Numerous studies have demonstrated that tumour cells have the ability to alter immune function to create an immune suppressed environment. This allows tumour cells to escape immune surveillance and consequently the tumour can progress. Dendritic and T cells have critical roles in immune activation and tolerance and are thus major targets of tumour-mediated immune suppression. Understanding the mechanism(s) by which tumour cells modulate the immune system will facilitate the development of immune system-based therapies for cancer treatments. In this study we sought to determine the nature of, and cellular and molecular mechanisms underlying, changes in immune status during tumour progression using mouse models of prostate cancer. Detailed analysis of the immunological status in a mouse prostate dysplasia model (12T-7slow) revealed that immune suppression accompanied tumour progression. We found that T cells isolated from tumour-bearing hosts were hypo-responsive to antigen stimulation. Furthermore, we demonstrated that CD4+CD25+ regulatory T cells were responsible, at least in part, for this alteration. Anti-CD25 antibody treatment reduced, but did not prevent, tumour growth in either a transplanted prostate tumour model or a spontaneously developing prostate tumour model. In addition, an altered dendritic cell phenotype and an elevated frequency of CD4+CD25+ regulatory T cells were observed within the tumour mass. Similar alterations were observed in the prostate-specific Pten knockout mice which develop advanced prostate adenocarcinoma. Interestingly, evidence of immune activation, such as an increased frequency of activated T cells, was detected in the tumour microenvironment in both mouse prostate tumour models. To identify factors that may play critical roles in the altered immune cell phenotype observed in the tumour microenvironment, a global gene expression profiling analysis was carried out to evaluate the changes in immune-related gene expression patterns. This analysis provided additional evidence for the co-existence of immune suppression and immune activation. Moreover, subsequent analyses suggested that one differentially expressed transcript, interferon regulatory factor 7, and its target genes might be involved in modulating immune cells and/or tumour progression. Taken together, these studies have important implications for designing specific and effective anti-tumour immune therapy strategies that involve manipulation of tumour cells, dendritic cells and regulatory T cells. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Solid organ transplantation is now an established and effective treatment option for end-stage organ failure. Whilst early outcomes have improved significantly over recent decades, longer-term outcomes have changed little. Despite advances in immunosuppression, most transplanted organs suffer an inevitable decline in function attributed to chronic rejection. It is evident that the alloimmune response remains incompletely characterised. Crucially, despite description several decades ago, the precise contribution that the direct (recognition of intact allogeneic MHC) and indirect (recognition of self-MHC restricted allopeptide) pathways make to allograft rejection remains incompletely understood. In this thesis, murine models of heterotopic cardiac transplantation have been utilised to analyse these pathways. The key findings of this work are as follows: 1) If able to evade NK cell killing, passenger donor CD4 T cells can make cognate, direct-pathway, interactions with recipient B cells. This interaction results in augmentation of all arms of the alloimmune response and acceleration of allograft rejection. 2) Direct-pathway CD4 T cell allorecognition is restricted to the immediate post transplantation period. Donor APCs are the major source of MHC class II for direct-pathway priming, and these are cleared rapidly by both innate and adaptive responses of the recipient, effectively limiting the longevity of direct allorecognition. 3) The duration of indirect-pathway responses against different alloantigens is variable, limited by availability of donor antigen. Expression of donor MHC class II is restricted to APCs and possibly endothelium (where expression is transient) limiting the duration of indirect-pathway allorecognition against MHC class II alloantigen. Indirect-pathway CD4 T cell responses targeted against parenchymal alloantigen are long-lived, and can provide help for generating alloantibody against different MHC alloantigens. 4) In response to continual presentation of target epitope indirect-pathway CD4 T cell responses against parenchymal expressed alloantigen are long-lived. The continual division of these cells results in greatly increased numbers of alloantigen-specific CD4 T cells in the chronic phase of the response, but despite this, memory responses are impaired. 5) Generating indirect-pathway regulatory T cells specific for parenchymal expressed alloantigen appears to be the most effective strategy to ameliorating chronic rejection.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

HIV Tat protein downregulates surface expression of the interleukin-7 receptor alpha-chain (CD127) on CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat binds directly to the cytoplasmic tail of CD127 inducing receptor internalization and degradation. Given the important roles of CD127 in proper immune function, the Tat/CD127 interactions were characterized and the mechanisms required to induce receptor loss from the surface of CD8 T cells were investigated. Tat deletion mutants were generated each sequentially lacking a region of the protein. CD8 T cells isolated from HIV negative volunteers were exposed to exogenous or intracellular Tat proteins before surface CD127 expression was analyzed by flow cytometry. To characterize Tat/CD127 physical interactions, wild type Tat and Tat mutants were incubated with lysates from a CD127+ Jurkat cell line followed by CD127/Tat co-immunoprecipitation. The effect of Tat on CD127 post-translational modifications was also investigated. Removal of the N-terminus of Tat (aa 1-10 or aa 17-21) prevented Tat from downregulating CD127 and prevented Tat from binding CD127 as assessed by co-immunoprecipitation. Deletion of the basic region (aa 48-59) also prevented Tat from downregulating CD127 but did not prevent Tat from interacting physically as demonstrated by co-immunoprecipitation. Strikingly, endogenously expressed Basic Tat acted as a dominant negative mutant, causing an accumulation of CD127 at the cell surface. These observations suggest that Tat may bind CD127 via its N-terminus to disrupt the normal recycling of the receptor, and then recruit cellular endocytic machinery to the receptor via it’s basic region, to remove the receptor from the cell surface and target it for degradation. Furthermore, Tat encourages the ubiquitination of CD127 by recruiting the cytokine-inducible SH2 containing (CIS) protein to the receptor, possibly leading to accelerated CD127 internalization and proteasomal degradation. I propose a model whereby Tat binds CD127 via its N-terminal region then recruits CIS via its basic region. CIS in turn recruits a cellular E3 ubiquitin ligase to ubiquitin tag the receptor for internalization and proteasome degradation. This research may lead to novel treatments designed to maintain IL-7 signalling and strengthen CD8 T cell function in HIV+ persons.

Embryonic stem cells (ESCs) possess certain immunomodulatory properties; the defined components in ESCs, however, are largely unknown. Based on proteomic database, I report here that milk fat globule epidermal growth factor 8 (MFG-E8) is a key component in ESCs to suppress T cell activation and regulate T cell polarization. MFG-E8 is enriched in undifferentiated ESCs while diminishing in differentiated ESCs. Neutralizing ESC-derived MFG-E8 substantially ameliorates the suppressive effects of ESCs on T cell activation and proliferation. Additionally, MFG-E8 in ESCs is capable of up-regulating T regulatory cells. I further prove that MFG-E8 suppresses T cell activation and regulates T cell polarization through inhibiting PKCθ phosphorylation. In vivo teratoma formation assay reveals an increase in ESC engraftments across allogeneic barriers with less immunologic rejection by up-regulation of MFG-E8 expression in ESCs, further validating the immunosuppressive properties of MFG-E8. Identifying an important immunoregulatory component in ESCs will greatly facilitate stem cell-based therapies.

La tolérance des lymphocytes TCD8+ est essentielle pour empêcher l'apparition d'auto-immunité mais représente un obstacle pour le développement de réponses cytotoxiques contre les tumeurs. La lymphodéplétion est utilisée comme adjuvant pour l'immunothérapie par transfert adoptif de cellules T cytotoxiques car elle améliore leur efficacité en favorisant la rupture de la tolérance périphérique. En condition lymphopénique aiguë, les lymphocytes T naïfs prolifèrent en absence apparente de stimulation antigénique et vont acquérir un phénotype et une fonctionnalité semblable aux cellules mémoires (LIP pour Lymphopenia Induced Proliferation). Les cellules mémoires ayant un seuil d'activation inférieur cellules naïves, il a été proposé que la différentiation des cellules TCD8+ potentiellement autoréactives en cellules de type mémoire dans des conditions lymphopéniques pouvait conduire à la rupture de la tolérance. Pendant ma thèse, j'ai étudié si la LIP est nécessaire pour la rupture de la tolérance croisée des cellules TCD8+ chez des souris transgéniques irradiées exprimant un antigène modèle dans les cellules β du pancréas. De manière surprenante, nous avons constaté que le blocage de la LIP ne permet pas d'inhiber l'apparition d'auto-réactivité : les lymphocytes TCD8+ qui ne transitent pas par un stade de différenciation de type mémoire parviennent à se différencier en cellules effectrices suite à la présentation croisée de l'antigène et à migrer vers le pancréas. Néanmoins, la LIP est requise pour induire de l'auto-réactivité lorsque la fréquence de lymphocytes T CD8+ est faible ; non pas à cause du déséquilibre de la population de cellules T régulatrices, mais dû à une nette augmentation du nombre de cellules TCD8+ autoréactives. Ainsi, bien que la LIP améliore les réponses auto- réactives des cellules TCD8+, la différentiation en cellules de type mémoire n'est pas indispensable pour la rupture de la tolérance croisée en condition lymphopénique induite par irradiation. / The immune system has evolved multiple mechanisms of peripheral tolerance to control CD8+ T cell responses and to prevent autoimmunity. However, they also represent a barrier for the development of cytotoxic responses against tumors. Lymphodepleting protocols are currently used as adjuvants for adoptive cytotoxic T cell immunotherapy because they enhance their potency. These protocols are thought to promote the breakdown of peripheral CD8+ T cell tolerance. Under acute lymphopenic conditions, naive T cells proliferate, in the apparent absence of antigenic stimulation at least in part due to a greater availability of the cytokine IL-7. Proliferating CD8+ T cells acquire a phenotype and functionality that is similar to memory cells and are termed memory-like cells. Since memory cells have a lower activation threshold than naïve cells, it has been proposed that differentiation of potentially autoreactive CD8+ T cells into memory-like cells under lymphopenic conditions could drive the breakdown of peripheral tolerance. Here we studied whether lymphopenia induced proliferation and differentiation are required for the breakdown of CD8+ T cell cross-tolerance in irradiated transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that blocking lymphopenia-induced proliferation and differentiation into memory-like cells did not prevent self-reactivity. CD8+ T cells that did not differentiate into memory-like cells still became effectors upon antigen cross-presentation and migrated to the site of antigen expression. Nonetheless, LIP did enhance CD8+ T cell mediated self-reactivity at low T cell frequencies. This effect could not be explained by a Treg imbalance but by a net increase in autoreactive CD8+ T cell numbers. Thus, although LIP enhances CD8+ T cell anti-self responses, differentiation into memory-like cells is not essential for the breakdown of cross-tolerance under the lymphopenic conditions provided by irradiation.

As células T regulatórias (Tregs) são essenciais para a manutenção da tolerância periférica, prevenção de doenças autoimunes e limitantes nas doenças inflamatórias crônicas. Além disso, essas células exercem um papel fundamental no controle da rejeição de transplantes. Diferentes protocolos mostraram que é possível obter Tregs a partir de células T naive CD4+ in vitro. Para tal, é consenso que o TGF-? e a interleucina-2 (IL-2) são capazes de direcionar as células T naive CD4+ a se tornarem regulatórias após um estímulo antigênico (anti-CD3/CD28). Nosso grupo recentemente notou que, durante a imunomodulação de linfócitos T pelas células estromais mesenquimais (CTMs), estas eram capazes de produzir adenosina que, por sua vez, participa do processo de imunorregulação. Outros trabalhos indicam que as CTMs suprimem a proliferação dos linfócitos T pela geração de Tregs e que as CTMs induzem a geração de Tregs através da regulação negativa da via TCR e da via AKTmTOR. Evidências apontam que a adenosina pode atuar regulando negativamente a via mTOR. Portanto, acredita-se que a adenosina possa participar do processo de geração de Tregs através da modulação da via mTOR. Além disso, estudos recentes indicam que a ativação de receptores de adenosina, mais especificamente A2a, com agentes agonistas, leva ao aumento da produção de células Tregs, enquanto que a utilização de agentes antagonistas destes receptores leva à diminuição da diferenciação de Tregs. Porém, estes estudos mostram a geração de Tregs a partir de células T naive de camundongos. Visto a grande importância das Tregs no contexto imunológico, a produção eficiente de Tregs in vitro tem importância fundamental para o desenvolvimento de novos protocolos terapêuticos para o tratamento de doenças autoimunes e no combate à rejeição de transplantes. Assim, o objetivo central deste trabalho foi avaliar a participação de agonistas e antagonistas de receptores de adenosina na indução de células T regulatórias geradas in vitro (iTreg) pela ativação de células T CD4+ naive isoladas de sangue de cordão umbilical (SCU) humano. Para isso, células mononucleares foram isoladas de bolsas de SCU e as células T naive foram isoladas imunomagnéticamente. Essas células foram ativadas com beads ligadas a anticorpos anti-CD2/CD3/CD28 e cultivadas por cinco dias na presença de IL-2 e diferentes concentrações de drogas agonistas e antagonistas de receptores de adenosina. Em seguida, foram avaliados os principais marcadores de células T regulatorias por meio de citometria de fluxo e o meio de cultura foi coletado ao final da geração para quantificação de citocinas. Além disso, o RNA total foi extraído de todas as condições de cultivo para a análise da expressão de genes envolvidos na geração e desenvolvimento das Tregs, por PCR quantitativo. O potencial de supressão de células T efetoras também foi avaliado. / Regulatory T cells (Tregs) are essential for the maintenance of peripheral tolerance, prevention of autoimmune and limiting diseases in chronic inflammatory diseases. In addition, these cells play a key role in the control of transplant rejection. Different protocols have shown that it is possible to obtain Tregs from naive CD4+ T cells in vitro. To this end, there is consensus that TGF-? and interleukin-2 (IL-2) are capable of directing the naive CD4 + T cells to become regulatory following an antigenic stimulus (anti-CD3/CD28).. Our group recently noted that during the immunomodulation of T lymphocytes by mesenchymal stromal cells (MSCs), they were able to produce adenosine which in turn participates in the immunoregulation process. Other studies indicate that MSCs suppress the proliferation of T lymphocytes by generation of Tregs and that MSCs induce generation of Tregs by downregulation of the TCR pathway and the AKT-mTOR pathway. Evidence indicates that adenosine may act by downregulating the mTOR pathway. Therefore, it is believed that adenosine may participate in the generation of Tregs by modulating the mTOR pathway. In addition, recent studies indicate that activation of adenosine receptors, more specifically A2a, with agonist agents, leads to increased production of Treg cells, whereas the use of antagonistic agents of these receptors leads to a decrease in Treg differentiation.. However, these studies show the generation of Tregs from naive T cells of mice. In view of the great importance of Tregs in the immunological context, the efficient production of Tregs in vitro is of fundamental importance for the development of new therapeutic protocols for the treatment of autoimmune diseases and in the fight against transplant rejection. Thus, the central objective of this study was to evaluate the participation of adenosine receptor agonists and antagonists in induction of regulatory T cells generated in vitro (iTreg) by the activation of naive CD4+ T cells isolated from human umbilical cord blood (SCU). For this, mononuclear cells were isolated from SCU and naive T cells were immunomagnetic isolated. These cells were activated with beads bound to anti-CD2/CD3/CD28 antibodies and cultured for five days in the presence of IL-2 and different concentrations of agonist drugs and antagonists of adenosine receptors. Next, the major regulatory T-cell markers were assessed by flow cytometry and the culture medium was collected at the end of the generation for quantification of cytokines. In addition, total RNA was extracted from all culture conditions for the analysis of the expression of genes involved in the generation and development of Tregs by quantitative PCR. The potential for suppression of effector T cells was also evaluated.

T cells are central regulators of adaptive immunity in infectious and pathophysiological diseases. The activation of T cells is regulated by the T cell antigen receptor (TCR) and co-stimulatory receptors like toll-like receptor 2 (TLR2). These receptors activate distinct and overlapping intracellular signaling pathways that ultimately shape T cell responses. Therefore, studies that elucidate the molecular mechanisms of signal transduction downstream of receptors like the TCR and TLR2 will highlight key pathways required for T cell activation. These pathways could then be clinically targeted to alter dysfunctional T cell responses that promote many human diseases. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are two tyrosine kinases activated by multiple surface receptors expressed on T cells. FAK and Pyk2 signaling regulate cell morphology, migration, adhesion, proliferation, and survival in other cell types. However, their functions in T cells are not well-described. Because FAK and Pyk2 functions are dysregulated in many disease states, it is important to understand their function in human T cells so that clinicians can safely target these kinases to treat various disorders. The studies described in this dissertation aim to more fully elucidate how FAK and Pyk2 control T cell activation mediated by the TCR. We used recombinant microRNAs or kinase inhibitors to transiently suppress FAK and Pyk2's expression or enzymatic function in human T cells. In doing so, several novel functions of FAK and Pyk2 were uncovered. In Chapter III, we revealed that FAK is a negative regulator of TCR signal transduction and function. Interestingly, in contrast to its function in other immune cell lineages, the work described in Chapters III and IV demonstrates that FAK is not required to regulate actin cytoskeletal responses downstream of the TCR. The data presented in Chapter IV demonstrate that Pyk2 regulates TCR-mediated actin cytoskeleton reorganization. This function appears to be linked to Pyk2's scaffolding function and not its enzymatic activity. In Chapter V, we demonstrated that the catalytic function of Pyk2 controls phosphatidylinositol-3-kinase (PI3K) activation in human T cells. Together, these data revealed that FAK and Pyk2 serve distinct functions in TCR signal transduction, actin cytoskeletal rearrangement, and effector responses. TCR-driven cytokine production and proliferation are enhanced when T cells are concurrently activated via TLR2 ligands. In Chapter VI, we describe the signaling pathways that TLR2 activates in human T cells, and we characterize how TCR and TLR2 signals converge to augment T cell responses. In contrast to studies performed using isolated murine T cells, we demonstrated that TLR2 does not activate nuclear-factor kappa B in human T cells. Instead, we found that TCR and TLR2 co-ligation selectively augments extracellular signal-related kinase 1 (Erk1)/Erk2 and Akt activation in human T cells. Thus, TLR2 co-stimulates murine and human T cells via distinct signaling mechanisms.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22347号 / 医博第4588号 / 新制||医||1042(附属図書館) / 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 江藤 浩之, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23079号 / 医博第4706号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 稲垣 暢也, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM