appendix
summary
SUMÁRIO
SAMENVATTING
LIST OF PUBLICATIONS
ACKNOWLEDGEMENTS
appendix
SUMMARY
Lymphoid tissue: from Ontogeny to Function
Secondary lymphoid organs (SLOs), such as lymph nodes, spleen and Peyer’s patches, play a
critical role in the homeostasis of the immune system. On the one hand, they form nuclei
of immunesurveillance, where danger signals and antigens are scrutinized by the innate and
adaptive immune system, and where immune responses are initiated. On the other hand, they
ensure that unwanted immune responses do not take place as they purge the lymphocyte pool
of autoreactive T cells and increase the threshold for B and T cell activation.
These seemingly antagonistic functions are particularly relevant at the body’s interfaces
with the external world, given the continuous exposure to both harmless as well as pathogenic
microorganisms and xenobiotics requiring disparate immune responses. In the present work,
we have analyzed the development of SLOs at one of such interfaces – the colon. First, we
describe a new methodology based on α-smooth muscle actin (αSMA) staining to discriminate
between colonic patches and colonic solitary intestinal lymphoid tissue (SILTs) and show that
the development of these tissues is independently and differentially regulated. Whereas colonic
patch development during embryogenesis depended on CXCL13-mediated lymphoid tissue
inducer (LTi) cell clustering followed by lymphotoxin α (LTα)-mediated consolidation, post-natal
colonic SILT development seemed, as far as analyzed, chemokine-independent. Differentiation of
gp38+VCAM1+ lymphoid tissue organizer (LTo) cells and dendritic cell accumulation within SILTs
was, however, LTα- dependent; and B cell recruitment and follicular dendritic cell development
dependent on microflora-independent MyD88 signaling. We subsequently extend our findings
by showing that, in contrast to colonic patches, colonic SILT maturation (i.e. B cell recruitment)
was dependent on Caspase1 activity (IL1 processing), IL1R/MyD88 signaling. This molecular axis
was responsible for the accumulation of NKp46+ innate lymphoid cells (ILCs) within the maturing
SILT. The presence of NKp46+ ILCs correlated with B-cell activating factor of tumor necrosis factor
family (BAFF) expression, which may support the accumulation and survival of the SILT-infiltrating
B cells. Focusing on the triggers that drive the early clustering of LTi cells at the colonic SILT
anlagen, we determined that SILT development critically depended on the proper differentiation
of the colonic epithelium. The combined action of repulsive and attractive factors secreted by
differentiated villus intestinal epithelial cells and undifferentiated crypt intestinal epithelial cells,
respectively, directed LTi cell clustering to the bottommost part of colonic intestinal crypts.
In vivo interference with such migratory vector by conditional deletion of differentiated secretory
intestinal epithelial cells or undifferentiated intestinal epithelial stem cells led to the development
of reduced numbers of SILTs.
SLOs are structured by a population of non-hematopoietic stromal cells that was previously
shown to strongly contribute to the function of the immune cells that reside within those SLOs.
In this work, we present additional data implicating these scarce cells in both immune tolerance
maintenance as well as the induction of immune responses. We show, that in addition to
mediating tolerance within the CD8+ T cell repertoire, lymph node stromal cells also tolerize CD4 +
T cells. MHC-II-mediated antigen presentation by lymph node stromal cells was required for the
homeostatic maintenance of regulatory T cells (Tregs), and consequently for the maintenance of
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immune quiescence and the enforcement of immune tolerance. In the selective absence of MHC-II
expression on lymph node stromal cells, which was achieved by means of MHC-II -/- lymph node
transplantation, the CD4 + and CD8+ T cell compartments became activated, ultimately resulting
in transplant rejection. Conversely, Toll-like receptor (TLR) expression on lymph node stromal
cells contributed to enhancement of immune responses. Stimulation of lymph node stromal
cells with TLR ligands increased the expression of chemokines and adhesion molecules, and
modified the patterns of immune cell recirculation leading to lymphocyte accumulation within
the reactive lymph node. As a result, higher numbers of antigen-specific T cells were recruited
into the developing immune responses, increasing its overall magnitude.
In conclusion, this work highlights the diversity of developmental pathways governing the
formation of SLOs and the critical engagement of their stromal cell constituents in shaping
immune function. The data presented opens new perspectives for the therapeutic manipulation
of the immune system by identifying aspects of lymphoid tissue organogenesis that are unique for
the development of some, but not other, SLOs. This may allow the targeting of local pathological
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processes while leaving systemic immunity unaltered. Furthermore, the identification of distinct
imunomodulatory properties of lymph node stromal cells could be targeted to prevent unwanted
immune responses, such as those occurring during auto-immune diseases, or to enhance desirable
immune responses, such as those elicited by vaccination.
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SUMÁRIO
Tecido Linfóide: do Desenvolvimento à Função
Os órgãos linfóides secondários (OLSs), tais como os gânglios linfáticos, o baço ou as placas
de Peyer, desempenham um papel crucial na homeostasia do sistema imunitário. Por um lado,
estes orgãos formam núcleos de vigilância imunitária, onde sinais de perigo e antigénios são
avaliados pelos sistemas imunitários inato e adaptativo e onde respostas imunitárias têm início.
Por outro lado, eles asseguram a não ocorrência de respostas imunitárias indesejadas, uma
vez que eliminam células T auto-reactivas e aumentam o limiar de excitabilidade necessário à
activação de linfócitos B e T.
Estas funções aparatemente antagónicas adquirem particular relevância nas interfaces entre
o nosso meio interno e mundo externo que nos rodeia, devido à presença em simultâneo de
microorganismos (e xenobióticos) inócuos e patogénicos que requerem respostas imunes díspares.
Neste trabalho, analizámos o desevolvimento de OLSs numa dessas interfaces – o cólon. Em
primeiro lugar, descrevemos uma nova metodologia para discriminar colonic patches de colonic
solitary intestinal lymphoid tissues (SILTs) baseada na marcação de α-smooth muscle actin (αSMA).
Com esta técnica, demonstrámos que o desenvolvimento destes tecidos linfóides ocorre de modo
independente e diverso. Enquanto o desenvolvimento embrionário de colonic patches depende
da agregação de células lymphoid tissue inducer (LTi) cells mediada por CXCL13 seguida por um
processo de consolidação mediado por lymphotoxin-α (LTα), o desenvolvimento de colonic SILTs
parece ocorrer independentemente de quimiocinas. Não obstante, a differenciação de células
lymphoid tissue organizer (LTo) gp38 +VCAM1+ e a acumulação de células dendríticas continuam
a depender de LTα; e o recrutamento de linfócitos B e a diferenciação de células foliculares
dendríticas de vias de sinalização dependentes de MyD88 mas independentes da microflora
intestinal. Estes dados foram posteriormente ampliados pela demonstração de que, ao contrário
do que sucede com as colonic patches, a maturação de colonic SILTs (i.e. o recrutamento de
linfócitos B) depende da actividade do enzyma Caspase1 (processamento enzimático de IL1)
e sinais com origem em IL1R/MyD88. Este eixo molecular é responsável pela acumulação de
innate lymphoid cells (ILCs) NKp46+ no colonic SILT. A presença destas células parece levar à
produção local de B-cell activating factor of the tumor necrosis factor family (BAFF), que por
sua vez permite a acumulação e a sobrevivência locais de células B. Focando a nossa atenção
nos mecanismos que levam à agregação inicial de células LTi no primórdio do SILT colónico,
determinámos que para o desenvolvimento destes tecidos a diferenciação adequada do epitélio
intestinal é fundamental. A acção combinada de factores repulsivos e atractivos secretados por
células epiteliais diferenciadas do vilo intestinal e células epiteliais intestinais indiferenciadas da
cripta intestinal, respectivamente, direciona a acumulação de células LTi para o fundo da cripta
intestinal. Interferência com este vector migratório in vivo pela eliminação condicional de células
diferenciadas da linhagem epitelial secretória ou células epiteliais estaminais indiferenciadas
provoca uma redução no número de SILTs colónicos formados.
Os OLSs são estruturados por uma população de células estromais não-hematopoiéticas, a
qual contribui significativamente para a actividade das células imunitárias presentes nesses órgãos.
Neste trabalho, apresentamos dados novos implicando esta pequena população na manutenção
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de tolerância imunitária e na indução de respostas imunitárias. Mostramos, que para além de
mediarem tolerância no seio do repertório de linfócitos T CD8+, células do estroma do gânglio
linfático também impõem tolerância sobre linfócitos T CD4 +. A apresentação de antigénios no
contexto de moléculas MHC-II por células do estroma do gânglio linfático é essencial para a
homeostasia de células T reguladoras (regulatory T cells (Tregs)) e consequentemente para a
manutenção de um estado de quiescência e tolerância imunes. A falta de moléculas MHC-II no
estroma do gânglio linfático, conseguida através do transplante de gânglios linfáticos deficientes
em MHC-II, leva à activacão de linfócitos T CD4 + e CD8+ e à rejeição dos transplantes. Por outro
lado, a expressão de Toll-like receptors (TLRs) nas células do estroma do gânglio linfático contribui
para o desenvolvimento de respostas imunes. A estimulação destas células com ligandos de TLR
aumenta a expressão de quimiocinas e moléculas de adesão, levando a alterações no padrão de
recirculação linfocitária e a uma consequente acumulação de linfócitos no gânglio linfático. Estas
alterações levam a um aumento no recrutamento de células T antigénio-especificas para o gânglio
linfático reactivo, o qual resulta num aumento da magnitude da resposta imune desencadeada.
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Em conclusão, este trabalho valoriza a diversidade de processos que coordena a formação de
SLOs e o papel dos seus componentes estromais na regulação da função imunitária. Os dados
apresentados abrem novas perspectivas para a intervenção terapêutica sobre o sistema imunitário
pela identificação de aspectos da organogénese de tecidos linfóides que afectam de mode
diverso o desenvolvimento de diferentes SLOs. Esta constatação poderá levar ao aparecimento
de intervenções destinadas a combater processos patológicos locais sem interferência sobre o
sistema imunitário sistémico. Além disso, a identificação de propriedades imunomodulatórias
de células do estroma do gânglio linfático distintas poderá ser usada para prevenir respostas
imunitárias indesejadas, tais como as que acontecem no seio de doenças autoimunes, ou para
aumentar respostas imunitárias úteis, tais como as desencadedas por vacinação.
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SAMENVATTING
Lymfoïde weefsel: van ontogenese tot functie
Secundaire lymfoïde organen (SLO) zoals lymfeklieren, de milt en de platen van Peyer, spelen
een belangrijke rol in de homeostase van het immuunsysteem. Deze kernen vormen de
‘bewakingsposten’ van het immuunsysteem waar ‘gevaar’ signalen en antigenen onderzocht
worden en het immuunsysteem aangezet wordt om te reageren.
Daarnaast voorkomen de lymfoïde organen een onnodig respons van het immuunsysteem
die het aantal auto-reactieve T-cellen in de lymfocyten ‘pool’ kan verminderen, en de drempel
kan verhogen voor B -en T-cell activiteit.
Deze schijnbaar antagonistische functies zijn vooral van belang in de interface van het lichaam
en de buitenwereld, aangezien de buitenwereld een overvloed heeft aan zowel goedaardige
als kwaadaardige micro-organismen en xenobiotica. In dit proefschrift hebben we dan ook
de ontwikkeling van de SLO onderzocht in één zo’n interface, namelijk, de dikke darm. Ten
eerste, beschrijven we een nieuwe methode die gebaseerd is op α-smooth muscle actin (αSMA)
aankleuring en discrimineert tussen colonic patches en colonic solitary intestinal lymphoid tissue
(SILT). Daarnaast tonen we aan dat de ontwikkeling van dit weefsel onafhankelijk en differentieel
gereguleerd wordt. Terwijl de ontwikkeling van colonic patches tijdens embryogenese
afhankelijk is van CXCL13-gemedieerde lymphoid tissue inducer (LTi) cel clustering gevolgd
door lymphotoxin α (LTα)-gemedieerde consolidatie, lijkt de post-natale ontwikkeling van de
SILT, tot dusver, chemokine-onafhankelijk. Differentiatie van gp38+VCAM1+lymphoid tissue
organizer (LTo) cellen en de accumulatie van dendritische cellen in de SILT was, hoe dan ook, LTαafhankelijk; B-cel werving en de ontwikkeling van folliculaire dendritische cellen was daarentegen
afhankelijk van microflora-onafhankelijke MyD88 signalering. Wij wijden ons onderzoek verder uit
door aan te tonen dat in tegenstelling tot de colonic patches, de maturatie van SILT (d.i. werving
van B-cellen) berust op Caspase1 activiteit (IL1 verwerking), IL1R/MyD88 signalering. Deze
moleculaire as is verantwoordelijk voor de accumulatie van NKp46+ innate lymphoid cells (ILCs)
binnenin de maturerende SILT. De aanwezigheid van NKp46+ ILCs correleerde aan de expressie
van B-cell activating factor of tumor necrosis factor family (BAFF), wat mogelijk ondersteuning
biedt aan de accumulatie en overleving van SILT-infiltrerende B- cellen. Met als focus de ‘triggers’
die vroegtijdige clustering van LTi cellen in de SILT anlagen, hebben we vastgesteld dat de
ontwikkeling van SILT in de dikke darm sterk afhankelijk is op de volmaakte differentiate van
de dike darm epithelium. De samenwerking van zowel aantrekkende en afstotende factoren
afgescheiden door gedifferentieerde villus intestinale epitheel cellen en ongedifferentieerde crypt
intestinale epitheel cellen, respectievelijk, dirigeerde LTi cell clustering naar de bodem van de dikke
darm crypten. Verstoring van dit trekkende vector in vivo door conditionele deletie van de genen
verbonden aan gedifferentieerde afscheidende intestinale epitheel cellen of ongedifferentieerde
intestinale epitheel stam cellen, zorgden voor een afname van de hoeveelheid ontwikkelde SILT.
SLO worden gestructureerd door een populatie van niet-hematopoïetische stromale cellen
waarvan voorheen aangetoond was dat ze sterk bijdragen aan de functie van immuuncellen die
zich bevinden in de SLO. In dit proefschrift presenteren we bijkomende data dat aangeeft dat deze
schaarse cellen betrokken zijn bij zowel immuuntolerantie als de inductie van immuunrespons. Wij
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laten zien dat lymfeklieren stromale cellen niet alleen tolerantie mediëren binnenin de CD8+T-cel
repertoire, maar ook de CD4 + T-cellen. MHC-II-gemedieerde antigeen presentatie door stromale
cellen van de lymfeklieren was nodig voor het homeostatische behoud van de regulatorische
T-cellen (T-regs), en zodoende voor het behoud van immune quiescence en toepassing van
immuuntolerantie. Bij selectieve afwezigheid van MHC-II expressie op lymfeklieren stromale
cellen, bewerkstelligd door MHC-II -/- lymfeklier transplantatie, werden de CD4 + and CD8+ T-cel
compartimenten geactiveerd, wat uiteindelijk resulteerde in de afstoting van de getransplanteerde
lymfeklieren. Toll-like receptor (TLR) expressie op lymfeklieren stromale cellen, daarentegen,
droeg juist bij aan een verbeterd immuun respons. Stimulatie van de stromale cellen met TLR
liganden verhoogde de expressie van chemokines en adhesie moleculen, en modificeerde de
patronen van immuuncel recirculatie wat leidde tot de accumulatie van lymfocyten binnenin de
reactieve lymfeklier. Als gevolg werd een groter aantal antigeen-specifieke T cellen gerecruteerd
door de ontwikkelende immuunresponsen, wat zorgde voor een algeheel vergroot respons.
Samenvattend belicht het onderzoek, beschreven in dit proefschrift, de diversiteit van de
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ontwikkelings pathways nodig voor de formatie van SLO en de hoognodige betrokkenheid van de
bestanddelen van stromale cellen voor het schapen van immuunfunctie. De gepresenteerde data
biedt nieuwe perspectieven op mogelijke therapeutische manipulatie van het immuunsysteem
door aspecten van lymfeorgaan formatie te identificeren die uniek zijn aan de ontwikkeling
van sommige, maar niet andere, SLO. Hierdoor kan men zich richten op lokale pathologische
processen, terwijl systemische immuniteit ongewijzigd blijft. Bovendien kan de identificatie
van verschillende immunomodulatoire eigenschappen van lymfeklieren stromale cellen leiden
tot gerichte preventie van ongewenste immuunresponsen, zoals die o.a. voorkomen bij
auto-immuunziektes; of de intensivering van gewenste immuunresponsen, die bijvoorbeeld
teweeggebracht worden door vaccinatie.
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List of publications
Cavaleiro R, Tendeiro R, Foxall RB, Soares RS, Baptista AP, Gomes P, Valadas E, Victorino RM,
Sousa AE (2013) Monocyte and myeloid dendritic cell activation occurs throughout the course
of HIV-2 infection, an attenuated form of HIV disease. J Infect Dis 207(11):1730-1742
Baptista AP, Olivier BJ, Goverse G, Greuter M, Knippenberg M, Kusser K, Domingues RG,
Veiga-Fernandes H, Luster AD, Lugering A, Randall TD, Cupedo T, Mebius RE (2013) Colonic
patch and colonic SILT development are independent and differentially regulated events. Mucosal
Immunol 6:511-521
Tendeiro R, Albuquerque AS, Foxall RB, Cavaleiro R, Soares RS, Baptista AP, Soares MV, Gomes P,
Sousa AE (2013) Preserved CD4 T-cell telomere length during long-lasting HIV-2 infection. AIDS
27(2):289-292
Tendeiro R, Fernandes S, Foxall RB, Taveira N, Soares RS, Baptista AP, Cavaleiro R, Gomes P,
Victorino RM, Sousa AE (2012) Memory B-cell depletion is a feature of HIV-2 infection even in
the absence of detectable viremia. AIDS 26(13):1607-1617
Tendeiro R, Foxall RB, Baptista AP, Pinto F, Soares RS, Cavaleiro R, Valadas E, Gomes P, Victorino
RM, Sousa AE (2012) PD-1 and its ligand PD-L1 are progressively up-regulated on CD4 and CD8
T-cells in HIV-2 infection irrespective of the presence of viremia. AIDS 26(9):1065-1071
Foxall RB, Albuquerque AS, Soares RS, Baptista AP, Cavaleiro R, Tendeiro R, Gomes P, Victorino
RM, Sousa AE (2011) Memory and naive-like regulatory CD4+ T cells expand during HIV-2 infection
in direct association with CD4+ T-cell depletion irrespectively of viremia. AIDS 25(16):1961-1970
Soares RS, Tendeiro R, Foxall RB, Baptista AP, Cavaleiro R, Gomes P, Camacho R, Valadas E,
Doroana M, Lucas M, Antunes F, Victorino RM, Sousa AE (2011) Cell-associated viral burden
provides evidence of ongoing viral replication in aviremic HIV-2 infected patients. J Virol
85(5):2429-2438
Cavaleiro R, Baptista AP, Soares RS, Tendeiro R, Foxall RB, Gomes P, Victorino RMM and Sousa
AE (2009) Major depletion of plasmacytoid dendritic cells in HIV-2 infection, an attenuated form
of HIV disease. PLoS Pathog 5 (11), e1000667
Cavaleiro R, Baptista AP, Foxall RB, Victorino RMM and Sousa AE (2009) Dendritic cells
differentiation and maturation in the presence of HIV type 2 envelope. AIDS Res Hum Retroviruses
25(4): 425-231
Pereira-Santos MC, Baptista AP, Melo A, Alves RR, Soares RS, Pedro E, Pereira-Barbosa M,
Victorino RMM and Sousa AE (2008) Expansion of circulating Foxp3+CD25bright CD4+ T cells
during specific venom immunotherapy. Clin Exp Allergy 38:291-297
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appendix
Acknowledgements
I would like to express my gratitude to those who have contributed to this thesis. To those
who have directly worked with me and to those who have shared their wisdom, enthusiasm,
and friendship.
To my promoter Prof. Reina E. Mebius. Reina, it has been a pleasure working with you, discussing
and sketching our ideas. I will make great use of everything you taught me during the years we
spent together.
To Prof. Georg Kraal. I enjoyed your stories and learned from you to look at science with
different eyes.
To the past and present members of group Yellow. Thank you for sharing ideas. To Brenda, in
particular, for inadvertently introducing me to the world of mucosal immunology, which ended
up becoming a substantial part of this thesis.
To the MCBI. Thank you for your support over the years. To my office friends, for the coffee
breaks and for trying to introduce me to the Dutch lifestyle. To the expat community, for keeping
me safe in the Mediterranean environment.
To Jamie and Marieke, for making my life easier and for translating my mail.
To the Juans and their families, Petros, the Portuguese clan Ana, Cláudia and Inês. Thank you
for being so good to me.
To my paranymphs Inês and Melissa. Thanks for your friendship and for going through all this
with me.
To the GABBA PhD program. Thank you for giving me the opportunity to perform my PhD in the
lab that I reckoned ideal for my career. To Prof. Maria de Sousa, for always questioning my results.
Last but not least, a big thanks to my family. To my parents, my sister, and my uncles for being
always present despite the geographical distance and for recharging my batteries every time I
go home. To Ale, for your love.
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