution
was observed between groups. For the leprosy and coinfected groups, 80% of the patients had a paucibacillary presentation at the time of diagnosis. Co-infected
patients were treated with the appropriate MDT for
paucibacillary and multibacillary leprosy. Median CD4+
T-cell counts in both HIV-infected groups were
matched (949 cells/ll, IQR 7275–1465 for controls; 297
cells/ll, IQR 265–410 for HIV-infected patients; and
236 cells/ll, IQR 161–390 for co-infected patients
Table 1).
Leprosy and HIV-1 infections lead to marked
disturbances of T-lymphocyte distribution
The CD4 : CD8 T-cell ratio was decreased in both HIV-1infected groups, but more in co-infected patients
compared with controls (016, IQR 009–020; and
130, IQR 095–19, respectively, P < 0001, Fig. 1a).
Although not statistically significant, co-infected patients
exhibited lower CD4 : CD8 ratios than HIV-1 monoinfected subjects, suggesting more severe immunopathology in the former group, despite similar CD4+ T-cell
counts (Fig. 1a).
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
209
K. I. Carvalho et al.
2·0
1·5
1·0
300
200
0
Control
Control
HIV HIV–leprosy Leprosy
P < 0·01
(d)
Plasmocytoid DC (%)
70
60
Vδ1 : Vδ2 ratio
400
100
0·5
0·0
(c)
P < 0·01
(b) 500
P < 0·01
HLA-DR MFI
CD4 : CD8 ratio
(a) 2·5
50
40
30
20
10
HIV HIV–leprosy Leprosy
P < 0·01
0·7
0·6
0·5
0·4
0·3
0·2
0·1
0·0
0
Control
HIV HIV–leprosy Leprosy
Control
Surface activation markers were evaluated in all four
groups. Marked HLA-DR up-regulation on CD8+ T cells
was observed in both HIV/M. leprae co-infected and HIV
mono-infected groups compared with controls [mean fluorescence intensities of 165 (IQR 94–271), 134 (IQR 114–
249) and 46 (IQR 21–68), respectively, P < 005, Fig. 1b],
but this was not seen in CD4+ T cells. There was a nonsignificant trend towards increased HLA-DR expression in
the co-infected compared with the HIV-1 mono-infected
group (Fig. 1b). No differences were observed in the
expression of CD69, CD25 and CD38 on CD4+ and
CD8+ T lymphocytes (data not shown).
Dendritic cells and Vd2 T lymphocytes are
proportionately diminished in co-infected patients
The Vd2 T-cell subset was decreased in the co-infection
group when compared with the control group (median
153%, IQR 073–24, P < 005), whereas the percentage
of Vd1 T cells was similar in all groups. There was a statistically significant overall difference in the Vd1 : Vd2 cell
ratio, exaggeratedly inverted in the co-infected group
compared with subjects infected with HIV-1 only
(co-infection, 303%, IQR 99–357; HIV, 59%, IQR 38–
137; controls, 063%, IQR 025–31; and leprosy, 286%,
IQR 044–1084, P < 005, Fig. 1c).
The percentages of plasmacytoid DC in total PBMC
were diminished in co-infected patients when compared
with controls (co-infected, 001%, IQR 0005–002; HIV,
002%, IQR 0005–018; control, 013%, IQR 009–018;
and leprosy, 003%, IQR 00–015, P < 005, Fig. 1d).
On the other hand, no significant differences in the
percentages of myeloid DC were observed (data not
shown).
210
HIV HIV–leprosyLeprosy
Figure 1. Several cellular immunological markers obtained using flow cytometry were evaluated and compared among the four groups of
volunteers. These markers comprised (a) the
CD4 : CD8 ratio, (b) cellular activation of
CD8+ T cells, measured by human leucocyte antigen (HLA)-DR expression, (c) the
Vd1 : Vd2 ratio and (d) the percentage of plasmacytoid dendritic cells among total peripheral
blood mononuclear cells (PBMC). Comparisons were carried out using the Kruskal–Wallis
non-parametric test followed by intergroup
comparisons by the Dunnet test. HIV, human
immunodeficiency virus; MFI, mean fluorescence intensity.
HIV-1 and leprosy drives the maturation of
T lymphocytes
CD4+ T cells were stained for surface expression of
CD45RA and CCR7. Phenotypic nomenclature was based
on that proposed by Sallusto et al., where CCR7+
CD45RA+ are described as naı̈ve cells, CCR7+ CD45RA)
as central memory cells and CCR7) CD45RA) as effector
memory cells 31. Control subjects had higher percentages
of naı̈ve and central memory cells compared with the
other groups, with a corresponding decrease in the proportion of effector memory cells (Fig. 2). The most pronounced difference in these maturation subsets was seen
when control subjects were compared with leprosy
patients (CCR7+ CD45RA+ naı̈ve: 547%, IQR 166–182
for controls and 056%, IQR 022–184 for leprosy;
CCR7+ CD45RA) central memory: 338%, IQR 281–357
for controls and 1505%, IQR 86–22 for leprosy;
CCR7) CD45RA) effector: 499%, IQR 476–64 for controls and 826%, IQR 7515–873 for leprosy). For CD8+
T-cell subsets, the only statistically significant difference
was observed when comparing central memory CCR7+
CD45RA) cells from control subjects (177%, IQR 1315–
30) with co-infected (428%, IQR 263–132) and leprosy
(567%, IQR 479–1045) patients.
Both pathogens tend to direct the immune response
towards IL-4 production
Next, we assessed cytokine production after PMA and
ionomycin stimulation. No differences were observed in
the production of TNF-a and IFN-c between CD4+ and
CD8+ T lymphocytes. On the other hand, IL-4 production, determined by high expression of IL-4 in gated
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
Immunity in M. leprae and HIV-1 co-infection
Control
(a)
Naïve
(a)
SSC
3000
0·42%
4·25%
0
10
0 102
103
104 105
0 102
103
104
105
IL-4
(b) 7
r = –0·5861; P = 0·0003
Control
HIV
HIV–leprosy
Leprosy
Central memory
(b)
+
P < 0·01
40
6
5
4
3
2
1
0
30
0
5
10
15
20
–
25
–
30
35
40
+
CCR7 CD45RA among CD4 T cells (%)
20
(c) 7
10
0
Control
HIV
HIV–leprosy
Leprosy
Effector memory
(c)
+
P < 0·01
CD4 T cells producing IL-4 (%)
CCR7+ CD45RA– % in CD4+ T cells
2000
1000
CD4 T cells producing IL-4 (%)
CCR7+ CD45RA+ % in CD4+ T cells
20
0
95
CCR7– CD45RA– % in CD4+ T cells
Co-infected
4000
P < 0·01
90
6
5
4
3
2
1
0
30
85
r = 0·4791; P = 0·0041
40
50
–
60
–
70
80
90
100
+
CCR7 CD45RA among CD4 T cells (%)
80
75
70
65
60
55
50
45
Control
HIV
HIV–leprosy
Leprosy
Figure 2. Distribution of cellular maturation markers of CD4+ T
cells. Cellular subpopulations were determined by the expression of
CCR7 and CD45RA after gating on CD3+ CD4+ cells. The percentage of (a) naı̈ve (CCR7+ CD45RA+), (b) central memory (CCR7+
CD45RA)) and (c) effector memory (CCR7) CD45RA)) cells are
depicted for all groups of subjects. HIV, human immunodeficiency
virus.
Figure 3. Interleukin-4 (IL-4) production was determined by intracellular staining and flow cytometry after stimulation with ionomycin and phorbol 12-myristate 13-acetate for 16 hr. (a) The IL-4+
gate was set for cells producing high levels of cytokine. The level of
IL-4 production was negatively correlated with the percentage of
central memory (CCR7+ CD45RA)) CD4+ T cells (b) and positively
correlated with effector memory (CCR7) CD45RA)) CD4+ T cells
(c). The results for all four study subject groups are shown: solid circles, healthy controls; open circles, co-infection; open triangles,
human immunodeficiency virus-1; open squares, leprosy. Correlations were assessed using the non-parametric Spearman’s test. SSC,
side scatter.
CD4+ T cells, was statistically lower in controls (057%,
IQR 033–093) when compared with the other three
groups (109%, IQR 062–285; P = 003). No statistically
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
211
K. I. Carvalho et al.
significant differences of cytokine production by CD8+ T
cells were observed.
The IL-4 production by CD4+ T lymphocytes was negatively correlated with the percentage of central memory
cells (r = )059, P < 001) and positively correlated with
the percentage of effector CD4+ T cells (r = 048,
P < 001) (Fig. 3). As shown in Fig. 3, the frequency of
IL-4+ T cells in control subjects clustered tightly, whereas
those in the three infected groups were increased and in
an overlapping distribution.
Discussion
There is considerable epidemiological overlap between
M. tuberculosis and HIV epidemics, so that co-infections
may be common in certain areas. In contrast, at present,
rather distinct populations tend to be infected with
M. leprae and HIV, so co-infections are much less common. However, future projections of spread of the HIV
epidemic into areas with more prevalent M. leprae infection may change the co-infection epidemiological characteristics. The importance of tuberculosis and HIV
co-infection as a public health problem is obvious, but
this is less clear for M. leprae and HIV co-infections.32,33
However, the special nature of M. leprae stimulates
unique questions about the possible consequences of
co-infection. Infection with M. leprae differs in several
ways from that with M. tuberculosis – there is a much
more gradual evolution of disease, a classic spectrum of
clinical manifestations related to Th1 and Th2 responsiveness by the host, often huge antigenic burdens that are
slow to clear, and pathogenesis that is largely caused by
spontaneous shifts in host immune responsiveness, resulting in inflammatory lepra reactions.
We set out to compare cellular immune parameters in
HIV-1-infected patients with and without leprosy. A
limitation of the present study was the small sample size,
owing to the relative rarity of HIV-1/M. leprae co-infected patients. Moreover, the challenge of interpreting
results from this cohort was compounded by the variability of HIV disease, according to stage of progression,
superimposed on the spectral nature of leprosy. In an
attempt to derive meaningful data from the present
sample, we endeavoured to match HIV-1 and M. leprae
co-infected patients with HIV-1 and M. leprae monoinfected subjects, for CD4 and bacillary index, respectively (Table 1).
In the present study, we confirmed that leprosy
mono-infection is associated with increased IL-4 production by CD4+ T cells (Fig. 3). A similar increase was
observed in HIV-1 mono-infection, as has been reported
by others,34 but no apparent additive or synergistic effect
was seen in HIV-1/M. leprae co-infected patients. Our
data suggest that leprosy co-infection may aggravate,
rather than ameliorate, HIV pathogenesis, as indicated
212
by the decreased ratio of CD4 : CD8 T cells, higher
frequency of activated CD8+ T cells and loss of plasmacytoid DC, all recognized features of progressive HIV-1
disease. This is in contrast to the observation of Gormus
et al., who made the unexpected observation of SIV
disease amelioration in the setting of experimental M. leprae co-infection of rhesus macaques.12 In the latter
studies, we speculate that the immunologic environment
associated with a high M. leprae antigenic burden might
have attenuated the immune activation-driven pathogenesis of SIV disease. However, our data do not support
the hypothesis that M. leprae co-infection can attenuate
the immunopathogenesis of human HIV-1 disease. On
the contrary, the results suggest that M. leprae co-infection may exacerbate HIV-1 pathogenesis. Clearly, there
are important differences between the macaque model
system and natural human infections. Macaques are
natural hosts of neither M. leprae nor SIV, and the animals were infected with a large intravenous inoculum of
bacilli. Perhaps most importantly, no inflammatory manifestations of leprosy were described in the experimental
animals. On the other hand, inflammatory lepra
reactions can complicate up to half of human cases of
leprosy, and this immunopathology may indeed account
for much of the nerve damage and morbidity of this
disease. Cutaneous and systemic expression of proinflammatory cytokines, such as TNF-a, have been
extensively documented in lepra reactions35,36 and may
be expected to promote HIV-1 replication. Indeed, cytokine-driven enhancement of viral replication has been
invoked to explain the aggravation of HIV-1 disease in
patients with concurrent tuberculosis.37 Thus, in HIV-1/
M. leprae co-infection, inflammation associated with
clinical or subclinical lepra reactions may offset the
potential for any beneficial immune-modulatory effects
of M. leprae on HIV-1 disease progression.
Sallusto et al. described that immunological memory is
displayed by distinct T-cell subsets: lymph node-homing
CCR7+ CD45RA) (central memory T cells, TCM) and tissue-homing cells CCR7) CD45RA) (effector memory
T cells, TEM).31 Our results suggest that leprosy patients
have a decreased number of naı̈ve cells when compared
with healthy controls, together with a decreased percentage of TCM and an increased percentage of TEM, mostly
in co-infected patients. We hypothesize that the
imbalance in the percentage distribution seen in leprosy
and co-infected patients reflects a switch from naı̈ve to
memory CD4+ T lymphocytes, as a result of continuous
antigenic stimulation and cellular activation, as also seen
in the context of tuberculosis.38 This finding may well
represent a reactive expansion of ‘protective memory’
TEM cells in response to M. leprae and HIV as a result of
differentiation of TCM to combat the pathogen, especially
in the tissues, considering the high antigenic burden
observed in both diseases.39
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
Immunity in M. leprae and HIV-1 co-infection
Curiously, a positive correlation was observed between
the proportional expansion of circulating TEM CD4+ T
cells and the percentage of IL-4+-producing CD4+ T cells
after stimulation with PMA and ionomycin (Fig. 3b).
Although our analysis did not permit us to ascertain
directly whether TEM are actually the producers of IL-4, it
is likely that these Th2-differentiated cells are indeed antigen-experienced members of the CD4+ TEM population.
This interpretation is consistent with previous reports of
higher IL-4 production in the context of M. leprae40,41
and HIV-142 infections. As increased frequencies of these
cells were observed in chronic HIV-1 and/or M. leprae
infections, there is clearly an association between IL-4
production and the presence of antigen. However, our
approach did not address the antigen specificity of the
IL-4-producing T cells. Others have demonstrated expression of Th2 cytokines in leprosy lesions,35,40,43 which may
represent antigen-driven or cytokine-driven expansion of
M. leprae-specific T cells.44 These responses may be influenced by the genetic background of the individual as well
as by environmental factors.44 We suggest that the continuing production of IL-4 by HIV-1 and M. leprae-specific
T cells may create a ‘Th2 environment’ in which the
priming of T cells to heterologous antigens is biased
towards IL-4 production.45 Exploring the association of
higher IL-4 production after PMA and ionomycin stimulation, and expansion of TEM, may present an opportunity to elucidate the mechanisms involved in the possibly
deleterious effect of M. leprae infection in HIV-1-infected
patients observed in our study.
In conclusion, this initial exploration of the cellular
immune interactions of leprosy and HIV-1 disease suggests that chronic infection with M. leprae might exacerbate the immunopathogenesis of HIV-1 disease. We
speculate that this may be the result of a combination of
inflammatory lepra reactions and the aggravated Th2
environment induced by M. leprae antigens. Prospective
longitudinal studies are needed to address the questions
raised in this work.
Acknowledgements
This work was partially supported by Fundação Paulista
contra a Hansenı́ase, National Institutes of Health, grant
#R01-AI052731-06, and The Fogarty International Center,
grant #D43 TW00003; KCS’s PhD scholarship was provided by the Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico (CNPq), Brazilian Ministry of
Science and Technology. We are also thankful for support
from the Heiser Program for Research in Leprosy and
Tuberculosis of The New York Community Trust.
Conflicts of interests
References
1 Lockwood DN, Kumar B. Treatment of leprosy. BMJ 2004;
328:1447–8.
2 Meima A, Smith WC, van Oortmarssen GJ, Richardus JH,
Habbema JD. The future incidence of leprosy: a scenario analysis. Bull World Health Organ 2004; 82:373–80.
3 Pereira GA, Stefani MM, Araujo Filho JA, Souza LC, Stefani GP,
Martelli CM. Human immunodeficiency virus type 1 (HIV-1)
and Mycobacterium leprae co-infection: HIV-1 subtypes and clinical, immunologic, and histopathologic profiles in a Brazilian
cohort. Am J Trop Med Hyg 2004; 71:679–84.
4 Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman
RL. Two types of murine helper T cell clone. I. Definition
according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136:2348–57.
5 Modlin RL. Th1-Th2 paradigm: insights from leprosy. J Invest
Dermatol 1994; 102:828–32.
6 Bloom BR. Learning from leprosy: a perspective on immunology
and the Third World. J Immunol 1986; 137:i–x.
7 Ustianowski AP, Lawn SD, Lockwood DN. Interactions between
HIV infection and leprosy: a paradox. Lancet Infect Dis 2006;
6:350–60.
8 Toossi Z, Mayanja-Kizza H, Hirsch CS et al. Impact of tuberculosis (TB) on HIV-1 activity in dually infected patients. Clin Exp
Immunol 2001; 123:233–8.
9 Whalen C, Horsburgh CR, Hom D, Lahart C, Simberkoff M,
Ellner J. Accelerated course of human immunodeficiency virus
infection after tuberculosis. Am J Respir Crit Care Med 1995;
151:129–35.
10 Nath I, Vemuri N, Reddi AL, Jain S, Brooks P, Colston MJ,
Misra RS, Ramesh V. The effect of antigen presenting cells on
the cytokine profiles of stable and reactional lepromatous leprosy patients. Immunol Lett 2000; 75:69–76.
11 Sampaio EP, Caneshi JR, Nery JA et al. Cellular immune
response to Mycobacterium leprae infection in human immunodeficiency virus-infected individuals. Infect Immun 1995;
63:1848–54.
12 Gormus BJ, Murphey-Corb M, Baskin GB, Uherka K, Martin
LN, Marx PA, Xu K, Ratterree MS. Interactions between Mycobacterium leprae and simian immunodeficiency virus (SIV) in
rhesus monkeys. J Med Primatol 2000; 29:259–67.
13 Eberl M, Hintz M, Reichenberg A, Kollas AK, Wiesner J, Jomaa
H. Microbial isoprenoid biosynthesis and human gammadelta
T cell activation. FEBS Lett 2003; 544:4–10.
14 Barnes PF, Grisso CL, Abrams JS, Band H, Rea TH, Modlin RL.
Gamma delta T lymphocytes in human tuberculosis. J Infect Dis
1992; 165:506–12.
15 Fujita M, Miyachi Y, Nakata K, Imamura S. Appearance of
gamma delta T cell receptor-positive cells following alpha beta
T cell receptor-positive cells in the lepromin reaction of human
skin. Immunol Lett 1993; 35:39–44.
16 Poccia F, Gougeon ML, Agrati C et al. Innate T-cell immunity
in HIV infection: the role of Vgamma9Vdelta2 T lymphocytes.
Curr Mol Med 2002; 2:769–81.
17 Poles MA, Barsoum S, Yu W et al. Human immunodeficiency
virus type 1 induces persistent changes in mucosal and blood
gammadelta T cells despite suppressive therapy. J Virol 2003;
77:10456–67.
The authors declare no competing conflicts of interests.
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
213
K. I. Carvalho et al.
18 Pulendran B. Modulating TH1/TH2 responses with microbes,
dendritic cells, and pathogen recognition receptors. Immunol Res
2004; 29:187–96.
19 Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA,
Bazan F, Liu YJ. Subsets of human dendritic cell precursors
express different toll-like receptors and respond to different
microbial antigens. J Exp Med 2001; 194:863–9.
20 Krutzik SR, Ochoa MT, Sieling PA et al. Activation and regulation of Toll-like receptors 2 and 1 in human leprosy. Nat Med
2003; 9:525–32.
21 Bafica A, Scanga CA, Feng CG, Leifer C, Cheever A, Sher A.
TLR9 regulates Th1 responses and cooperates with TLR2 in
mediating optimal resistance to Mycobacterium tuberculosis.
J Exp Med 2005; 202:1715–24.
22 Levy JA, Scott I, Mackewicz C. Protection from HIV/AIDS: the
importance of innate immunity. Clin Immunol 2003; 108:167–
74.
23 Organizaton WH. Chemotherapy of leprosy for control programme, report of WHO study group. WHO TechRepSer 1982;
675:1–33.
24 Saúde MD. Recomendações para Terapia Anti-Retroviral em Adultos e Adolescentes Infectados pelo HIV. Brası́lia-DF: Ministério da
Saúde, 2006:1–85.
25 Ridley DS, Jopling WH. Classification of leprosy according to
immunity. A five-group system. Int J Lepr Other Mycobact Dis
1966; 34:255–73.
26 Hirsch HH, Kaufmann G, Sendi P, Battegay M. Immune reconstitution in HIV-infected patients. Clin Infect Dis 2004; 38:1159–
66.
27 Goebel FD. Immune reconstitution inflammatory syndrome
(IRIS) – another new disease entity following treatment initiation of HIV infection. Infection 2005; 33:43–5.
28 Couppie P, Abel S, Voinchet H, Roussel M, Helenon R, Huerre
M, Sainte-Marie D, Cabie A. Immune reconstitution inflammatory syndrome associated with HIV and leprosy. Arch Dermatol
2004; 140:997–1000.
29 Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 2001; 45:194–
205.
30 Kallas EG, Gibbons DC, Soucier H, Fitzgerald T, Treanor JJ,
Evans TG. Detection of intracellular antigen-specific cytokines in
human T cell populations. J Infect Dis 1999; 179:1124–31.
31 Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two
subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999; 401:708–12.
32 Stenger S, Modlin RL. T cell mediated immunity to Mycobacterium tuberculosis. Curr Opin Microbiol 1999; 2:89–93.
33 Antas PR, Sales JS, Pereira KC, Oliveira EB, Cunha KS, Sarno
EN, Sampaio EP. Patterns of intracellular cytokines in CD4 and
214
34
35
36
37
38
39
40
41
42
43
44
45
CD8 T cells from patients with mycobacterial infections. Braz J
Med Biol Res 2004; 37:1119–29.
Sousa AE, Chaves AF, Doroana M, Antunes F, Victorino RM.
Bulk cytokine production versus frequency of cytokine-producing cells in HIV1 infection before and during HAART. Clin
Immunol 2000; 97:162–70.
Yamamura M, Wang XH, Ohmen JD, Uyemura K, Rea TH,
Bloom BR, Modlin RL. Cytokine patterns of immunologically
mediated tissue damage. J Immunol 1992; 149:1470–5.
Barnes PF, Abrams JS, Lu S, Sieling PA, Rea TH, Modlin RL.
Patterns of cytokine production by mycobacterium-reactive
human T-cell clones. Infect Immun 1993; 61:197–203.
de Castro Cunha RM, Kallas EG, Rodrigues DS, Nascimento
Burattini M, Salomao R. Interferon-gamma and tumour necrosis
factor-alpha production by CD4+ T and CD8+ T lymphocytes
in AIDS patients with tuberculosis. Clin Exp Immunol 2005;
140:491–7.
Rodrigues DS, Medeiros EA, Weckx LY, Bonnez W, Salomao R,
Kallas EG. Immunophenotypic characterization of peripheral
T lymphocytes in Mycobacterium tuberculosis infection and disease. Clin Exp Immunol 2002; 128:149–54.
Sallusto F, Geginat J, Lanzavecchia A. Central memory and
effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 2004; 22:745–63.
Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH,
Bloom BR, Modlin RL. Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 1991; 254:277–
9.
Salgame P, Abrams JS, Clayberger C, Goldstein H, Convit J,
Modlin RL, Bloom BR. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science
1991; 254:279–82.
Galli G, Annunziato F, Mavilia C et al. Enhanced HIV expression during Th2-oriented responses explained by the opposite
regulatory effect of IL-4 and IFN-gamma of fusin/CXCR4. Eur J
Immunol 1998; 28:3280–90.
Sieling PA, Abrams JS, Yamamura M, Salgame P, Bloom BR,
Rea TH, Modlin RL. Immunosuppressive roles for IL-10 and
IL-4 in human infection. In vitro modulation of T cell responses
in leprosy. J Immunol 1993; 150:5501–10.
Mitra DK, De Rosa SC, Luke A et al. Differential representations
of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases. Int Immunol 1999; 11:1801–
10.
Stutz A, Graf P, Beinhauer B, Hammerschmid F, Neumann C,
Woisetschlager M, Jung T. CD45 isoform expression is associated with different susceptibilities of human naive and effector
CD4+ T cells to respond to IL-4. Eur J Immunol 2005; 35:575–
83.
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
Carvalho KI
Trabalho 2
O HIV-1 tem vários mecanismos para tentar abolir a atividade antiviral das
células NKT, incluindo induzir menor expressão de CD1d em células
apresentadoras de antígeno. Para evitar este efeito e obter novos
conhecimentos da resposta ex vivo das células NKT, células de pacientes
infectados pelo HIV-1 e indivíduos controle saudáveis foram estimulados com
alfa-galactosil-ceramida conjugado com CD1d para analisar a secreção de
citocinas pelas células NKT. A frequência de células NKT que secretaram
IFN-γ e TNF-α estava significantemente diminuída em infectados pelo HIV-1
quando comparados com os controles. A magnitude da resposta de IFN-γ
tem correlação inversa com o número de anos de infecção, sugerindo que a
função das células NKT são diminuídas progressivamente ao longo do
tempo. Não houve alteração na resposta das células NKT nos indivíduos
infectados pelo HIV após tratamento com PMA e ionomicina, sugerindo um
defeito no sinal do TCR que prejudica a produção de citocinas. Foi observado
uma diminuição na magnitude da resposta de citocinas Th1 nas células NKT
quando correlacionado com a expressão de CD161, sugerindo um
mecanismo inibitório deste receptor na regulação da resposta de células
NKT.
31
Carvalho KI
Trabalho 2
O HIV-1 tem vários mecanismos para tentar abolir a atividade antiviral das
células NKT, incluindo induzir menor expressão de CD1d em células
apresentadoras de antígeno. Para evitar este efeito e obter novos
conhecimentos da resposta ex vivo das células NKT, células de pacientes
infectados pelo HIV-1 e indivíduos controle saudáveis foram estimulados com
alfa-galactosil-ceramida conjugado com CD1d para analisar a secreção de
citocinas pelas células NKT. A frequência de células NKT que secretaram
IFN-γ e TNF-α estava significantemente diminuída em infectados pelo HIV-1
quando comparados com os controles. A magnitude da resposta de IFN-γ
tem correlação inversa com o número de anos de infecção, sugerindo que a
função das células NKT são diminuídas progressivamente ao longo do
tempo. Não houve alteração na resposta das células NKT nos indivíduos
infectados pelo HIV após tratamento com PMA e ionomicina, sugerindo um
defeito no sinal do TCR que prejudica a produção de citocinas. Foi observado
uma diminuição na magnitude da resposta de citocinas Th1 nas células NKT
quando correlacionado com a expressão de CD161, sugerindo um
mecanismo inibitório deste receptor na regulação da resposta de células
NKT.
31
Carvalho KI
Trabalho 2
O HIV-1 tem vários mecanismos para tentar abolir a atividade antiviral das
células NKT, incluindo induzir menor expressão de CD1d em células
apresentadoras de antígeno. Para evitar este efeito e obter novos
conhecimentos da resposta ex vivo das células NKT, células de pacientes
infectados pelo HIV-1 e indivíduos controle saudáveis foram estimulados com
alfa-galactosil-ceramida conjugado com CD1d para analisar a secreção de
citocinas pelas células NKT. A frequência de células NKT que secretaram
IFN-γ e TNF-α estava significantemente diminuída em infectados pelo HIV-1
quando comparados com os controles. A magnitude da resposta de IFN-γ
tem correlação inversa com o número de anos de infecção, sugerindo que a
função das células NKT são diminuídas progressivamente ao longo do
tempo. Não houve alteração na resposta das células NKT nos indivíduos
infectados pelo HIV após tratamento com PMA e ionomicina, sugerindo um
defeito no sinal do TCR que prejudica a produção de citocinas. Foi observado
uma diminuição na magnitude da resposta de citocinas Th1 nas células NKT
quando correlacionado com a expressão de CD161, sugerindo um
mecanismo inibitório deste receptor na regulação da resposta de células
NKT.
31
Snyder-Cappione et al
Lower Th1 cytokine secretion ex vivo by CD1d-restricted NKT cells in HIV-1-infected
individuals is associated with high CD161 expression.
Jennifer E. Snyder-Cappione,1* Christopher P. Loo,1 Karina I. Carvalho,2 Carlotta
Kuylenstierna,3 Steven G. Deeks,4 Frederick M. Hecht,4 Michael G. Rosenberg,5
Johan K. Sandberg,3 Esper G. Kallas,2,6 and Douglas F. Nixon1
1
Division of Experimental Medicine, Department of Medicine, University of California, San
Francisco, CA, USA.
2
Infectious Diseases Division, Federal University of Sao Paulo/SP and Brazil Laboratório de
Imunologia II Disciplina de Infectologia, Universidade Federal de São Paulo, Rua Mirassol
207
04044-010, Sao Paulo, SP Brazil.
3
CIM, Department of Medicine, F59, Karolinska Institute, Karolinska University Hospital,
Huddinge, 14186 Stockholm, Sweden.
4
Positive Health Program, San Francisco General Hospital, San Francisco, California, USA.
5
Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, New York, USA.
6
Clinical Immunology and Allergy Division, University of São Paulo, São Paulo, Brazil.
Short Title: NKT cell functions in HIV
*
Address correspondence to:
Jennifer E. Snyder-Cappione, Ph.D.
University of California San Francisco
1001 Potrero Avenue, Building 3, Room 607
San Francisco, CA 94110
Tel: (415) 206-4981
Fax: (415) 206-8091
Email: [email protected]
Snyder-Cappione et al. 2
Nonstandard abbreviations used: Elispot, enzyme-linked immunosorbent spot; α-GalCer,
alpha-galactosyl ceramide; DX-αGalCer, DimerX-CD1d reagent loaded with α-GalCer;
ART, anti-retroviral treatment, TCR, T cell receptor
Snyder-Cappione et al. 3
Abstract
HIV-1 has several mechanisms to abrogate the anti-viral activity of NKT cells, including the
down-regulation of CD1d on antigen presenting cells. To circumvent this effect and gain new
understanding of the ex vivo NKT cell response, we measured cytokines from NKT cells of
HIV-1 infected and healthy individuals after stimulation with alpha-galactosyl ceramideloaded CD1d dimers. The frequencies of NKT cells secreting IFN-gamma and TNF-alpha
were significantly lower in HIV-1-infected subjects than healthy controls. The magnitude of
the IFN-gamma response correlated inversely with the number of years of infection,
suggesting NKT cell functions are progressively lost over time. NKT cell responses in HIVinfected subjects after treatment with PMA and ionomycin were essentially normal,
suggesting that defective TCR-signaling was underlying the impaired cytokine production.
Lower magnitude of the NKT Th1 response correlated with higher CD161 expression,
suggesting a role for this inhibitory receptor in regulating NKT cell responsiveness.
Key Words: NKT cell, ex vivo, HIV, CD161, IFN-gamma, TNF-alpha
Snyder-Cappione et al. 4
Introduction
NKT cells are a unique subset of T cells thought to bridge the innate and adaptive arms of the
immune response (1). Human invariant NKT cells express a canonical TCR incorporating
Vα24JαQ with a limited Vβ repertoire, predominantly Vβ11 (2, 3). TCRs of human NKT
cells recognize glycolipid antigens on the nonclassical MHC molecule CD1d. NKT cells
recognize both self and nonself glycolipids, including alpha-galactosyl ceramide (αGalCer)
(4), which is derived from the marine sponge Agelas mauritianus. NKT cells secrete a variety
of Th1, Th2, and Th17 cytokines (5-14) and contribute to immune responses against foreign,
self, and tumor antigens.
Considerable evidence suggests that NKT cells contribute to an effective immune
response in HIV-1 infection (15). A subset of NKT cells expresses CD4, the co-receptor for
HIV entry. These CD4+ NKT cells are selectively infected and depleted by the virus (16-18).
HIV-1 also down-regulates CD1d in infected cells (19-21). Thus, HIV-1 appears to have
evolved distinct NKT evasion mechanisms to ensure its propagation in the host. While it is
suggested that ART may increase NKT cell functions (22), the effect of HIV-1 on the
functional capacity of circulating NKT cells is largely unknown.
In this study, we measured the ex vivo effector functions of NKT cells from healthy and
HIV-1infected individuals after stimulation with DimerX, a human CD1d-Ig recombinant
fusion protein used for targeted NKT cell stimulation (22) or PMA and Ionomycin. We also
compared the disease status of the infected individuals and subsets of the NKT compartment
with the magnitude of the cytokine response to DimerX-αGalCer stimulation.
Snyder-Cappione et al. 5
Methods
Subjects. Healthy volunteers and HIV-1-infected subjects were recruited at the University of
California, San Francisco (San Francisco, CA) and the JACOBI Medical Center (Bronx,
NY). All samples were obtained according to protocols approved by the Research Subjects
Review Board at each institution. Informed consent was obtained from all subjects. The
characteristics of the subjects are summarized in Supplemental Table 1.
Human lymphocyte preparation. Blood was drawn into ACD or EDTA tubes. PBMCs were
frozen in heat-inactivated FBS containing 10% DMSO at a concentration of 1 x 106 to 10 x
106/ml and stored in liquid nitrogen until use.
Antigens. For dimer loading, 20 µg of human CD1d-Ig recombinant fusion proteins (BD
DimerX; BD Biosciences) was mixed with 5 µg of αGalCer (Kirin Brewery, Japan) in a final
volume of 100 µl and incubated overnight at 37oC. PBS was used a loading (vehicle) control
for all αGalCer stimulation assays. After overnight incubation, an additional 320 µl of PBS
was added. DimerX complexes were added to culture wells at a final concentration of 15
µl/ml. Titration was performed to ensure this concentration provided maximal stimulation of
all NKT cells in PBMC cultures. Cells were also stimulated with PMA (50 ng/ml), and
ionomycin (500 ng/ml).
Fluorescent antibodies and tetramers. CD1d-tetramer loaded with PBS57 PE was from the
NIH Tetramer Facility (Emory University, Atlanta, GA). Other reagents include: anti-CD3
ECD (Beckman Coulter); anti-IL4 FITC, anti-IFNγ PE-Cy7, anti-TNFα Alexa 700, antiCD69 APC-Cy7, anti-CD4 Alexa 700, anti-CD56 PE-Cy7, and anti-CD161 APC (all from
BD Biosciences); Amine Aqua for live/dead discrimination (Invitrogen), anti-CD8 Qdot 605
(University of California, San Francisco), and anti-vα24 biotin (Beckman Coulter).
Snyder-Cappione et al. 6
Flow cytometry. For phenotypic staining, PBMCs were incubated with CD1d-tetramerPBS57, anti-CD3, anti-CD56, anti-CD161, anti-CD4, anti-CD69, Amine Aqua, anti-CD8,
and anti-vα24 for 30 minutes at 4oC. Cells were then washed twice with FACS buffer (PBS
with 0.5% BSA and 2 mM EDTA), resuspended in 2% paraformaldehyde, and run on a LSRII Flow Cytometer (BD Biosciences). For measurement of NKT cell cytokine production by
intracellular cytokine staining, PBMCs were cultured with DimerX-PBS, DimerX-αGalCer,
or PMA and ionomycin as described above. After 1 hour at 37oC in 5% CO2 brefeldin A (5
µg/ml) was added. After incubation for 12–16 hours, the cells were washed and CD1dtetramer-PBS57, anti-CD4, anti-CD69, anti-CD8, anti-vα24, and Amine Aqua were added
for 30 minutes at 4oC in the dark. The cells were washed and incubated with 2%
paraformaldehyde and permeabilization buffer (BD Biosciences) for 20 minutes at room
temperature. The cells were washed and anti-IL4, anti-CD3, anti-IFNγ, anti-TNFα, and Qdot
655 streptavidin (Invitrogen) were added. After incubation for 30 minutes at 4oC in the dark,
the cells were washed, resuspended in 2% paraformaldehyde, and run on a LSR-II Flow
Cytometer. The data were analyzed with FlowJo software (version 8.5.2, Tree Star). Gating
and analysis were performed without knowledge of the subjects’ characteristics, including
HIV status.
Statistical analysis. The two-tailed t test with Welsh's correction was used to compare the
different groups in Figures 1C, and 1D. Linear regression analysis was used in Figures 1E
and 2A.
Results and Discussion
Circulating NKT cells from HIV-infected subjects exhibit weak Th1 cytokine responses to
DX-αGalCer. We measured the ex vivo effector functions of NKT cells from 24 HIV-1-
Snyder-Cappione et al. 7
infected subjects and 10 healthy controls by intracellular cytokine staining. Due to the
variability of NKT cell frequencies and limitations of available PBMC, a subject's data were
included in this study if greater than 20 events were collected within the NKT gate. After
completion of blinded analysis from all samples, data from 11 of the HIV-1-infected subjects
and six of the healthy controls met this criterion (Supplemental Table 1). Seven of the HIV-1infected subjects had suppressed virus at the time of sample collection, with five on ART
(mean CD4+ T cell count, 561 cells/µl; mean plasma HIV-1 RNA level, 136 copies/ml). Four
subjects were defined as viremic (with viral loads greater than 1,000 copies/ml), and three of
these subjects were on ART (mean CD4+ T cell count, 555 cells/µl; mean plasma HIV-1
RNA level, 15,032 copies/ml). NKT cells were identified as CD3+, Vα24+, and CD1dtetramer+ (Figure 1A). The mean NKT cell frequencies were lower in the HIV-1 infected
subjects than in healthy controls (Supplemental Table 1 and data not shown).
After DX-αGalCer stimulation, the percentage of NKT cells that produced TNF-α or IFN-γ
was significantly higher in the healthy controls than in the HIV-1-infected subjects (p <
0.0001 for TNF-α; p = 0.0016 for IFN-γ) (Figure 1B, 1C), but both groups exhibited similar
NKT responses to PMA + ionomycin (Figure 1C). HIV-1 may render the Th1-biased NKT
subsets unresponsive to CD1-TCR signaling but not PMA and Ionomycin, as has been
observed by mouse NKT cells after exposure to bacterial products in vivo (23). Among HIV1 infected subjects, the TNF-α and IFN-γ responses to DX-αGalCer were higher, on average,
in those with suppressed virus than in virologic subjects; however, the difference was not
statistically significant (Figure 1D). Neither the CD4+ T cell counts nor the viral loads of the
infected subjects correlated with the percentage of NKT cells producing IFN-γ or TNF-α
in
response to αGalCer (data not shown). However, there was a significant negative correlation
between the years since infection and the magnitude of the IFN-γ response to DX-αGalCer.
This suggests NKT cells undergo exhaustion due to chronic stimulation and/or exposure to
inflammatory cytokines.
Snyder-Cappione et al. 8
The frequency of NKT cells secreting Th1 cytokines in response to DX-aGalCer correlates
with surface expression of CD161 in HIV-infected subjects. We compared the distribution of
NKT cell subsets and Th1 cytokines induced by DX-αGalCer in HIV-infected subjects. The
percentages of IFN-γ or TNF-α secreting NKT cells did not correlate with expression of
CD56, CD69, or all combinations of CD4 and CD8 antigens (data not shown). However, the
percentage of NKT cells expressing CD161, a reported NKT cell maturation marker (Since
we were the first to publish this in PNAS 2004 I think it would be better to cite our own
paper) (24), correlated negatively with the production of both TNF-α and IFN-γ (Figure 2).
The percentage of circulating CD161+ NKT cells was higher in the healthy controls than in
the HIV-1 infected subjects (Figure 2B). Therefore, it was surprising that a lack of CD161
expression by NKT cells from HIV-1 infected subjects correlated with stronger Th1
responses. CD161 is often used to define the maturation state of NKT cell populations, with
higher expression reflecting a more mature phenotype (24). Interestingly, anergic CD161+
human T cells have been reported (25).
These data suggest chronic HIV-1 infection disables the ability of NKT cells to respond to
MHC-antigen stimulation in vivo. Future longitudinal studies comparing the DX-αGalCer
response of NKT cells with rates of disease progression may elucidate the role of these cells
in viral control.
Acknowledgements
Support for this work was provided by National Institute of Allergies and Infectious Diseases
Snyder-Cappione et al. 9
(NIAID R37-A152731, the NIH (AI060379), the UCSF CFAR (P30 AI27763, P30
MH59037), NIAID (AI055273), the Center for AIDS Prevention Studies (P30 MH62246),
and the UCSF Clinical and Translational Science Institute (UL1 RR024131-01). Additional
support was provided by the Brazilian Program for STD and AIDS, Ministry of Health
(914/BRA/3014 – UNESCO / Kallas), the São Paulo City Health Department (20040.168.922-7/Kallas), Fundação de Amparo a Pesquisa do Estado de São Paulo (04/158569/Kallas), the John E. Fogarty International Center (D43 TW00003), the AIDS Research
Institute of the AIDS Biology Program at UCSF. KIC was supported by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian Ministry of Education.
Snyder-Cappione et al. 10
References
1.
Benlagha, K., and Bendelac, A. 2000. CD1d-restricted mouse V alpha 14 and human
V alpha 24 T cells: lymphocytes of innate immunity. Semin Immunol 12:537-542.
2.
Lee, P.T., Benlagha, K., Teyton, L., and Bendelac, A. 2002. Distinct functional
lineages of human V(alpha)24 natural killer T cells. J Exp Med 195:637-641.
3.
Prussin, C., and Foster, B. 1997. TCR V alpha 24 and V beta 11 coexpression defines
a human NK1 T cell analog containing a unique Th0 subpopulation. J Immunol
159:5862-5870.
4.
Kronenberg, M., and Gapin, L. 2002. The unconventional lifestyle of NKT cells. Nat
Rev Immunol 2:557-568.
5.
de Lalla, C., Galli, G., Aldrighetti, L., Romeo, R., Mariani, M., Monno, A., Nuti, S.,
Colombo, M., Callea, F., Porcelli, S.A., et al. 2004. Production of profibrotic
cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral
hepatitis. J Immunol 173:1417-1425.
6.
Exley, M., Porcelli, S., Furman, M., Garcia, J., and Balk, S. 1998. CD161 (NKRP1A) costimulation of CD1d-dependent activation of human T cells expressing
invariant V alpha 24 J alpha Q T cell receptor alpha chains. J Exp Med 188:867-876.
7.
Gumperz, J.E., Miyake, S., Yamamura, T., and Brenner, M.B. 2002. Functionally
distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer
staining. J Exp Med 195:625-636.
8.
Kim, C.H., Johnston, B., and Butcher, E.C. 2002. Trafficking machinery of NKT
cells: shared and differential chemokine receptor expression among V alpha 24(+)V
beta 11(+) NKT cell subsets with distinct cytokine-producing capacity. Blood 100:1116.
9.
Spada, F.M., Koezuka, Y., and Porcelli, S.A. 1998. CD1d-restricted recognition of
synthetic glycolipid antigens by human natural killer T cells. J Exp Med 188:15291534.
10.
Wilson, S.B., Kent, S.C., Patton, K.T., Orban, T., Jackson, R.A., Exley, M., Porcelli,
S., Schatz, D.A., Atkinson, M.A., Balk, S.P., et al. 1998. Extreme Th1 bias of
invariant Valpha24JalphaQ T cells in type 1 diabetes. Nature 391:177-181.
11.
Coquet, J.M., Chakravarti, S., Kyparissoudis, K., McNab, F.W., Pitt, L.A., McKenzie,
B.S., Berzins, S.P., Smyth, M.J., and Godfrey, D.I. 2008. Diverse cytokine production
by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell
population. Proc Natl Acad Sci U S A 105:11287-11292.
12.
Lee, K.A., Kang, M.H., Lee, Y.S., Kim, Y.J., Kim, D.H., Ko, H.J., and Kang, C.Y.
2008. A distinct subset of natural killer T cells produces IL-17, contributing to airway
infiltration of neutrophils but not to airway hyperreactivity. Cell Immunol.
13.
Pichavant, M., Goya, S., Meyer, E.H., Johnston, R.A., Kim, H.Y., Matangkasombut,
P., Zhu, M., Iwakura, Y., Savage, P.B., DeKruyff, R.H., et al. 2008. Ozone exposure
in a mouse model induces airway hyperreactivity that requires the presence of natural
killer T cells and IL-17. J Exp Med 205:385-393.
14.
Rachitskaya, A.V., Hansen, A.M., Horai, R., Li, Z., Villasmil, R., Luger, D.,
Nussenblatt, R.B., and Caspi, R.R. 2008. Cutting Edge: NKT Cells Constitutively
Express IL-23 Receptor and ROR{gamma}t and Rapidly Produce IL-17 upon
Receptor Ligation in an IL-6-Independent Fashion. J Immunol 180:5167-5171.
15.
Unutmaz, D. 2003. NKT cells and HIV infection. Microbes Infect 5:1041-1047.
16.
Sandberg, J.K., Fast, N.M., Palacios, E.H., Fennelly, G., Dobroszycki, J., Palumbo,
P., Wiznia, A., Grant, R.M., Bhardwaj, N., Rosenberg, M.G., et al. 2002. Selective
loss of innate CD4(+) V alpha 24 natural killer T cells in human immunodeficiency
Snyder-Cappione et al. 11
17.
18.
19.
20.
21.
22.
23.
24.
25.
virus infection. J Virol 76:7528-7534.
Motsinger, A., Haas, D.W., Stanic, A.K., Van Kaer, L., Joyce, S., and Unutmaz, D.
2002. CD1d-restricted human natural killer T cells are highly susceptible to human
immunodeficiency virus 1 infection. J Exp Med 195:869-879.
van der Vliet, H.J., von Blomberg, B.M., Hazenberg, M.D., Nishi, N., Otto, S.A., van
Benthem, B.H., Prins, M., Claessen, F.A., van den Eertwegh, A.J., Giaccone, G., et al.
2002. Selective decrease in circulating V alpha 24+V beta 11+ NKT cells during HIV
type 1 infection. J Immunol 168:1490-1495.
Chen, N., McCarthy, C., Drakesmith, H., Li, D., Cerundolo, V., McMichael, A.J.,
Screaton, G.R., and Xu, X.N. 2006. HIV-1 down-regulates the expression of CD1d
via Nef. Eur J Immunol 36:278-286.
Cho, S., Knox, K.S., Kohli, L.M., He, J.J., Exley, M.A., Wilson, S.B., and
Brutkiewicz, R.R. 2005. Impaired cell surface expression of human CD1d by the
formation of an HIV-1 Nef/CD1d complex. Virology 337:242-252.
Hage, C.A., Kohli, L.L., Cho, S., Brutkiewicz, R.R., Twigg, H.L., 3rd, and Knox,
K.S. 2005. Human immunodeficiency virus gp120 downregulates CD1d cell surface
expression. Immunol Lett 98:131-135.
Vasan, S., Poles, M.A., Horowitz, A., Siladji, E.E., Markowitz, M., and Tsuji, M.
2007. Function of NKT cells, potential anti-HIV effector cells, are improved by
beginning HAART during acute HIV-1 infection. Int Immunol 19:943-951.
Kim, S., Lalani, S., Parekh, V.V., Vincent, T.L., Wu, L., and Van Kaer, L. 2008.
Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant
NKT cells in mice. J Clin Invest 118:2301-2315.
Berzins, S.P., Cochrane, A.D., Pellicci, D.G., Smyth, M.J., and Godfrey, D.I. 2005.
Limited correlation between human thymus and blood NKT cell content revealed by
an ontogeny study of paired tissue samples. Eur J Immunol 35:1399-1407.
Takahashi, T., Dejbakhsh-Jones, S., and Strober, S. 2006. Expression of CD161
(NKR-P1A) defines subsets of human CD4 and CD8 T cells with different functional
activities. J Immunol 176:211-216.
Snyder-Cappione et al/
A
B
10
CD1d-Tetramer
200K
5
100K
50K
0
0
10
2
10
3
CD3
10
4
10
10
3
10
2
0
5
0
10
2
10
3
10
4
10
% of NKT cells
75
Vα24
9.7
0.87
93
0.43
100
25
75
50
25
0
TNFα
25.6
5.65
PMA and Ionomycin
50
0
60.1
IFNγ
p<.0016
p<.0001
4.57
5
DX-αGalCer
C
HIV-Infected
TNFα
10 4
150K
% of NKT cells
Side Scatter
250K
Healthy
TNFα
IFNγ
IFNγ
Healthy Control
HIV-infected
D
E
% of NKT cells
50
40
TNFα
30
IFNγ
40
30
p=.0260
r2=.6620
20
20
20
10
10
0
TNFα
IFNγ
Suppressed
Virologic
0
0
0
10
20
0
10
20
Years Since Infection
Figure 1. NKT cells from HIV-infected subjects exhibit low IFNγ and TNFα production in response
to DimerX-αGalCer. (A) NKT cell gating included CD3+, Vα24+, CD1d-tetramer+ cells. (B) Percentages
of NKT cells secreting TNFα and IFNγ in response to DX-αGalCer; sample plots from one healthy control
subject and one HIV-infected subject with similar NKT cell levels are shown. (C) Percentage of NKT cells
producing IFNγ, and TNF - α in response to stimulation with DX- αGalCer or PMA + ionomycin. (D)
Cytokine secretion in response to DX-αGalCer in cells from HIV-infected subjects with or without suppressed
virus. The differences were not statistically significant. (E) Association between the NKT cytokine response to
DX-aGalCer and the years since HIV infection.
Snyder-Cappione et al/
p = 0.0457
r = .3735
90
80
70
0
25
% of IFNγ+ NKT cells
50
% of CD161+ NKT cells
% of CD161+ NKT cells
100
100
p = 0.0094
r = .5462
85
70
0
20
% of TNFα+ NKT cells
40
% of CD161+ NKT cells
B
A
100
90
80
70
60
Healthy
HIV-infected
Figure 2. Th1 cytokine secretion by NKT cells correlates inversely with CD161 expression in HIV-infected
subjects. (A) Association between the percentage of CD161+ NKT cells and production of TNFα and IFNγ by
NKT cells in response to αGalCer in HIV-infected subjects. (B) CD161 expression on NKT cells of healthy
controls and HIV-infected subjects. There was not a statistically significant difference between the groups.
Carvalho KI
Trabalho 3
As células NKT constituem uma população de linfócitos amplamente definida
pela expressão de TCR e marcadores de células NK, que tem atraído
bastante atenção devido a seu papel potencial de ligar a resposta
imunológica inata à adaptativa. Células NKT na periferia apresentam fenótipo
de memória e podem secretar altos níveis de citocinas após o estímulo com
antígenos, incluindo IFN-γ, TNF-α, IL-4 e IL-13. Neste estudo, nós avaliamos
as células NKT nos grupos de pacientes coinfectados pelo HIV e pelo M.
leprae.
Os voluntários foram divididos em quatro grupos: 27 controles
saudáveis, 17 indivíduos infectados pelo HIV-1, 17 pacientes com
hanseníase, e 23 pacientes coinfectados pelo HIV e pelo M. leprae. Foram
realizados ensaios de citometria de fluxo e ELISPOT em células
mononucleares
do
sangue
periférico
(CMSP)
congeladas.
Nós
demonstramos que pacientes coinfectados têm redução de células NKT no
sangue periférico quando comparados com indivíduos saudáveis e pacientes
monoinfectados pelo M. lepare. Por outro lado, as células NKT de pacientes
coinfectados secretam mais IFN-γ quando comparadas com pacientes
monoinfectados pelo M. leprae. Estes resultados sugerem que as células
NKT têm atividade aumentada em pacientes coinfectados, porém com
frequência diminuída no sangue periférico.
46
Title: NKT cells profile in HIV and leprosy coinfected patients.
Suggested list of authors: Karina I. Carvalho1, Fernanda R. Bruno1, Jennifer SnyderCappione2, Solange Maeda1, Jane Tomimori1, Marilia B. Xavier4, Patrick Hasllet5,
Douglas Nixon2, Esper G. Kallas1,3
1 – Federal University of São Paulo, São Paulo, Brazil
2 – Division of Experimental Medicine, San Francisco General Hospital, Department
of Medicine, University of California, San Francisco, USA
3 – Division of Clinical Immunology and Allergy, University of São Paulo, São
Paulo, Brazil
4 – Federal University of Pará, Pará, Brazil
5 – University of Miami, Florida, USA
Running title: NKT cells in HIV and Leprosy coinfection (40 characters)
Word count: 2.230
Keywords: HIV, Mycobacterium leprae, coinfection, NKT cell, activation, CD4
Carvalho KI et al
Footnote page:
Corresponding author:
Esper Georges Kallas, M.D., Ph.D.
Universidade de São Paulo
Laboratório de Investigação Médica 60
Av. Dr. Arnaldo 455, terceiro andar
São Paulo – SP 01246-903
Phone: (+55-11) 3061-8395
Fax: (+55-11) 3061-8392
[email protected]
Funding
This work was partially supported by the National Institutes of Health, grant #R01-AI
52731 (Nixon) and The Fogarty International Center, grant #D43 TW00003; KIC’s
Ph.D. scholarship has been provided by the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Brazilian Ministry of Science and Technology. We
are also thankful for support from the Heiser Program for Research in Leprosy and
Tuberculosis of The New York Community Trust.
2
Carvalho KI et al
Abstract (180)
NKT cells are a heterogeneous population of lymphocytes loosely defined as
cells that express a TCR in addition to NK cell markers, which have attracted a great
deal of attention due to their potential role linking innate and adaptive immune
responses. Peripheral NKT cells display a memory activated phenotype and can
rapidly secrete large amounts of cytokines including IFN-γ, TNF-α, IL-4, and IL-13
on antigenic activation. In this study, we evaluated NKT cells in the context of HIV
and M. leprae coinfection. The volunteers were enrolled into 4 groups: 27 healthy
controls, 17 HIV seropositive patients, 17 patients with leprosy, and 23 co-infected
patients with leprosy and HIV-1 infection. Flow cytometric and ELISPOT assays
were performed in stored PBMC. We demonstrated that coinfected patients have
reduced NKT cells in the peripheral blood when compared to healthy subjects and
leprosy monoinfected patients.
On the other hand, NKT cells from coinfected
patients secrete more IFN-γ when compared to leprosy monoinfected patients. These
results suggest that NKT cells are highly active in coinfected patients, although
occurring in decreased frequency in the peripheral blood.
3
Carvalho KI et al
Introduction
Natural killer T cells are a specialized T cell lineage with unique functional
characteristics that distinguish them from conventional T lymphocytes [1]. Their
regulatory role in immune responses that require opposite regulatory pathways has
been attributed to an apparent flexibility of NKT cells with regard to their
predominant cytokine profile [2]. Peripheral NKT cells display a memory activated
phenotype and can rapidly secrete large amounts of cytokines including IFN-γ, TNFα, IL-4, and IL-13 on antigenic activation [3].
NKT cells are a heterogeneous population of lymphocytes loosely defined as
cells that express a TCR in addition to NK cell markers [4], which have attracted a
great deal of attention due to their potential to link the innate and adaptive arms of the
immune system. Characteristically, they respond very rapidly to certain stimulus and
are then able to activate a number of immune effectors [5]. Presentation of αgalactosylceramide (α-GalCer) by CD1d-expressing APC, such as dendritic cells
(DC), results in rapid activation of NKT cells.
It is clear that the capacity to
participate in early immune responses and to modulate both innate and adaptative
immunity confers NKT cells the potential to mediate important activities in the
control of pathogens and subsequent clearance of infections [6].
Gansert et al., provided evidence that α-GalCer can activate antimicrobial
pathways in a CD1d-restricted manner in humans [7]. The protection conferred by
NKT cells could be a result of the fact that the cytokines they produce are not only
critical in activating early innate immune responses, but also favor the development of
the classical virus-specific T-cell responses that are ultimately responsible for clearing
the infection [8].
4
Carvalho KI et al
Leprosy is a debilitating chronic, infectious disease caused by Mycobacterium
leprae that involves skin and peripheral nerves [9]. Most individuals infected with M.
leprae do not manifest leprosy, but a few manifest the disease depending on their
immunological status [10]. Of major concern has been the prevalence of HIV is
increasing in many countries where leprosy is endemic [11], and the possibility that
HIV coinfection might shift the clinical spectrum of leprosy paucibacillary to
multibacillary forms, enhancing the transmission of M. leprae in the community [12].
Frommel et al. confirmed the overall rise in HIV seropositivity and the increase of
coinfection by M. leprae and HIV [13]. Since HIV-1 compromises the cell-mediated
immune response, the HIV-1-positive individual infected with M. leprae might be
expected to manifest the lepromatous form of the disease or, alternatively, develop
rapid progression from tuberculoid to lepromatous form, as HIV-1 infection impairs
the cellular immune response [14]. In this study we demonstrated that coinfected
patients have reduced NKT cells in the peripheral blood when compared to healthy
subjects and leprosy monoinfected patients. On the other hand, NKT cells from
coinfected patients secrete more IFN-γ when compared to leprosy monoinfected
patients.
5
Carvalho KI et al
Materials and Methods
Subjects and sample collection
Volunteers were recruited at the Federal University of Sao Paulo and the
Federal University of Pará, Brazil. Written, informed consent was obtained from all
volunteers according to the guidelines of the Brazilian Ministry of Health, approved
by the Institutional Review Board. The study subjects were divided into 4 groups:
twenty-seven healthy controls and seventeen HIV seropositive patients, most of
whom had CD4+ T cell counts of less than 400 cells/µL, were identified at the
Federal University of Sao Paulo; seventeen patients with leprosy were enrolled and
classified according to the bacillary load [15, 16], as well as twenty-three co-infected
patients with leprosy and HIV-1 infection, all recruited at Leprosy Clinics in both
sites. Leprosy patients were matched for bacillary load with the cases in the coinfected group.
Flow cytometry
PBMC were isolated from volunteers, the cells were cryopreserved and stored
in liquid nitrogen until used in the assays. The following monoclonal antibodies were
used in the FACS assays: anti-HLA-DR-peridin chlorophyll protein (PerCP) (clone
L243), from BD Biosciences (San Jose, CA); CD4- phycoerythrin–cyanine (PE-Cy7)
(clone SK3), CD3 allophycocyanin cyanine-7 (APC-Cy7) (clones SK7), and CD161APC (clone DX12), from BD PharMingen (San Jose, CA); Vα24 phycoerythrin (PE)
(clone C15), Vβ11-Fluorescein isothiocyanate (FITC) (clone C21) from Immunotech
6
Carvalho KI et al
(BC). All the antibodies were used for cell-surface staining. Fluoresce minus one
(FMO) was used for gate strategy.
After thawing, cells were centrifuged at 1,500 rpm for 5 min and transferred
into 96 V bottom well plates (Nunc, Denmark) in 100 µL of staining buffer (PBS
supplemented with 0.1% sodium azide [Sigma] and 1% FBS, pH 7.4-7.6) with the
surface monoclonal antibodies panel. Cells were incubated at 4°C in darkness for 30
minutes, washed twice, and re-suspended in 100 µL of fixation buffer (1%
paraformaldehyde [Polysciences, Warrington, PA] in PBS, pH 7.4-7.6).
Samples were acquired on a FACSCanto, using FACSDiva software (BD
Biosciences), and then analyzed with FlowJo software (Tree Star, San Carlo, CA).
Fluorescence voltages were determined using matched unstained cells. Compensation
was carried out with CompBeads (BD Biosciences) single-stained with CD3-PerCP,
CD4-FITC, CD8-APC-Cy7, CD4-PE-Cy7, CD3-PE, and CD3-APC. Samples were
acquired until at least 800,000 events in a live lymphocyte gate.
Measurement of cytokine-producing cells by Elispot
To determine the amount of IFNγ and IL-4 secreting cells, MAIP Elispot
plates (Millipore) were coated with either anti-IFN-γ (10µg/ml) and anti-IL-4
(15µg/ml) (Mabtech), in PBS, 50 µl/well, either overnight at 4°C or for one hour at
room temperature. After three washes with complete medium, PBMC (3x105) and the
presence or absence of a synthetic glicolipid α-glactosyl-ceramide (α-GalCer)
(AXXORA), at a final volume of 200 µl/well. Plates were incubated at 37 °C in 5%
CO2 for 16-20 hours. After washing with phosphate-buffered saline (PBS) plus 0.1%
7
Carvalho KI et al
Tween 20 (PBST), the following biotinylated antibodies were added to the
appropriate wells: anti-IL-4 (1µg/ml) (Mabtech) and anti-IFN-γ (1µg/ml) (Mabtech),
in PBS 0.1% tween 1% BSA (PBSTB) for 30 minutes at room temperature. The
plates are washed again three times with PBST, and alkaline phosphatase-conjugated
streptavidin (Jackson Immunoresearch) was added (50 µl of 1:1,000 dilution in
PBSTB) and incubated for 30 min at room temperature. Plates were washed in PBST,
incubated with blue substrate (Vector Labs, # SK-5300) until spots were clearly
visible, then rinsed with tap water (submitted Snyder-Cappione). Colored spots were
counted with an Immunospot S5 Analyser (CTL, LLC).
Statistical analysis
Groups were compared using non-parametric models; data are reported as median and
interquartile range. Comparisons among groups were carried out using KruskalWallis non-parametric test. p values were considered significant if <0.05. Results are
expressed in medians and interquartile ranges (IQR).
8
Carvalho KI et al
Results
Characteristics of the HIV-M. leprae co-infected patient
The median age of all participants was 37 years (IQR, 35-48) and most were
male (81%), although no difference in gender distribution was observed between
groups.
For the leprosy and co-infected groups, 77% of the patients had a
multibacillary presentation at the time of diagnosis. Co-infected patients were treated
with the appropriate multi drug therapy (MDT) for paucibacillary (PB) and
multibacillary (MB) leprosy, when indicated. Our results demonstrated that healthy
controls (Median: 916.5, IQR, 687-1170) have higher CD4+ T cell counts (cells/mm3)
when compared with HIV patients (Median: 391, IQR, 272-536) and co-infected
patients (Median: 285, IQR, 235-480]), p<0.001. Leprosy patients (Median: 733,
IQR, 699-870]), show higher numbers of CD4+ T cell counts (cells/mm3) when
compared with co-infected patients (p<0.001). For CD8+ T cell counts healthy
controls (Median: 556, IQR, 735-376) have lower numbers when compared with coinfected patients (Median: 806, IQR, 1548-578), p<0,05 (Table 1).
Measurement of NKT cell frequencies in peripheral blood
NKT cells represent a subset of lymphocytes that is defined operationally as
bearing both the TCR and the NK receptor, CD161 [17]. We defined NKT cells as
those with the CD3+Vα24+Vβ11+ phenotype. Berzins et al. described that adults
blood NKT cells frequency range from 0.006 to 0.78% [18].
Our results
demonstrated that healthy controls (median: 0.0890 [IQR: 0.032 – 0,370]) and leprosy
monoinfected patients (median: 0.060 [IQR: 0.034 – 0.125]) have in the periphery
9
Carvalho KI et al
higher NKT cells when compared with coinfected patients (median: 0.017 [IQR:
0.004 – 0.032]), p<0.001 (Figure 1A).
Regarding to M. tuberculosis, Snyder-
Cappione et al. demonstrated that the percentages of circulating NKT cells are
selectively and significantly lower in patients with active pulmonary tuberculosis than
those in uninfected individuals were lower in frequency [19].
Subset of CD4+ NKT cells
Expression of CD4 distinguishes two phenotypically and functionally distinct
subsets of NKT cells. CD4+ NKT cells were found to produce both Th1 and Th2
cytokines, whereas CD4- NKT cells were found mainly to produce Th1 cytokines [20,
21].
In adult peripheral blood, close to 50% of NKT cells are CD4- with low
expression of CD8+ [22]. We observed that leprosy patients have more CD4 NKT
cells positive when compared with HIV positive patients (p< 0.05) (Figure 1B).
Activation marker in NKT cells
We used CD161 and HLA-DR as activation markers in NKT cells. Although
5 to 10% of human peripheral blood T cells express CD161 [5, 23], constitutive
expression of CD161+NKT cells along with CD1b and CD1d expression on
monocytes-macrofages confirms the presence of NKT cells in peripheral blood of
leprosy patients [24].
Our results could not detect any significant difference in
CD161 expression on NKT cells in all groups. NKT cells can became activated
during a variety of infections and inflammatory responses [25]. These data indicate
that activation marker HLA-DR of NKT cells in coinfected patients (median: 61.75
[IQR: 25.95 – 79.60]) were higher expressing when compared with leprosy patients
10
Carvalho KI et al
(median: 8.16 [IQR: 0.91 – 30]) and healthy controls (median: 13 [IQR: 3.20 –
35.30]), p<0.005 (Figure 1C).
NKT cells from coinfected patients produce IFN-γ in response to α-GalCer
It is well established that NKT cells are activated in response to the marine
sponge-derived glycolipid antigen α-GalCer and antigen presentation occurs through
CD1d [7]. ELISPOT is a very sensitive method to detect the frequency of antigenreactive cells in a population of lymphocytes with multiple specificities [26]. Our
results demonstrated that the NKT cells, when stimulated with specific antigen αGalCer, have higher secretion IFN-γ in coinfected patients (median: 51.80 [IQR:
18.60 – 67.40]) when compared to leprosy monoinfected patients (median: 38.30
[IQR: 22.90 – 51.35]), p<0.05 (Figure 2B). No difference in IL-4 secretion by NKT
cells was detected comparing the different groups of participants (Figure 2A).
11
Carvalho KI et al
Discussion
The importance of NKT cells might depend on their ability to be activated
early during viral infection and to produce cytokines such as IFN-γ quickly, thus
initiating the activation of NK cells, which then exert the primary antiviral effects,
either directly by cytolysis or indirectly through the further release of cytokines [8].
NKT cells participate in host defense against microbial infection [7]. Several groups
demonstrated that NKT cells were lower in peripheral blood in Mycobacterium
infections [19, 27]. We demonstrated that coinfected patients show lower NKT cells
in peripheral blood when compared with healthy subjects and leprosy monoinfected
patients. One important effector mechanism by which NKT cells may contribute to
host defense against infection in humans is the production of cytokines [7]. Having
originally been recognized for their ability to produce the Th2 cytokine IL-4, NKT
cells take on a Th1 phenotype during development and produce significant amounts
IFN-γ [28]. In leprosy patients we observed an increased percentage of NKT cells
expressing IFN-γ, compared to a lower frequency expressing IL-4 [24].
The
differential expression of IL-4 cytokine expression by NKT cells is critical for their
function in many different disease states. The finding that NKT cells recognize αGalCer presented by dendritic cells in a CD1d-dependent manner represents a novel
recognition mechanism in the immune system [29]. In this study, we observed that
coinfected patients produced higher amount of IFN-γ when stimulated with specific
antigen of NKT cells α-GalCer, but did not produce IL-4 in the same subjects.
Mempel et al. described the presence of NKT cells in leprous granulomas, and the
presence of this distinctive T-cell subset is clearly associated with T-cell forms of
12
Carvalho KI et al
leprosy [30]. One hypothesis is that the NKT cells can be in the site of infection and
in the peripheral blood is activated in these patients.
Human NKT cells could be involved in the initial stages of HIV-1 infection
and in the spread of the virus throughout the host. Although many studies have
attributed beneficial antiviral responses to NKT cells have also been implicated in
detrimental immune responses that lead to immunopathology and disease [8]. HIV
entry into target cells requires concomitant expression of CD4 and chemokine
receptors. CD4+ T cells are suitable targets for HIV-1 infection because they express
CD4 as well as high levels of the chemokine receptor CCR5 [31]. Recent findings
show that NKT cells in PBMCs of HIV-1 infected individuals are dramatically
reduced compared with healthy donors [2, 32, 33]. Therefore, the loss of NKT cells
in HIV+ individuals may lead to autoimmunity or autoimmune-like conditions.
Diminished NKT cell-mediated anti-tumor responses could also contribute to
increased incidence of tumors such as Kaposi’s sarcoma and non-Hodgkin’s
lymphoma in AIDS patients [23]. In this study, we observed a trend to a decreased
NKT cells percentage in the peripheral blood. We also found that CD4+ NKT cells
were significantly lower in HIV+ subjects when compared with leprosy monoinfected
patients. Thus, selective loss of the CD4+ subpopulation of NKT cells may have
significant effects on the function of the NKT-cell compartment in HIV-infected
individuals. In conclusion, we demonstrated, for the first time, a preferential depletion
of the immunoregulatory Vα24+Vβ11+ during HIV/leprosy coinfection and a
decreased in the CD4+ subset of NKT cell population in HIV+ subjects when
compared to leprosy subjects.
These findings also raise the possibility that
mycobacterial infections are associated with depletion of CD4+ NKT cells.
13
Carvalho KI et al
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Crowe, N. Y., Uldrich, A. P., Kyparissoudis, K., Hammond, K. J.,
Hayakawa, Y., Sidobre, S., Keating, R., Kronenberg, M., Smyth, M. J.
and Godfrey, D. I., Glycolipid antigen drives rapid expansion and sustained
cytokine production by NK T cells. J Immunol 2003. 171: 4020-4027.
van der Vliet, H. J., von Blomberg, B. M., Hazenberg, M. D., Nishi, N.,
Otto, S. A., van Benthem, B. H., Prins, M., Claessen, F. A., van den
Eertwegh, A. J., Giaccone, G., Miedema, F., Scheper, R. J. and Pinedo, H.
M., Selective decrease in circulating V alpha 24+V beta 11+ NKT cells during
HIV type 1 infection. J Immunol 2002. 168: 1490-1495.
Thedrez, A., de Lalla, C., Allain, S., Zaccagnino, L., Sidobre, S.,
Garavaglia, C., Borsellino, G., Dellabona, P., Bonneville, M., Scotet, E.
and Casorati, G., CD4 engagement by CD1d potentiates activation of CD4+
invariant NKT cells. Blood 2007. 110: 251-258.
Godfrey, D. I., Hammond, K. J., Poulton, L. D., Smyth, M. J. and Baxter,
A. G., NKT cells: facts, functions and fallacies. Immunol Today 2000. 21:
573-583.
Kronenberg, M., Toward an understanding of NKT cell biology: progress
and paradoxes. Annu Rev Immunol 2005. 23: 877-900.
McNab, F. W., Berzins, S. P., Pellicci, D. G., Kyparissoudis, K., Field, K.,
Smyth, M. J. and Godfrey, D. I., The influence of CD1d in postselection
NKT cell maturation and homeostasis. J Immunol 2005. 175: 3762-3768.
Gansert, J. L., Kiessler, V., Engele, M., Wittke, F., Rollinghoff, M.,
Krensky, A. M., Porcelli, S. A., Modlin, R. L. and Stenger, S., Human
NKT cells express granulysin and exhibit antimycobacterial activity. J
Immunol 2003. 170: 3154-3161.
Van Dommelen, S. L. and Degli-Esposti, M. A., NKT cells and viral
immunity. Immunol Cell Biol 2004. 82: 332-341.
Murray, R. A., Siddiqui, M. R., Mendillo, M., Krahenbuhl, J. and
Kaplan, G., Mycobacterium leprae inhibits dendritic cell activation and
maturation. J Immunol 2007. 178: 338-344.
Makino, M., Maeda, Y. and Ishii, N., Immunostimulatory activity of major
membrane protein-II from Mycobacterium leprae. Cell Immunol 2005. 233:
53-60.
Ustianowski, A. P., Lawn, S. D. and Lockwood, D. N., Interactions between
HIV infection and leprosy: a paradox. Lancet Infect Dis 2006. 6: 350-360.
Orege, P. A., Fine, P. E., Lucas, S. B., Obura, M., Okelo, C., Okuku, P.
and Were, M., A case control study on human immunodeficiency virus-1
(HIV-1) infection as a risk factor for tuberculosis and leprosy in western
Kenya. Tuber Lung Dis 1993. 74: 377-381.
Frommel, D., Tekle-Haimanot, R., Verdier, M., Negesse, Y., Bulto, T. and
Denis, F., HIV infection and leprosy: a four-year survey in Ethiopia. Lancet
1994. 344: 165-166.
Sampaio, E. P., Caneshi, J. R., Nery, J. A., Duppre, N. C., Pereira, G. M.,
Vieira, L. M., Moreira, A. L., Kaplan, G. and Sarno, E. N., Cellular
immune response to Mycobacterium leprae infection in human
14
Carvalho KI et al
15
16
17
18
19
20
21
22
23
24
25
26
27
28
immunodeficiency virus-infected individuals. Infect Immun 1995. 63: 18481854.
Ridley, D. S. and Jopling, W. H., Classification of leprosy according to
immunity. A five-group system. Int J Lepr Other Mycobact Dis 1966. 34: 255273.
Saúde, M. d., Plano Nacional de Eliminação da Hanseníase em Nível
Municipal 2006-2010 2006.
Wilson, S. B. and Byrne, M. C., Gene expression in NKT cells: defining a
functionally distinct CD1d-restricted T cell subset. Curr Opin Immunol 2001.
13: 555-561.
Berzins, S. P., Cochrane, A. D., Pellicci, D. G., Smyth, M. J. and Godfrey,
D. I., Limited correlation between human thymus and blood NKT cell content
revealed by an ontogeny study of paired tissue samples. Eur J Immunol 2005.
35: 1399-1407.
Snyder-Cappione, J. E., Nixon, D. F., Loo, C. P., Chapman, J. M.,
Meiklejohn, D. A., Melo, F. F., Costa, P. R., Sandberg, J. K., Rodrigues,
D. S. and Kallas, E. G., Individuals with pulmonary tuberculosis have lower
levels of circulating CD1d-restricted NKT cells. J Infect Dis 2007. 195: 13611364.
Gumperz, J. E., Miyake, S., Yamamura, T. and Brenner, M. B.,
Functionally distinct subsets of CD1d-restricted natural killer T cells revealed
by CD1d tetramer staining. J Exp Med 2002. 195: 625-636.
Lee, P. T., Benlagha, K., Teyton, L. and Bendelac, A., Distinct functional
lineages of human V(alpha)24 natural killer T cells. J Exp Med 2002. 195:
637-641.
Sandberg, J. K., Stoddart, C. A., Brilot, F., Jordan, K. A. and Nixon, D.
F., Development of innate CD4+ alpha-chain variable gene segment 24
(Valpha24) natural killer T cells in the early human fetal thymus is regulated
by IL-7. Proc Natl Acad Sci U S A 2004. 101: 7058-7063.
Unutmaz, D., NKT cells and HIV infection. Microbes Infect 2003. 5: 10411047.
Chattree, V., Khanna, N., Bisht, V. and Rao, D. N., Inhibition of apoptosis,
activation of NKT cell and upregulation of CD40 and CD40L mediated by M.
leprae antigen(s) combined with Murabutide and Trat peptide in leprosy
patients. Mol Cell Biochem 2008. 309: 87-97.
Van Kaer, L., NKT cells: T lymphocytes with innate effector functions. Curr
Opin Immunol 2007. 19: 354-364.
Sieling, P. A., Torrelles, J. B., Stenger, S., Chung, W., Burdick, A. E., Rea,
T. H., Brennan, P. J., Belisle, J. T., Porcelli, S. A. and Modlin, R. L., The
human CD1-restricted T cell repertoire is limited to cross-reactive antigens:
implications for host responses against immunologically related pathogens. J
Immunol 2005. 174: 2637-2644.
Montoya, C. J., Catano, J. C., Ramirez, Z., Rugeles, M. T., Wilson, S. B.
and Landay, A. L., Invariant NKT cells from HIV-1 or Mycobacterium
tuberculosis-infected patients express an activated phenotype. Clin Immunol
2008. 127: 1-6.
Hammond, K. J., Pelikan, S. B., Crowe, N. Y., Randle-Barrett, E.,
Nakayama, T., Taniguchi, M., Smyth, M. J., van Driel, I. R., Scollay, R.,
Baxter, A. G. and Godfrey, D. I., NKT cells are phenotypically and
functionally diverse. Eur J Immunol 1999. 29: 3768-3781.
15
Carvalho KI et al
29
30
31
32
33
Porcelli, S. A., Segelke, B. W., Sugita, M., Wilson, I. A. and Brenner, M.
B., The CD1 family of lipid antigen-presenting molecules. Immunol Today
1998. 19: 362-368.
Mempel, M., Flageul, B., Suarez, F., Ronet, C., Dubertret, L., Kourilsky,
P., Gachelin, G. and Musette, P., Comparison of the T cell patterns in
leprous and cutaneous sarcoid granulomas. Presence of Valpha24-invariant
natural killer T cells in T-cell-reactive leprosy together with a highly biased T
cell receptor Valpha repertoire. Am J Pathol 2000. 157: 509-523.
Kinter, A., Arthos, J., Cicala, C. and Fauci, A. S., Chemokines, cytokines
and HIV: a complex network of interactions that influence HIV pathogenesis.
Immunol Rev 2000. 177: 88-98.
Motsinger, A., Haas, D. W., Stanic, A. K., Van Kaer, L., Joyce, S. and
Unutmaz, D., CD1d-restricted human natural killer T cells are highly
susceptible to human immunodeficiency virus 1 infection. J Exp Med 2002.
195: 869-879.
Sandberg, J. K., Fast, N. M., Palacios, E. H., Fennelly, G., Dobroszycki,
J., Palumbo, P., Wiznia, A., Grant, R. M., Bhardwaj, N., Rosenberg, M.
G. and Nixon, D. F., Selective loss of innate CD4(+) V alpha 24 natural killer
T cells in human immunodeficiency virus infection. J Virol 2002. 76: 75287534.
16
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Table 1. Demographic, clinical, and laboratory characteristics of participants.
Healthy
Leprosy
HIV
HIVLeprosy
Gender
Age
(years)
mediam
(n=26)
Female
42%
Male
58%
(n=17)
Female
12%
Male
88%
(n=17)
(n=25)
33.54
(n=26)
972.92
(n=25)
556
(n=14)
35.43
(n=7)
672.5
(n=17)
40.59
(n=23)
39.87
Male
100%
(n=23)
Female
22%
Male
78%
CD4+ T
CD8+ T
cells/mm3 cells/mm3
Viral
load
(HIVRNA
log10)
ND
Leprosy
clinical
presentation
(n=5)
511
ND
(n=17)
402.27
(n=16)
1028
(n=16)
2.58
(n=14)
TT 7%
LL 36%
BT 21%
BL 36%
ND
(n=22)
341.52
(n=23)
806
(n=21)
3.06
ND
(n=23)
TT 13%
LL 0%
BT 48%
BL 39%
ND: not done.
17
Carvalho KI et al
Figure legends
Figure 1. Distribution of NKT cells in the periphery in four groups. (A) Log10
expression of NKT cells (**p < 0.01). (B) The frequency of CD4+ NKT
cells substes (*p < 0.05). (C) Activation marker anti-HLADR in NKT cells
(**p < 0.01) Group comparisons were carried out using Kruskal-Wallis nonparametric test.
Figure 2. Enumeration of cytokine producing cells following stimulation with
antigen-loaded CD1d dimers. Human PBMC were added to cytokine
Elispot assays directly ex vivo with αGalCer-specific cytokine producing IL4 (not significant) and INF-γ (*p < 0.05).
18
Carvalho KI et al
Figure 1.
A
100
**
**
10
1
0.1
0.01
0.001
Healthy
HIV
Leprosy
B
Dual
C
**
*
100
100
**
75
50
50
25
0
0
Healthy
HIV
Leprosy
Dual
Healthy
HIV
Leprosy
Dual
19
Carvalho KI et al
Figure 2
A
30
ns
20
10
0
Healthy
HIV
Leprosy
B
Dual
*
30
20
10
0
Healthy
HIV
Leprosy
Dual
20
Carvalho KI et al
21
Carvalho KI
Trabalho 4
Neste estudo, foram examinados os fenótipos de ativação e a secreção de
citocinas pelas células NKT em pacientes com CVID. Este é o primeiro
estudo da frequência e função de células NKT nestes pacientes. Os
resultados demonstraram que a frequência de células NKT no sangue
periférico está diminuída nos que apresentam CVID (media: 0.02 [IQR: 0.01 –
0.05]) quando comparados com controles saudáveis (media: 0.28 [IQR: 0.13
– 0.48]); p < 0.001. Foi demonstrada uma correlação inversa entre a
secreção de IL-4 pelas células NKT e níveis da expressão de CXCR6. Os
resultados sugerem que talvez possamos auxiliar o tratamento dos pacientes
CVID se fosse possível obter um aumento no número de células NKT através
de uma terapia modulatória.
80
Title:
Decreased frequency of circulating activated natural killer T cells in
patients with common variable immunodeficiency disorders (CVID).
Karina I. Carvalho1, Karina Melo1, Fernanda R. Bruno1, Jennifer E. SnyderCappione2, Douglas F. Nixon2, Beatriz T. Costa-Carvalho1, Esper G. Kallas1-3
1
Federal University of São Paulo, São Paulo, Brazil; 2Division of Experimental
Medicine, San Francisco General Hospital, University of California, San Francisco,
USA; 3Division of Clinical Immunology and Allergy, University of São Paulo, São
Paulo, Brazil
Running title: NKT cells in CVID
Key words:
NKT cells, common variable immunodeficiency disorder, CVID,
CXCR6, CCR5, IL-4
Word count: 1196 words (excluding abstract, references, and figure legends).
Carvalho et al.
Abstract (113 words)
In this study, we examined the phenotype, activation markers, and cytokine
secretion from NKT cells in patients with CVID. To our knowledge, this is the first
study of the function and frequency of NKT cells in such patients. Our results
showed that the frequency of NKT cells in peripheral blood is markedly lower in
CVID patients (median 0.02 [IQR: 0.01 - 0.05]) when compared to healthy controls
(median 0.28 [IQR: 0.13 - 0.48]; Figure 1B), p < 0.001. We also showed an inverse
correlation between IL-4 secretion by NKT cells and their level of CXCR6
expression. Boosting of NKT cell numbers through therapeutic modulation might be
a valuable adjunctive treatment in CVID subjects.
2
Carvalho et al.
Introduction
Common variable immunodeficiency disorder (CVID) is comprised of 20-30
different
immunodeficiencies
with
similar
characteristics,
including
hypogammaglobulinemia and impaired B cell functions (1-3). CVID is the most
common form of significant clinical primary antibody failure in adults and children
(4). Although CVID is characterized as a hyporesponsive immune disorder, many
subjects develop autoimmune diseases (5).
Natural killer T (NKT) cells are lymphocytes that express a rearranged Vα24Jα18 semi-invariant TCR in humans, and recognize a glycolipid (for example the
prototypic α-galactosyl ceramide (αGalCer)), presented in the context of the nonclassical MHC molecule, CD1d (6). Upon TCR stimulation, NKT cells are able to
rapidly secrete both Th1 and Th2 cytokines (7), and appear to be an integral
component of the suppression of autoreactive T cells (8).
with
αGalCer
enhances
T-dependent
humoral
Activation of NKT cells
immune
responses
against
coadministered T-dependent Ag, and this involves interaction with CD1d-expressing
B cells (13). NKT cells can also help B lymphocyte responses, and mice immunized
with proteins and αGalCer develop antibody titers 1-2 log10 higher than those induced
by proteins alone (14).
Because of the important interactions of B cells with NKT cells, we measured
the frequencies, chemokine receptor patterns, and ex vivo effector functions of NKT
cells in CVID patients compared with healthy controls.
We hypothesized that
prevalence of autoimmune symptoms/ diseases in CVID was due to a yet unknown
defect in NKT cells. Our results show that NKT cells circulate at lower frequencies
in the peripheral blood of CVID subjects compared to controls. However, these NKT
3
Carvalho et al.
cells are highly activated with marked upregulation of the expression of CCR5 and
CXCR6.
Study Desing
Subjects and sample collection
This study was reviewed and approved by the local Institutional Review
Board, and informed consent was obtained from all participants. Diagnosis of CVID
was established according to the criteria by the Pan-American Group for
Immunodeficiency (PAGID). Seven healthy controls and seventeen CVID subjects
were identified at the Division of Pediatric Clinical Immunology located at the
Federal University of São Paulo. PBMC were isolated from volunteers and the cells
were cryopreserved until the day of the assay.
Flow cytometry
The following monoclonal antibodies were used: CD3-peridin chlorophyll
protein (PerCP), CD8-allophycocyanin (APC) and, CD4-PE-Cy7, from BD
Biosciences (San Jose, CA); CCR5- phycoerythrin–cyanine (PE-Cy7), and CD161APC, from BD PharMingen ; Vα24 phycoerythrin (PE), Vβ11-Fluorescein
isothiocyanate (FITC) from Immunotech; CXCR6-APC, and CD69 allophycocyanin
cyanine-7(APC-Cy7).
Fluorescence minus one (FMO) was used to gate (15).
Samples were acquired on a FACSCanto, using FACSDiva software (BD
Biosciences), and analyzed with FlowJo software (Tree Star, San Carlo, CA).
Elispot
4
Carvalho et al.
To determine the NKT cell secretion of interferon γ (IFNγ) and interleukin 4 (IL-4),
we used ELISPOT kits for detection of human IFNγ and IL-4 (Mabtech). PBMC
cells (3 x 105/ well) were incubated for 18 hours or overnight in the presence or
absence of an α-glactosyl-ceramide (α-GalCer) (AXXORA). Cells were removed
and plates processed according to the to the manufacturer’s instructions. Colored
spots were counted with an Immunospot S5 Analyser (CTL, LLC) (Snyder-Cappione.
et al., submitted).
Statistical analysis
Groups were compared using non-parametric models; data are reported as
median and interquartile ranges (IQR). Comparisons among groups used the MannWhitney non-parametric test. Correlations were performed using Spearman nonparametric test. p values were considered significant if <0.05.
5
Carvalho et al.
Results and Discussion
In this study, we examined the phenotype, activation markers, and cytokine
secretion from NKT cells in patients with CVID. To our knowledge, this is the first
study of the function and frequency of NKT cells in such patients. Our results
showed that the frequency of NKT cells in peripheral blood is markedly lower in
CVID patients (median: 0.02 [IQR: 0.01 - 0.05]) when compared to healthy controls
(median: 0.28 [IQR: 0.13 - 0.48]) (Figure 1B), p < 0.001. We also showed an inverse
correlation between IL-4 secretion by NKT cells and their level of CXCR6
expression.
NKT cells also display a partially activated/memory phenotype reflected by
the expression of molecules such as CD69 and CD44 found in human cord blood (16),
and share several phenotypic features with NK cells, such as surface expression of
CD161 (17). In this study CD69 expression on NKT cells was markedly elevated in
CVID subjects (median: 32.90 [IQR: 19.30 - 71.40]), suggesting an increased
activation state, or NKT cells at a late stage of differentiation. NKT cells from mice
spleen express CD69 at different stages, indicating that after NKT cells exit the
thymus they undergo further maturation by up-regulating CD69 (18).
CXCR6 expression is associated with the function and fate of NKT cells by
controlling their survival, cytokine production, and ability to induce tissue damage
(19). Previous studies describe that mice NKT cells were able to express CXCR6 (11,
19). Interestingly, this chemokine receptor is expressed in humans on Th1 and Tc1
memory CD4+ and CD8+ T lymphocytes (20), and CXCR6 preferentially express
double negative and CD8+ subsets in NKT cells (9, 21). It has been known that
CXCR6 is expressed at a high level on NKT cells even under physiological
conditions, as compared with other lymphocytes (22). Our results suggest that NKT
6
Carvalho et al.
cells express higher chemokine receptors, such as CCR5 and CXCR6, in CVID
subjects subjects (median: 70.80 [IQR: 63.30 – 95.30], and median: 18.70 [IQR:
14.20 – 27.20], respectively, p < 0.01), and this increased expression of the CXCR6 is
negatively correlated with IL-4 expression in total PBMC stimulated with αGalCer.
It is likely that the higher expression of these chemokines receptors by NKT cells
demonstrates a homing potential to nonlymphoid tissues and is highly associated with
inflammation. Together with the higher expression of CD69 on these NKT cells, the
cumulative data suggests a model of low numbers of highly activated circulating NKT
cells in CVID patients.
A recent study has shown that dendritic cells (DCs) grown in the presence of
low immunoglobulin levels preferentially present antigens to group I CD1-restricted T
cells (CD1a, b, and c), known to play an important role in antimicrobial responses,
whereas DCs grown with high immunoglobulin levels are superior in presenting
glycolipids to NKT cells (23). From our results in the CVID patients presented here,
who have low levels of circulating immunoglobulins, we can hypothesize that the
reduced frequency of NKT cells is related to lower CD1d expression on DCs. As
treatment with immunoglobulin is used for CVID patients, it will also be possible to
monitor longitudinally the frequency of NKT cells and how it relates to CD1d
expression on dendritic cells in future studies.
There are some limitations to this study. It is a cross sectional study, and NKT
cell frequencies may change over time, although we have previously shown a stability
of NKT cell numbers in healthy individuals (24). CIVD represents a spectrum of
diseases, and different genetic causes might lead to differences in NKT cell
frequencies. Also, we sampled NKT cells only in peripheral blood. Nevertheless, we
do not believe that these caveats would change the conclusions, based on the strong
statistical differences.
7
Carvalho et al.
In summary, CVID subjects have a lower frequency of activated homing NKT
cells in peripheral blood, with a decreased ability to secrete IFNγ and IL-4. Boosting
of NKT cell numbers through therapeutic modulation might be a valuable adjunctive
treatment in CVID subjects.
Acknowledgments
This work was partially supported by the National Institutes of Health (grants #R37AI52731 and AI060379) (Nixon); KIC’s Ph.D. scholarship has been provided by the
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazilian
8
Carvalho et al.
Ministry of Science and Technology. We also thank Helena Tomiyama and Tania
Maria da Silva for continued laboratory support.
Contribution: K.I.C., designed the research, performed the experiments, analyzed the
data, wrote the manuscript; K.M., evaluated the participants and collected the clinical
samples, supported the experiments; F.R.B., performed the experiments, supported
the data analyses; J.E.S-C., developed the NKT protocols, wrote the paper; D. F. N.,
obtained funds for the project, designed the research, wrote the paper; B.C-C.,
obtained funds for the project, supervised the clinical activities, revised the paper;
E.G.K., obtained funds for the project, designed the research, supervised the data
analyses, wrote the manuscript
Conflict-of-interet disclosure: The authors declare no competing conflicts of interests.
Corresponding author: Esper Georges Kallas, M.D., Ph.D., Universidade de São
Paulo, Laboratório de Investigação Médica 60, Av. Dr. Arnaldo 455, terceiro andar,
São Paulo – SP 01246-903, Phone: (+55-11) 3061-8395, Fax: (+55-11) 3061-8392 ;
e-mail : [email protected]
9
Carvalho et al.
References
1.
Haymore BR, Mikita CP, Tsokos GC. Common variable immune deficiency
(CVID) presenting as an autoimmune disease: role of memory B cells. Autoimmun
Rev 2008;7(4):309-12.
2.
Cunningham-Rundles C. Autoimmune Manifestations in Common Variable
Immunodeficiency. J Clin Immunol 2008.
3.
Lopes-da-Silva S, Rizzo LV. Autoimmunity in Common Variable
Immunodeficiency. J Clin Immunol 2008.
4.
Chapel H, Lucas M, Lee M, Bjorkander J, Webster D, Grimbacher B, et al.
Common variable immunodeficiency disorders: division into distinct clinical
phenotypes. Blood 2008.
5.
Cunningham-Rundles C, Bodian C. Common variable immunodeficiency:
clinical and immunological features of 248 patients. Clin Immunol 1999;92(1):34-48.
6.
Kronenberg M. Toward an understanding of NKT cell biology: progress and
paradoxes. Annu Rev Immunol 2005;23:877-900.
7.
Au-Yeung BB, Fowell DJ. A key role for Itk in both IFN gamma and IL-4
production by NKT cells. J Immunol 2007;179(1):111-9.
8.
Falcone M, Facciotti F, Ghidoli N, Monti P, Olivieri S, Zaccagnino L, et al.
Up-regulation of CD1d expression restores the immunoregulatory function of NKT
cells and prevents autoimmune diabetes in nonobese diabetic mice. J Immunol
2004;172(10):5908-16.
9.
Lee PT, Benlagha K, Teyton L, Bendelac A. Distinct functional lineages of
human V(alpha)24 natural killer T cells. J Exp Med 2002;195(5):637-41.
10
Carvalho et al.
10.
Thomas SY, Hou R, Boyson JE, Means TK, Hess C, Olson DP, et al. CD1d-
restricted NKT cells express a chemokine receptor profile indicative of Th1-type
inflammatory homing cells. J Immunol 2003;171(5):2571-80.
11.
Johnston B, Kim CH, Soler D, Emoto M, Butcher EC. Differential chemokine
responses and homing patterns of murine TCR alpha beta NKT cell subsets. J
Immunol 2003;171(6):2960-9.
12.
Germanov E, Veinotte L, Cullen R, Chamberlain E, Butcher EC, Johnston B.
Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of
CD1d-Restricted NKT Cells. J Immunol 2008;181(1):81-91.
13.
Lang GA, Devera TS, Lang ML. Requirement for CD1d expression by B cells
to stimulate NKT cell-enhanced antibody production. Blood 2008;111(4):2158-62.
14.
Galli G, Pittoni P, Tonti E, Malzone C, Uematsu Y, Tortoli M, et al. Invariant
NKT cells sustain specific B cell responses and memory. Proc Natl Acad Sci U S A
2007;104(10):3984-9.
15.
Roederer M. Spectral compensation for flow cytometry: visualization artifacts,
limitations, and caveats. Cytometry 2001;45(3):194-205.
16.
McNab FW, Berzins SP, Pellicci DG, Kyparissoudis K, Field K, Smyth MJ, et
al. The influence of CD1d in postselection NKT cell maturation and homeostasis. J
Immunol 2005;175(6):3762-8.
17.
Thedrez A, de Lalla C, Allain S, Zaccagnino L, Sidobre S, Garavaglia C, et al.
CD4 engagement by CD1d potentiates activation of CD4+ invariant NKT cells. Blood
2007;110(1):251-8.
18.
Kim PJ, Pai SY, Brigl M, Besra GS, Gumperz J, Ho IC. GATA-3 regulates the
development and function of invariant NKT cells. J Immunol 2006;177(10):6650-9.
19.
Seino K, Taniguchi M. Functionally distinct NKT cell subsets and subtypes. J
Exp Med 2005;202(12):1623-6.
11
Carvalho et al.
20.
Kim CH, Kunkel EJ, Boisvert J, Johnston B, Campbell JJ, Genovese MC, et
al. Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with
extralymphoid tissue homing potential. J Clin Invest 2001;107(5):595-601.
21.
Kim CH, Johnston B, Butcher EC. Trafficking machinery of NKT cells:
shared and differential chemokine receptor expression among V alpha 24(+)V beta
11(+) NKT cell subsets with distinct cytokine-producing capacity. Blood
2002;100(1):11-6.
22.
Jiang X, Shimaoka T, Kojo S, Harada M, Watarai H, Wakao H, et al. Cutting
edge: critical role of CXCL16/CXCR6 in NKT cell trafficking in allograft tolerance. J
Immunol 2005;175(4):2051-5.
23.
Smed-Sorensen A, Moll M, Cheng TY, Lore K, Norlin AC, Perbeck L, et al.
IgG regulates the CD1 expression profile and lipid antigen-presenting function in
human dendritic cells via FcgammaRIIa. Blood 2008;111(10):5037-46.
24.
Sandberg JK, Bhardwaj N, Nixon DF. Dominant effector memory
characteristics, capacity for dynamic adaptive expansion, and sex bias in the innate
Valpha24 NKT cell compartment. Eur J Immunol 2003;33(3):588-96.
12
Carvalho et al.
Figures legends
Figure 1: Expression of NKT cells in peripheral blood. (A) Representative flow
cytometric analyses on PBMC CD3+ T cells and Vα24+Vβ11+ for NKT cells in a
health subject (left) and in a CVID patient (right). (B) Log10 expression of NKT cells
in healthy subjects and CVID patients (**p < 0.001). Group comparisons were
carried out using Mann-Whitney non-parametric test.
Figure 2: Representative flow cytometric and percentage of activation,
chemokine receptors in NKT cells.
(A) Density plot showing expression of
CXCR6, CCR5, and CD69 marker from NKT cells gate (Vα24+Vβ11) in healthy
subject and (B) in a CVI patient. (C) Percentage of chemokine receptors CXCR6,
CCR5, and CD69 expression on NKT cells (p<0.01).
Group comparisons were
carried out using Mann-Whitney non-parametric test.
13
Carvalho et al.
Figure 1.A
A
Healthy control
CVID patient
4000
4000
3000
3000
S
S
C
2000
2000
67.2%
77.8%
1000
1000
0
0
102
103
104
0
105
0
102
CD3
104
105
CD3
105
105
104
Vβ
11
103
104
103
0.35%
103
102
0.04%
102
0
0
0
102
103
104
105
0
Vα24
102
103
104
105
Vα24
14
Carvalho et al.
Figure 1 B
B
100
10
1
0.1
0.01
0.001
Control
CVID
15
Carvalho et al.
Figure 2
A
Healthy control
400
0
400
0
400
0
300
0
300
0
300
0
200
0
200
0
200
0
100
0
100
0
100
0
7.1
0
0
1
0
2
1
0
3
1
0
4
1
0
2
0
5
0
1
0
2
1
0
3
1
0
4
1
0
24.
0
5
0
1
0
2
1
0
3
1
0
4
0
1
0
2
1
0
3
1
0
4
1
0
5
CVID patient
400
0
400
0
400
0
300
0
300
0
300
0
200
0
200
0
200
0
100
0
100
0
100
0
3
0
0
1
0
2
1
0
3
1
0
4
1
0
52.
0
5
0
1
0
2
CXCR6
1
0
3
1
0
4
1
0
5
46.
0
CCR5
1
0
CD69
B
100
75
100
75
50
50
50
25
25
0
0
Control
CVID
0
Control
CVID
Control
CVID
16
5
Carvalho KI
Conclusões
Destes trabalhos podemos concluir que:
1. A coinfecção pelo M. leprae e o HIV esta associada no sangue
periférico a:
a. Diminuição da razão de CD4:CD8;
b. Diminuição da frequência de células dendríticas plasmocitóides;
c. Aumento dos níveis de ativação das células T CD8+;
d. Aumento da razão de células T Vδ1:Vδ2;
e. A produção de IL-4 pelos células T CD4+ foi correlacionada
positivamente com a porcentagem de subpopulações de
memória efetora de células T CD4+.
2. Em pacientes infectados pelo HIV foi observado que:
a. A frequência de células NKT secretam baixos níveis de IFN-γ e
TNF-α quando comparadas com indivíduos saudáveis;
b. Ocorreu uma diminuição na magnitude da resposta de citocinas
Th1 pelas células NKT quando correlacionadas com a
expressão de CD161.
3. O estudo de células NKT em pacientes coinfectados pelo M. leprae e o
HIV mostrou que:
a. Estão diminuídas no sangue periférico quando comparadas com
indivíduos saudáveis e monoinfectados pelo M. leprae;
b. Secretam mais IFN-γ quando comparados com
indivíduos
monoinfectados pelo M.leprae.
67
Carvalho KI
Referências Bibliográficas
REFERÊNCIAS BIBLIOGRÁFICAS
Abel, L. and F. Demenais (1988). "Detection of major genes for susceptibility to
leprosy and its subtypes in a Caribbean island: Desirade island." Am J Hum
Genet 42(2): 256-66.
Abel, L., D. L. Vu, et al. (1995). "Complex segregation analysis of leprosy in
southern Vietnam." Genet Epidemiol 12(1): 63-82.
Alter, A., A. Alcais, et al. (2008). "Leprosy as a genetic model for susceptibility to
common infectious diseases." Hum Genet 123(3): 227-35.
Andrade, V. L., J. C. Avelleira, et al. (1991). "Leprosy as cause of false-positive
results in serological assays for the detection of antibodies to HIV-1." Int J
Lepr Other Mycobact Dis 59(1): 125-6.
Apostolou, I., Y. Takahama, et al. (1999). "Murine natural killer T(NKT) cells
[correction of natural killer cells] contribute to the granulomatous reaction
caused by mycobacterial cell walls." Proc Natl Acad Sci U S A 96(9): 5141-6.
Asselin-Paturel, C., A. Boonstra, et al. (2001). "Mouse type I IFN-producing cells are
immature APCs with plasmacytoid morphology." Nat Immunol 2(12): 114450.
Au-Yeung, B. B. and D. J. Fowell (2007). "A key role for Itk in both IFN gamma and
IL-4 production by NKT cells." J Immunol 179(1): 111-9.
Balabanian, K., J. Harriague, et al. (2004). "CXCR4-tropic HIV-1 envelope
glycoprotein functions as a viral chemokine in unstimulated primary CD4+ T
lymphocytes." J Immunol 173(12): 7150-60.
Barnes, P. F., A. B. Bloch, et al. (1991). "Tuberculosis in patients with human
immunodeficiency virus infection." N Engl J Med 324(23): 1644-50.
Barre-Sinoussi, F. (1996). "HIV as the cause of AIDS." Lancet 348(9019): 31-5.
Barre-Sinoussi, F., J. C. Chermann, et al. (1983). "Isolation of a T-lymphotropic
retrovirus from a patient at risk for acquired immune deficiency syndrome
(AIDS)." Science 220(4599): 868-71.
Batista, M., M. SM., et al. (2007). "Leprosy reversal reaction as immune
reconstitution inflammatory syndrome in AIDS patients." Clin Infect Dis in
press.
Bendelac, A. (1995). "Mouse NK1+ T cells." Curr Opin Immunol 7(3): 367-74.
68
Carvalho KI
Referências Bibliográficas
Bendelac, A., O. Lantz, et al. (1995). "CD1 recognition by mouse NK1+ T
lymphocytes." Science 268(5212): 863-5.
Bendelac, A., M. N. Rivera, et al. (1997). "Mouse CD1-specific NK1 T cells:
development, specificity, and function." Annu Rev Immunol 15: 535-62.
Bendelac, A., P. B. Savage, et al. (2007). "The biology of NKT cells." Annu Rev
Immunol 25: 297-336.
Benlagha, K., A. Weiss, et al. (2000). "In vivo identification of glycolipid antigenspecific T cells using fluorescent CD1d tetramers." J Exp Med 191(11): 1895903.
Bloom, B. R. (1986). "Learning from leprosy: a perspective on immunology and the
Third World." J Immunol 137(1): i-x.
Bochud, P. Y., T. R. Hawn, et al. (2003). "Cutting edge: a Toll-like receptor 2
polymorphism that is associated with lepromatous leprosy is unable to mediate
mycobacterial signaling." J Immunol 170(7): 3451-4.
Borgdorff, M. W., J. van den Broek, et al. (1993). "HIV-1 infection as a risk factor for
leprosy; a case-control study in Tanzania." Int J Lepr Other Mycobact Dis
61(4): 556-62.
Britton, W. J. and D. N. Lockwood (2004). "Leprosy." Lancet 363(9416): 1209-19.
Chaudhury, S., S. K. Hazra, et al. (1994). "An eight-year field trial on antileprosy
vaccines among high-risk household contacts in the Calcutta metropolis." Int J
Lepr Other Mycobact Dis 62(3): 389-94.
Chen, X., X. Wang, et al. (2007). "Modulation of CD1d-restricted NKT cell responses
by CD4." J Leukoc Biol 82(6): 1455-65.
Cicala, C., J. Arthos, et al. (2002). "HIV envelope induces a cascade of cell signals in
non-proliferating target cells that favor virus replication." Proc Natl Acad Sci
U S A 99(14): 9380-5.
Cole, S. T., K. Eiglmeier, et al. (2001). "Massive gene decay in the leprosy bacillus."
Nature 409(6823): 1007-11.
Convit, J., C. Sampson, et al. (1992). "Immunoprophylactic trial with combined
Mycobacterium leprae/BCG vaccine against leprosy: preliminary results."
Lancet 339(8791): 446-50.
Couppie, P., S. Abel, et al. (2004). "Immune reconstitution inflammatory syndrome
associated with HIV and leprosy." Arch Dermatol 140(8): 997-1000.
69
Carvalho KI
Referências Bibliográficas
Crowe, N. Y., D. I. Godfrey, et al. (2003). "Natural killer T cells are targets for
human immunodeficiency virus infection." Immunology 108(1): 1-2.
Douek, D. C., L. J. Picker, et al. (2003). "T cell dynamics in HIV-1 infection." Annu
Rev Immunol 21: 265-304.
Eberl, G., R. Lees, et al. (1999). "Tissue-specific segregation of CD1d-dependent and
CD1d-independent NK T cells." J Immunol 162(11): 6410-9.
Eckert, D. M. and P. S. Kim (2001). "Mechanisms of viral membrane fusion and its
inhibition." Annu Rev Biochem 70: 777-810.
Eger, K. A., M. S. Sundrud, et al. (2006). "Human natural killer T cells are
heterogeneous in their capacity to reprogram their effector functions." PLoS
ONE 1: e50.
Emerman, M. and M. H. Malim (1998). "HIV-1 regulatory/accessory genes: keys to
unraveling viral and host cell biology." Science 280(5371): 1880-4.
Emoto, M., Y. Emoto, et al. (1999). "Induction of IFN-gamma-producing CD4+
natural killer T cells by Mycobacterium bovis bacillus Calmette Guerin." Eur J
Immunol 29(2): 650-9.
Exley, M. A., Q. He, et al. (2002). "Cutting edge: Compartmentalization of Th1-like
noninvariant CD1d-reactive T cells in hepatitis C virus-infected liver." J
Immunol 168(4): 1519-23.
Ferreira, F. R., L. R. Goulart, et al. (2004). "Susceptibility to leprosy may be
conditioned by an interaction between the NRAMP1 promoter polymorphisms
and the lepromin response." Int J Lepr Other Mycobact Dis 72(4): 457-67.
Gadue, P. and P. L. Stein (2002). "NK T cell precursors exhibit differential cytokine
regulation and require Itk for efficient maturation." J Immunol 169(5): 2397406.
Gansert, J. L., V. Kiessler, et al. (2003). "Human NKT cells express granulysin and
exhibit antimycobacterial activity." J Immunol 170(6): 3154-61.
Gebre, S., P. Saunderson, et al. (2000). "The effect of HIV status on the clinical
picture of leprosy: a prospective study in Ethiopia." Lepr Rev 71(3): 338-43.
Giorgi, J. V., L. E. Hultin, et al. (1999). "Shorter survival in advanced human
immunodeficiency virus type 1 infection is more closely associated with T
lymphocyte activation than with plasma virus burden or virus chemokine
coreceptor usage." J Infect Dis 179(4): 859-70.
70
Carvalho KI
Referências Bibliográficas
Godfrey, D. I., K. J. Hammond, et al. (2000). "NKT cells: facts, functions and
fallacies." Immunol Today 21(11): 573-83.
Godfrey, D. I. and M. Kronenberg (2004). "Going both ways: immune regulation via
CD1d-dependent NKT cells." J Clin Invest 114(10): 1379-88.
Goebel, F. D. (2005). "Immune reconstitution inflammatory syndrome (IRIS)-another new disease entity following treatment initiation of HIV infection."
Infection 33(1): 43-5.
Gumperz, J. E. and M. B. Brenner (2001). "CD1-specific T cells in microbial
immunity." Curr Opin Immunol 13(4): 471-8.
Gumperz, J. E., S. Miyake, et al. (2002). "Functionally distinct subsets of CD1drestricted natural killer T cells revealed by CD1d tetramer staining." J Exp
Med 195(5): 625-36.
Gupte, M. D., R. S. Vallishayee, et al. (1998). "Comparative leprosy vaccine trial in
south India." Indian J Lepr 70(4): 369-88.
Hammond, K. J., S. B. Pelikan, et al. (1999). "NKT cells are phenotypically and
functionally diverse." Eur J Immunol 29(11): 3768-81.
Hammond, K. J., D. G. Pellicci, et al. (2001). "CD1d-restricted NKT cells: an
interstrain comparison." J Immunol 167(3): 1164-73.
Hansen, D. S., M. A. Siomos, et al. (2003). "Regulation of murine cerebral malaria
pathogenesis by CD1d-restricted NKT cells and the natural killer complex."
Immunity 18(3): 391-402.
Harboe, M. (1988). "Rheumatoid factors in leprosy and parasitic diseases." Scand J
Rheumatol Suppl 75: 309-13.
Hayakawa, Y., D. I. Godfrey, et al. (2004). "Alpha-galactosylceramide: potential
immunomodulatory activity and future application." Curr Med Chem 11(2):
241-52.
Heine, H. and E. Lien (2003). "Toll-like receptors and their function in innate and
adaptive immunity." Int Arch Allergy Immunol 130(3): 180-92.
Hirsch, H. H., G. Kaufmann, et al. (2004). "Immune reconstitution in HIV-infected
patients." Clin Infect Dis 38(8): 1159-66.
Hong, S., D. C. Scherer, et al. (1999). "Lipid antigen presentation in the immune
system: lessons learned from CD1d knockout mice." Immunol Rev 169: 3144.
71
Carvalho KI
Referências Bibliográficas
Ishihara, S., M. Nieda, et al. (1999). "CD8(+)NKR-P1A (+)T cells preferentially
accumulate in human liver." Eur J Immunol 29(8): 2406-13.
Kang, T. J., S. B. Lee, et al. (2002). "A polymorphism in the toll-like receptor 2 is
associated with IL-12 production from monocyte in lepromatous leprosy."
Cytokine 20(2): 56-62.
Kang, T. J., C. E. Yeum, et al. (2004). "Differential production of interleukin-10 and
interleukin-12 in mononuclear cells from leprosy patients with a Toll-like
receptor 2 mutation." Immunology 112(4): 674-80.
Kashala, O., R. Marlink, et al. (1994). "Infection with human immunodeficiency virus
type 1 (HIV-1) and human T cell lymphotropic viruses among leprosy patients
and contacts: correlation between HIV-1 cross-reactivity and antibodies to
lipoarabinomannan." J Infect Dis 169(2): 296-304.
Kawano, T., T. Nakayama, et al. (1999). "Antitumor cytotoxicity mediated by ligandactivated human V alpha24 NKT cells." Cancer Res 59(20): 5102-5.
Kenna, T., L. Golden-Mason, et al. (2003). "NKT cells from normal and tumorbearing human livers are phenotypically and functionally distinct from murine
NKT cells." J Immunol 171(4): 1775-9.
Kharkar, V., U. H. Bhor, et al. (2007). "Type I lepra reaction presenting as immune
reconstitution inflammatory syndrome." Indian J Dermatol Venereol Leprol
73(4): 253-6.
Kirchheimer, W. F. and E. E. Storrs (1971). "Attempts to establish the armadillo
(Dasypus novemcinctus Linn.) as a model for the study of leprosy. I. Report of
lepromatoid leprosy in an experimentally infected armadillo." Int J Lepr Other
Mycobact Dis 39(3): 693-702.
Klatser, P. R., S. van Beers, et al. (1993). "Detection of Mycobacterium leprae nasal
carriers in populations for which leprosy is endemic." J Clin Microbiol 31(11):
2947-51.
Korber, B., B. Gaschen, et al. (2001). "Evolutionary and immunological implications
of contemporary HIV-1 variation." Br Med Bull 58: 19-42.
Kronenberg, M. (2005). "Toward an understanding of NKT cell biology: progress and
paradoxes." Annu Rev Immunol 23: 877-900.
Kronenberg, M. and L. Gapin (2002). "The unconventional lifestyle of NKT cells."
Nat Rev Immunol 2(8): 557-68.
72
Carvalho KI
Referências Bibliográficas
Krutzik, S. R., M. T. Ochoa, et al. (2003). "Activation and regulation of Toll-like
receptors 2 and 1 in human leprosy." Nat Med 9(5): 525-32.
Lockwood, D. N. and S. Suneetha (2005). "Leprosy: too complex a disease for a
simple elimination paradigm." Bull World Health Organ 83(3): 230-5.
Lucas, S. B., P. E. Fine, et al. (1995). "Infection with human immunodeficiency virus
type 1 among leprosy patients in Zaire." J Infect Dis 171(2): 502-4.
Matsuda, J. L., O. V. Naidenko, et al. (2000). "Tracking the response of natural killer
T cells to a glycolipid antigen using CD1d tetramers." J Exp Med 192(5): 74154.
Meeran, K. (1989). "Prevalence of HIV infection among patients with leprosy and
tuberculosis in rural Zambia." Bmj 298(6670): 364-5.
Meima, A., W. C. Smith, et al. (2004). "The future incidence of leprosy: a scenario
analysis." Bull World Health Organ 82(5): 373-80.
Mercer, J. C., M. J. Ragin, et al. (2005). "Natural killer T cells: rapid responders
controlling immunity and disease." Int J Biochem Cell Biol 37(7): 1337-43.
Metelitsa, L. S., O. V. Naidenko, et al. (2001). "Human NKT cells mediate antitumor
cytotoxicity directly by recognizing target cell CD1d with bound ligand or
indirectly by producing IL-2 to activate NK cells." J Immunol 167(6): 311422.
Milanga, M., L. O. Kashala, et al. (1999). "Brief survey of leprosy situation in Congo:
sero-epidemiologic profile in correlation with some emerging viral
infections." Nihon Hansenbyo Gakkai Zasshi 68(2): 109-16.
Mira, M. T., A. Alcais, et al. (2004). "Susceptibility to leprosy is associated with
PARK2 and PACRG." Nature 427(6975): 636-40.
Moraes, M. O., E. N. Sarno, et al. (1999). "Cytokine mRNA expression in leprosy: a
possible role for interferon-gamma and interleukin-12 in reactions (RR and
ENL)." Scand J Immunol 50(5): 541-9.
Moses, A. E., K. A. Adelowo, et al. (2003). "Prevalence of HIV-1 infection among
patients with leprosy and pulmonary tuberculosis in a semi-arid region,
Nigeria." J R Soc Health 123(2): 117-9.
Motsinger, A., D. W. Haas, et al. (2002). "CD1d-restricted human natural killer T
cells are highly susceptible to human immunodeficiency virus 1 infection." J
Exp Med 195(7): 869-79.
73
Carvalho KI
Referências Bibliográficas
Munyao, T. M., J. J. Bwayo, et al. (1994). "Human immunodeficiency virus- 1 in
leprosy patients attending Kenyatta National Hospital, Nairobi." East Afr Med
J 71(8): 490-2.
Murdoch, D. M., W. D. Venter, et al. (2007). "Immune reconstitution inflammatory
syndrome (IRIS): review of common infectious manifestations and treatment
options." AIDS Res Ther 4: 9.
Murray, K. A. (1982). "Syphilis in patients with Hansen's disease." Int J Lepr Other
Mycobact Dis 50(2): 152-8.
Murray, R. A., M. R. Siddiqui, et al. (2007). "Mycobacterium leprae inhibits dendritic
cell activation and maturation." J Immunol 178(1): 338-44.
Nicol, A., M. Nieda, et al. (2000). "Human invariant valpha24+ natural killer T cells
activated by alpha-galactosylceramide (KRN7000) have cytotoxic anti-tumour
activity through mechanisms distinct from T cells and natural killer cells."
Immunology 99(2): 229-34.
Nuti, S., D. Rosa, et al. (1998). "Dynamics of intra-hepatic lymphocytes in chronic
hepatitis C: enrichment for Valpha24+ T cells and rapid elimination of
effector cells by apoptosis." Eur J Immunol 28(11): 3448-55.
Ohteki, T. and H. R. MacDonald (1994). "Major histocompatibility complex class I
related molecules control the development of CD4+8- and CD4-8- subsets of
natural killer 1.1+ T cell receptor-alpha/beta+ cells in the liver of mice." J Exp
Med 180(2): 699-704.
Organization, P. A. H. (2007). Situation Report: Leprosy in the Americas, Pan
American Health Organization.
Organizaton, W. H. (1982). "Chemotherapy of leprosy for control programme, report
of WHO study group." WHO Tech.Rep.Ser. 675: 1-33.
Ortaldo, J. R., H. A. Young, et al. (2004). "Dissociation of NKT stimulation, cytokine
induction, and NK activation in vivo by the use of distinct TCR-binding
ceramides." J Immunol 172(2): 943-53.
Ottenhoff, T. H., P. J. Converse, et al. (1987). "HLA antigens and neural reversal
reactions in Ethiopian borderline tuberculoid leprosy patients." Int J Lepr
Other Mycobact Dis 55(2): 261-6.
Ottenhoff, T. H. and R. R. de Vries (1987). "HLA class II immune response and
suppression genes in leprosy." Int J Lepr Other Mycobact Dis 55(3): 521-34.
74
Carvalho KI
Referências Bibliográficas
Pereira, G. A., M. M. Stefani, et al. (2004). "Human immunodeficiency virus type 1
(HIV-1) and Mycobacterium leprae co-infection: HIV-1 subtypes and clinical,
immunologic, and histopathologic profiles in a Brazilian cohort." Am J Trop
Med Hyg 71(5): 679-84.
Platt, E. J., D. M. Shea, et al. (2005). "Variants of human immunodeficiency virus
type 1 that efficiently use CCR5 lacking the tyrosine-sulfated amino terminus
have adaptive mutations in gp120, including loss of a functional N-glycan." J
Virol 79(7): 4357-68.
Prevention, C. f. D. C. a. (1983). "Immunodeficiency among female sexual partners
of males with acquired immune deficiency syndrome (AIDS) - New York."
MMWR Morb Mortal Wkly Rep 31(52): 697-8.
Prussin, C. and B. Foster (1997). "TCR V alpha 24 and V beta 11 coexpression
defines a human NK1 T cell analog containing a unique Th0 subpopulation." J
Immunol 159(12): 5862-70.
Quiroga, M. F., G. J. Martinez, et al. (2004). "Activation of signaling lymphocytic
activation molecule triggers a signaling cascade that enhances Th1 responses
in human intracellular infection." J Immunol 173(6): 4120-9.
Ramratnam, B., S. Bonhoeffer, et al. (1999). "Rapid production and clearance of
HIV-1 and hepatitis C virus assessed by large volume plasma apheresis."
Lancet 354(9192): 1782-5.
Rauch, J., J. Gumperz, et al. (2003). "Structural features of the acyl chain determine
self-phospholipid antigen recognition by a CD1d-restricted invariant NKT
(iNKT) cell." J Biol Chem 278(48): 47508-15.
Ray, N. and R. W. Doms (2006). "HIV-1 coreceptors and their inhibitors." Curr Top
Microbiol Immunol 303: 97-120.
Ridley, D. S. and W. H. Jopling (1966). "Classification of leprosy according to
immunity. A five-group system." Int J Lepr Other Mycobact Dis 34(3): 25573.
Rolf, J., E. Berntman, et al. (2008). "Molecular profiling reveals distinct functional
attributes of CD1d-restricted natural killer (NK) T cell subsets." Mol Immunol
45(9): 2607-20.
Roy, S., W. McGuire, et al. (1997). "Tumor necrosis factor promoter polymorphism
and susceptibility to lepromatous leprosy." J Infect Dis 176(2): 530-2.
75
Carvalho KI
Referências Bibliográficas
Sampaio, E. P., J. R. Caneshi, et al. (1995). "Cellular immune response to
Mycobacterium leprae infection in human immunodeficiency virus-infected
individuals." Infect Immun 63(5): 1848-54.
Sandberg, J. K., N. M. Fast, et al. (2002). "Selective loss of innate CD4(+) V alpha 24
natural killer T cells in human immunodeficiency virus infection." J Virol
76(15): 7528-34.
Sandberg, J. K., C. A. Stoddart, et al. (2004). "Development of innate CD4+ alphachain variable gene segment 24 (Valpha24) natural killer T cells in the early
human fetal thymus is regulated by IL-7." Proc Natl Acad Sci U S A 101(18):
7058-63.
Saúde, M. d. (2006). Recomendações para Terapia Anti-Retroviral em Adultos e
Adolescentes Infectados pelo HIV, Secretaria de Vigilância em Saúde,
Programa Nacional de DST e Aids: 1 - 85.
Saúde, M. d. (2007). Boletim Epidemiológico AIDS/DST, Ministério da Saúde: 1 48.
Schofield, L., M. J. McConville, et al. (1999). "CD1d-restricted immunoglobulin G
formation to GPI-anchored antigens mediated by NKT cells." Science
283(5399): 225-9.
Scollard, D. M., L. B. Adams, et al. (2006). "The continuing challenges of leprosy."
Clin Microbiol Rev 19(2): 338-81.
Seino, K., S. Motohashi, et al. (2006). "Natural killer T cell-mediated antitumor
immune responses and their clinical applications." Cancer Sci 97(9): 807-12.
Shields, E. D., D. A. Russell, et al. (1987). "Genetic epidemiology of the
susceptibility to leprosy." J Clin Invest 79(4): 1139-43.
ShivRaj, L., S. A. Patil, et al. (1988). "Antibodies to HIV-1 in sera from patients with
mycobacterial infections." Int J Lepr Other Mycobact Dis 56(4): 546-51.
Sidobre, S., O. V. Naidenko, et al. (2002). "The V alpha 14 NKT cell TCR exhibits
high-affinity binding to a glycolipid/CD1d complex." J Immunol 169(3):
1340-8.
Simon, V. and D. D. Ho (2003). "HIV-1 dynamics in vivo: implications for therapy."
Nat Rev Microbiol 1(3): 181-90.
Simon, V., D. D. Ho, et al. (2006). "HIV/AIDS epidemiology, pathogenesis,
prevention, and treatment." Lancet 368(9534): 489-504.
76
Carvalho KI
Referências Bibliográficas
Smith, C. and J. H. Richardus (2008). "Leprosy strategy is about control, not
eradication." Lancet 371(9617): 969-70.
Smyth, M. J. and D. I. Godfrey (2000). "NKT cells and tumor immunity--a doubleedged sword." Nat Immunol 1(6): 459-60.
Sridevi, K., K. Neena, et al. (2004). "Expression of costimulatory molecules (CD80,
CD86, CD28, CD152), accessory molecules (TCR alphabeta, TCR
gammadelta) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy
patients using Mycobacterium leprae antigen (MLCWA) with murabutide and
T cell peptide of Trat protein." Int Immunopharmacol 4(1): 1-14.
Sterne, J. A., A. C. Turner, et al. (1995). "Testing for antibody to human
immunodeficiency virus type 1 in a population in which mycobacterial
diseases are endemic." J Infect Dis 172(2): 543-6.
Stevenson, M. (2003). "HIV-1 pathogenesis." Nat Med 9(7): 853-60.
Taniguchi, M., M. Harada, et al. (2003). "The regulatory role of Valpha14 NKT cells
in innate and acquired immune response." Annu Rev Immunol 21: 483-513.
Thomson, M. M. and R. Najera (2005). "Molecular epidemiology of HIV-1 variants
in the global AIDS pandemic: an update." AIDS Rev 7(4): 210-24.
Tomura, M., W. G. Yu, et al. (1999). "A novel function of Valpha14+CD4+NKT
cells: stimulation of IL-12 production by antigen-presenting cells in the innate
immune system." J Immunol 163(1): 93-101.
Turk, J. L. and R. J. Rees (1988). "AIDS and leprosy." Lepr Rev 59(3): 193-4.
Unutmaz, D. (2003). "NKT cells and HIV infection." Microbes Infect 5(11): 1041-7.
Ustianowski, A. P., S. D. Lawn, et al. (2006). "Interactions between HIV infection
and leprosy: a paradox." Lancet Infect Dis 6(6): 350-60.
van den Broek, J., H. J. Chum, et al. (1997). "Association between leprosy and HIV
infection in Tanzania." Int J Lepr Other Mycobact Dis 65(2): 203-10.
van der Vliet, H. J., B. M. von Blomberg, et al. (2002). "Selective decrease in
circulating V alpha 24+V beta 11+ NKT cells during HIV type 1 infection." J
Immunol 168(3): 1490-5.
Wang, J. H., R. Meijers, et al. (2001). "Crystal structure of the human CD4 Nterminal two-domain fragment complexed to a class II MHC molecule." Proc
Natl Acad Sci U S A 98(19): 10799-804.
Wang, J. H. and E. L. Reinherz (2002). "Structural basis of T cell recognition of
peptides bound to MHC molecules." Mol Immunol 38(14): 1039-49.
77
Carvalho KI
Referências Bibliográficas
Wilson, M. T., C. Johansson, et al. (2003). "The response of natural killer T cells to
glycolipid antigens is characterized by surface receptor down-modulation and
expansion." Proc Natl Acad Sci U S A 100(19): 10913-8.
Wu, D. Y., N. H. Segal, et al. (2003). "Cross-presentation of disialoganglioside GD3
to natural killer T cells." J Exp Med 198(1): 173-81.
78