Immune responses to antigens presented at skin, or other epithelial surfaces such as the cervix, are important for the clearance of viral infections, such as human papillomavirus (HPV) that infect epithelial cells [13]. Elucidation of the components of an effective immune response to antigens presented in this manner will potentially aid in design of immune modulatory techniques or therapeutic vaccine strategies to treat conditions such as cervical cancer. This thesis addresses the role of CD4+ T lymphocytes in immune responses to antigens presented in skin. CD4+ T cells have a well established role in the priming of CD8+ T cells, such that priming without help results in defective CD8+ T cell memory response [15]. The role of CD4+ T cells in the immune response subsequent to priming is less well delineated [15, 16]. Murine skin grafting is a model of antigen presented at an epithelial surface. The model used in this thesis utilises grafts transgenically expressing neo-antigens (human growth hormone=hGH, ovalbumin=OVA) under the control of a keratin promoter (K14 or K5) in the graft. The corresponding mice are termed K14hGH and K5mOVA. With hGH as the antigen, rejection of such skin grafts were shown to require CD4+ T cells [1]. The most surprising finding was that this requirement for CD4+ T cells was maintained even in an antigen-experienced host (in the recall immune response to hGH). CD4+ T cells are required by graft-primed recipients to reject hGH-expressing grafts, but are not required to reject grafts expressing alternative antigens such as OVA. In an adoptive transfer model into lymphopaenic hosts, when high numbers of CD8+ T cells were transferred, any addition of CD4+ T cells was superfluous. However, with low numbers of OVA-specific CD8+ T cells, the addition of CD4+ T cells resulted in a significantly faster rate of K5mOVA skin graft rejection. This helper enhancement of K5mOVA skin graft rejection is maintained, even 7 when CD8+ T cells were previously activated to a memory phenotype prior to transfer, indicating that CD4+ T cells do have effects after CTL priming in vivo. The requirement for CD4+ T cells in the rejection of C57.K14hGH grafts is abrogated by the addition of a local inflammatory stimulus (TLR7 agonist, imiquimod). This is a local rather than systemic effect, suggesting an influence on trafficking or local effector function. Administration of agonist anti-CD40 antibody also partially abrogates the need for CD4+ T cells in rejection of C57.K14hGH grafts by primed hosts. Although CD40 has a well established role in priming of naïve CTL responses, our findings indicate that CD40 can alter events after priming, and suggests a possible mechanism for the role of CD4+ T cells in this system. With these data, we speculate that CD4+ T cells may provide help by altering the state of APC cross-presenting antigen to experienced CD8+ T cells, and that this can be substituted by TLR or CD40 mediated activation of APC. The result may be an increased number of effector CD8+ T cells, as we demonstrate that high numbers of antigen-specific CD8+ T cells can abrogate this effect.

O vírus linfotrópico de células T humanas (HTLV-1) foi o primeiro retrovírus descrito e está etiologicamente ligado a duas principais doenças: a leucemia/linfoma de célula T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). Apenas 0,3 a 5% dos indivíduos infectados desenvolvem essas doenças associadas, enquanto a maioria permanece assintomática. A HAM/TSP é uma manifestação inflamatória do sistema nervoso central e o mecanismo pelo qual o HTLV-1 induz o surgimento de HAM/TSP ainda não está totalmente esclarecido. Atualmente, uma abordagem promissora no entendimento de mecanismos, bem como na fisiopatogênese das infecções virais tem sido a avaliação da função de microRNAs (miRNAs). Há poucos dados na literatura envolvendo estas moléculas na infecção pelo HTLV-1 em linfócitos T CD4+ bem como no estabelecimento da doença HAM/TSP. No presente estudo, foi avaliada a expressão de miRNAs dos linfócitos T CD4+ isolados de portadores sem HAM/TSP (HAC), pacientes HAM/TSP e indivíduos sadios (CT) por meio de PCR em tempo real. A análise do perfil de expressão dos miRNAs nessas células revelou que 56 e 10 miRNAs apresentavamse mais 1,5 vezes aumentados no grupo HAM/TSP e HAC, respectivamente. O miR- 125b-1-1 apresentou expressão significamente maior no grupo HAC e o miR-146a, no grupo HAM/TSP. A análise in silico de predição de alvo demonstrou que o gene IFNG era potencialmente alvo do miR-125b-1-1 e os genes IRAK1 e TRAF6 do miR- 146a. Foi demonstrado que a expressão do IFNG no grupo HAC era 1,3 vezes mais elevado que o grupo CT e 1,8 vezes mais elevado no grupo HAM que no grupo CT. Houve um aumento na expressão de TRAF6 de 15,7 e 1,5 vezes nos grupos HAM/TSP e HAC, respectivamente. Não foi observada diferença na expressão de IRAK1 entre os três grupos. O ensaios de superexpressão do miR-125b-1-1 alterou a expressão do IFNG e do miR-146a alterou a expressão do gene IRAK1 e sua proteína. Os resultados evidenciados neste trabalho ressaltam a importância dos miRNAs na modulação de genes e proteínas importantes durante a infeção pelo HTLV-1. A correlação entre o miR-125b-1-1 e gene IFNG sugere que este miRNA esteja envolvido nos mecanismos de desenvolvimento de HAM/TSP. Além disso, a interação entre o miR-146a e os genes IRAK1 e TRAF6 sugerem que este miRNA esteja relacionado a mecanismos de persistência viral da infecção pelo HTLV-1 em linfócitos T CD4+. / Human T-cell lymphotropic vírus type 1 (HTLV-1) was the first human retrovirus discovered and it is related with two major diseases: adult T cell lymphoma/leukaemia (ATLL) and HTLV-1 -associated myelopathy/tropical spastic paraparesis (HAM/TS). About 0.3 to 5% of infected individuals will develop HTLV-1 related diseases, while the majority will remain life-long asymptomatic carriers of the virus. HAM/TSP is an inflammatory manifestation of central nervous system and the mechanism involved in HAM/TSP development is noy well elucidated. Currently, a promising approach on understanding the mechanisms as well as physiopathogenesis of viral infections has been the evaluation of the role of microRNAs (miRNAs) roles. There are few data involving CD4+ T cells miRNA expression in HTLV-1 infection as well as HAM/TSP establishment. To identify miRNAs differentially expressed in CD4+ T cells among non-infected individuals (CT), asymptomatic (HAC) and HAM/TSP patients we applied quantitative real time PCR. The analysis of miRNA expression profile in these cells showed 56 and 10 miRNAs upregulated 1.5 times in HAM/TSP and HAC groups, respectively. miR- 125b-1-1 was upregulated in HAC group and miR-146a in HAM/TSP. In silico analysis of target prediction showed that IFNG was a potentially miR-125b-1-1 target and IRAK1 and TRAF6 were miR-146a targets. IFNG expression was 1.3 higher in HAC than CT group and 1.8 higher in HAM/TSP than CT group. It was observed that TRAF6 expression was 15.7 and 1.5 times higher in HAM/TSP and HAC groups, respectively. There was no difference of IRAK1 expression among the three groups. Overexpression assays of miR-125b-1-1 altered IFNG expression and overexpression of miR-146a altered IRAK1 gene and protein expression. The results revealed that miRNAs modulate genes and proteins during HTLV-1 infection. miR- 125b-1-1 and IFNG gene correlation suggests that miR-125b-1-1 seems to contribute to HAM/TSP development. Besides, miR-146a and IRAK1 and TRAF6 interaction suggests that miT-146a seems to contribute to HTLV-1 establishment in CD4+ T cells.

Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized. Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining. Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints. Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.

Interleukin-7 is an essential cytokine that plays a major role in the development and homeostatic maintenance of T-cells. The presence of soluble forms of various cytokine receptors have been proposed to be involved in the endogenous regulation of cytokine activity. Due to the natural ability of soluble CD127 (sCD127) to bind to IL-7, there is an interest in its potential application as an immunotherapeutic agent in diseases, where IL-7 has been found to be relevant, including HIV infection. In this study, I hypothesize that by administering sCD127 to healthy mice, IL-7 activity should be enhanced, thus enhancing T cell proliferation in vivo. The work presented here focuses on three main objectives: 1) evaluating the effect of IL-7 with or without sCD127 on T cell proliferation in healthy mice; 2) validating a mouse model of T cell depletion using anti-CD4 and CD8 antibodies; and 3) determining the effect of sCD127 treatment with or without IL-7 on T cell reconstitution and proliferation in the T cell depletion model. To assess the effect of administering exogenous sCD127, IL-7 or the combination on T cell proliferation, peripheral blood mononuclear cells and spleen were isolated, and stained to characterize T cell number, proliferation, and surface CD127 expression by flow cytometry. For the T cell depletion model, wild type C57BL/6 mice were injected intra-peritoneally with 150 μg single dose of anti-CD4 and anti-CD8 depleting antibodies. Consequently, mice were bled weekly to demonstrate the kinetics of T cell reconstitution following depletion (from d7 to d63). Our results demonstrated that in healthy mice daily treatment with murine IL-7 significantly stimulated T cell proliferation and consequently increased cell number. This observation was further boosted by pre-complexing IL-7 with sCD127. For T cell depletion experiments, the kinetics of T-cell reconstitution was different between the CD4+ and CD8+ T cells. CD4+ T cell reconstitution was almost complete 6 weeks following T cell depletion, while CD8+ T cells were only partially reconstituted at this time point. Treatment with IL-7 or combined therapy had a transient and significant effect on T cell proliferation and reconstitution, and this influence was abrogated after treatment discontinuation. Interestingly, CD8+ T cells exert greater responses to our treatments in that a more pronounced proliferation and significant increase in cell number was observed relative to the effect seen on CD4+ T cells in both healthy and depleted mice. In conclusion, antibody-mediated T cell depletion is a potentially valuable tool to investigate lymphopenia-induced proliferation and potential therapies thereof. This study suggests that combining sCD127 and IL-7 therapies enhances IL-7-mediated T cell proliferation, and provides important information for the potential therapeutic use of sCD127 and its impact on IL-7 function.

Final publication is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38173/abstract;jsessionid=DA29F0C067C89EC1147E79EE7380D21A.f01t04?systemMessage=Wiley+Online+Library+will+be+disrupted+on+24th+October+2015+at+10%3A00-10%3A30+BST+%2F+05%3A00-05%3A30+EDT+%2F+17%3A00-17%3A30++SGT++for+essential+maintenance.++Apologies+for+the+inconvenience / 京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第13003号 / 論医科博第3号 / 新制||医科||5(附属図書館) / 32931 / (主査)教授 杉田 昌彦, 教授 生田 宏一, 教授 三森 経世 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20084号 / 医博第4177号 / 新制||医||1018(附属図書館) / 33200 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 山田 亮, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Au cours d’une réponse immunitaire primaire, les lymphocytes T CD8 mémoires émergent à partir d'un environnement de forte activation immunitaire. Les cellules régulatrices T CD4 FoxP3+ (LTregs) jouent un rôle clé de suppression de la réponse immunitaire. Nous montrons que les LTregs sont nécessaires pour la génération d’une réponse mémoire T CD8 fonctionnelle. En absence de LTregs lors du priming, les LT CD8 mémoires générées prolifèrent faiblement et ne parviennent pas à se différencier après une réactivation antigénique en effecteurs cytotoxiques secondaires fonctionnelles. Nous suggérons que les LTregs agissent tôt, lors de la phase d'expansion des LT CD8, en réduisant l’exposition des précurseurs mémoires T CD8 à l'interleukine-2. Ce nouveau rôle crucial des LTregs a des implications pour le développement optimal de vaccin.Chez les patients sous traitement antirétroviral efficace et prolongée (ART), le VIH peut persister dans un petit pool de cellules T CD4 mémoires quiescentes de longue durée de vie infectées par du virus latent intégré. Ce réservoir latent comprend différentes sous-populations mémoires. Nos résultats suggèrent une contraction progressive de la taille du réservoir latent autour d'un noyau formé de sous-populations T CD4 mémoires moins différenciées (centrales mémoires TCM et souches mémoires TSCM). Ce processus très lent semble dépendre de la taille initiale et du taux de décroissance qui diffère entre les sous-populations mémoires infectées de manière latente. Nos résultats suggèrent également une extrême stabilité du sous-réservoir TSCM, dont la taille est directement liée à l'exposition cumulée au virus plasmatique avant le début du traitement ART, soulignant l'importance d'une initiation précoce du traitement antirétroviral efficace. La présence de cette dynamique intrinsèque dans le réservoir latent peut avoir des implications pour la conception de stratégies optimales de purge thérapeutique contre le VIH. / During the primary immune response, CD8 memory emerges from an environment of strong immune activation. The FoxP3 regulatory CD4 T-cell subset (Treg) is known as a key suppressive component of the immune system. We report that Tregs are required for the generation of functional CD8 memory. In the absence of Tregs during priming, the resulting memory cells proliferate poorly and fail to differentiate into functional cytotoxic secondary effectors following antigen reactivation. We find that the Tregs act early, during the expansion phase of primary CD8 effectors, by fine tuning interleukin-2 exposure of CD8 memory precursors. This crucial new role of Tregs has implications for optimal vaccine development. In patients who are receiving prolonged antiretroviral treatment (ART), HIV can persist within a small pool of long-lived resting memory CD4 T cells infected with integrated latent virus. This latent reservoir involves distinct memory subsets. We provide results that suggest a progressive reduction of the size of the blood latent reservoir around a core of less-differentiated memory subsets (central memory and stem cell-like memory).This process appears to be driven by the differences in initial sizes and decay rates between latently infected memory subsets. Our results also suggest an extreme stability of the TSCM sub-reservoir, the size of which is directly related to cumulative plasma virus exposure before the onset of ART, stressing the importance of early initiation of effective ART. The presence of these intrinsic dynamics within the latent reservoir may have implications for the design of optimal HIV therapeutic purging strategies.

IL-23 is an essential mediator of chronic intestinal inflammation in experimental models of colitis. Polymorphisms in the IL23R locus are associated with IBD susceptibility in humans. The biological activity of IL-23 has been linked to Th17 cells but little is known about the cellular and molecular mechanism by which IL-23 drives intestinal inflammation. The work presented herein has identified that direct IL-23 signalling into CD4+ T cells was not only required for the accumulation of Th17 cells in the intestine but also modulated their phenotype. Through direct cell intrinsic effects on T cells, IL-23 drove the emergence of an IL-17A+IFN-γ+ population of T cells that co-expressed RORγ and T-bet. Interestingly, we found that expression of RORγ but not T-bet by T cells was required for the development of intestinal inflammation. Furthermore, colitis induced by T-bet deficient T cells was dependent on IL-17A, and showed a unique inflammatory phenotype, thus demonstrating that pathogenic intestinal Th17 responses can develop independently of T-bet. In addition, using transcriptional profiling we identified a core set of genes that is regulated by direct cell-intrinsic IL-23 signals into intestinal CD4+ T cells. This revealed a previously unrecognised role for IL-23 in suppressing Th2 associated genes, such as GATA3 and IL-33R. Functional experiments demonstrated that expression of GATA3 in CD4+ T cells limited their colitogenic potential, suggesting that IL-23-mediated inhibition of GATA3 might contribute to the development of intestinal inflammation. Finally, we described a novel function for IL-33 as a factor that promotes Foxp3+ iTreg differentiation in vitro and in vivo through direct effects on T cells. This activity of IL-33 was inhibited in the presence of IL-23, providing a mechanistic link for the known role of IL-23 in restraining iTreg generation. Collectively, these data suggest that IL-23 promotes acquisition of a pathogenic effector T cell phenotype through multiple mechanisms. This indicates that therapeutic blockade of IL-23 is likely to reduce pro-inflammatory mediators while also facilitating the expansion of regulatory pathways that might help to re-establish intestinal homeostasis.