L’échappement des cellules tumorales au processus d’immunosurveillance semble être une condition nécessaire au développement tumoral dans les modèles précliniques, comme chez l’homme. Les mécanismes par lesquels la tumeur parvient à médier une immunosubvertion sont multiples et font intervenir la plupart des cellules du système immunitaire, au sein desquelles, les cellules immunorégulatrices telles les cellules myéloides suppressives (MDSCs) ou les lymphocytes T régulateurs (Tregs, exprimant le facteur de transcription Foxp3), semblent jouer un rôle prépondérant. Les résultats présentés dans ce travail visent à mieux comprendre les rôles fonctionnels et pronostics des cellules myéloïdes suppressives et des Tregs dans le cancer, avec une attention plus particulière sur la façon dont ces cellules peuvent être modulées par la chimiothérapie. Concernant les MDSCs, nos travaux ont permis de mieux comprendre les mécanismes moléculaires présidant à leur accumulation d’une part, et d’autre part à l’acquisition de leur propriétés immunosuppressives, à travers une voie de signalisation impliquant les exosomes d’origine tumorale. Cette découverte, et la propriété d’une molécule d’usage thérapeutique courant, l’amiloride, de diminuer la production d’exosomes, y compris par les cellules tumorales, offrent une nouvelle possibilité de ciblage pharmacologique des MDSCs. Par ailleurs, l’étude des effets cytotoxiques sur les MDSCs de plusieurs molécules de chimiothérapie nous a permis de montrer que le 5-fluorouracile, probablement en raison d’un faible niveau d’expression de sa cible, la thymidilate synthase, dans les MDSCs, possédait une capacité sélective à éliminer ces cellules. Nos travaux d’immunohistochimie conduits sur des prélèvements tumoraux issus de patientes porteuses de cancers du sein localisés traitées par chimiothérapie néoadjuvante ont quand à eux permis de démontrer que la chimiothérapie néoadjuvante s’accompagne de modifications qualitatives de l’infiltration tumorale à la fois en lymphocytes T CD8+ et en lymphocytes T régulateurs Foxp3+. L’existence, après chimiothérapie néoadjuvante, d’une balance favorable de la réponse immunitaire, associant forte infiltration en CD8+ et faible infiltration en Foxp3+ s’accompagne d’une augmentation significative des marqueurs de cytotoxicité à médiation cellulaire, et est significativement corrélée à une éradication complète des cellules tumorales. Cette signature immunologique favorable se traduit également à long terme par une meilleure survie sans récidive et une meilleure survie globale, indépendamment du type de chimiothérapie reçue, de l’obtention ou non d’une réponse complète histologique, et du sous type moléculaire de cancer du sein. La combinaison de cette information immunologique avec la connaissance de la taille du résidu tumoral après traitement permet de considérablement affiner le pronostic des patientes. Enfin, nos travaux préliminaires semblent montrer que l’expression de Foxp3 dans les cellules cancéreuses de tumeurs du sein HER2+++ constitue un facteur de bon pronostic. Ces résultats illustrent donc l’importance non pas seulement des caractéristiques tumorales, mais aussi des caractéristiques de l’hôte, en particulier de la réponse immunitaire qu’il est capable de susciter, et de l’influence de la chimiothérapie sur cette dernière. / Evasion of immune surveillance by certain tumour cells seems to be a basic requirement for tumour development in preclinical models and in humans. The mechanisms by which the tumour mediates its immune evasion are manifold, and involve the majority of immune system cells. Among these, immunoregulatory cells such as myeloid-derived suppressor cells (MDSCs) or regulatory T lymphocytes (T-regs, which express the transcription factor Foxp3) appear to play a predominant role. The results presented in this work aim to improve our understanding of the functional and prognostic roles of myeloid suppressor cells and T-regs in cancer, focussing particularly on how these cells are modulated by chemotherapy. Regarding MDSCs, our work has made it possible to better understand on the one hand the molecular mechanisms underlying their accumulation, and on the other hand, their acquisition of immunosuppressive properties, through a signaling pathway involving exosomes of tumoral origin. This discovery, combined with the ability of amiloride, a molecule in frequent therapeutic use, to reduce the production of exosomes, even by tumour cells, offers new avenues for pharmacological targeting of MDSCs. Indeed, a study of the cytotoxic effects on MDSCs of several chemotherapy compounds made it possible to show that 5-fluorouracil has a selective capacity to eliminate MDSCs, probably due to the low level of expression of its target, thymidylate synthase, in MDSCs. Our immunohistochemical studies on tumour specimens resected from patients with localised breast cancer treated by neoadjuvant chemotherapy have shown that neoadjuvant chemotherapy is associated with qualitative changes in tumour infiltration by both CD8+ T-lymphocytes and Foxp3+ T-regs. The existence of a favourable immune response ratio after neoadjuvant chemotherapy, as defined by high infiltration by CD8+ and a low level of Foxp3+ infiltration, is associated with a significant increase in markers of cell-mediated cytotoxicity, and is also significantly correlated with complete eradication of tumour cells. This favourable immunological profile is reflected in the long-term by improved disease-free survival and better overall survival, regardless of the type of chemotherapy, the achievement or not of complete pathological response, and the molecular sub-type of breast cancer. Combining this immunological information with the data about the extent of tumour residue after treatment makes it possible to considerably refine prognosis in these patients. Finally, our preliminary work suggests that expression of Foxp3 in cancerous cells in HER2+++ breast tumours is a favourable prognostic factor. Overall, these results illustrate the importance not only of the tumour characteristics, but also of the host characteristics, in particular, the type of immune response that it is capable of eliciting, and the effect of chemotherapy on this immune response.

Les phosphoantigènes (P-Ag) de P. falciparum (P.f.) induisent une forte activation et une expansion des lymphocytes T (LT) Vγ9Vδ2 par un mécanisme encore mal décrit. Les LT Vγ9Vδ2 actives inhibent le cycle sanguin de P. f. par des médiateurs cytotoxiques solubles, inhibant ainsi la capacité invasive des mérozoites. Il a été montre in vitro que des LT Vγ9Vδ2 activés par les P-Ag peuvent présenter des antigènes et activer les LT αβ, agissant ainsi comme des cellules présentatrices d’antigènes (APC). Cette fonction n’a cependant pas été démontrée dans un contexte physiopathologique. Le but de ce projet est i) d’étudier les mécanismes d’activation des LT Vγ9Vδ2 par les stades sanguins P. f. et ii) d’evaluer le potentiel APC des LT Vγ9Vδ2 stimules par P. f. Nous montrons que l’activation des LT Vγ9Vδ2 par des globules rouges parasites par P. f. (GRP) intacts ne dépend ni d’un contact cellulaire, ni de l’expression de butyrophiline par le GRP. Les LT Vγ9Vδ2 sont activés par des molécules contenues dans les surnageants de culture de GRP, ayant les caractéristiques de P-Ags et étant libérées lors de la rupture des GRP. In vitro, les LT Vγ9Vδ2 stimules par les GRP expriment des marqueurs de surface associés à un rôle d’APC et cross-présentent un antigène modèle à une lignée T CD8 spécifique. In vivo, nous montrons une expression augmentée des marqueurs APC à la surface de LT Vγ9Vδ2 de patients infectés par P. falciparum. L’ensemble de ces données suggèrent que les P-Ag libérés par les GRP dans le milieu extracellulaire pourraient activer les LT Vγ9Vδ2 à distance, et ouvrent de nouvelles perspectives quant au rôle des LT Vγ9Vδ2 dans la réponse immunitaire adaptative anti-palustre. / P. falciparum derived phosphoantigens (P‐Ag) induce potent activation and expansion of Vγ9Vδ2 T-cells by a poorly described mechanism. Activated Vγ9Vδ2 T cells inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. In vitro, P-Ag activated Vγ9Vδ2 T lymphocytes have been shown to present antigens and induce αβ T lymphocyte responses, i.e. to act as an antigen presenting cell (APC). Whether this activity can be involved in a pathophysiological context is unknown. The aim of this PhD project is to a) investigate the mechanisms of Vγ9Vδ2 T cell activation by blood stage P. falciparum and b) assess the potential of P. falciparum activated Vγ9Vδ2 T cells to display APC functionality. We show that Vγ9Vδ2 T-cell activation by intact iRBCs is independent of iRBC contact and butyrophilin expression. Blood stage culture supernatants can potently activate Vγ9Vδ2 T-cells and bioactivity is found to be attributable to P-Ags released at the time of parasite egress from the RBC. In vitro iRBC stimulated Vγ9Vδ2 T cells up-regulate surface expression of APC associated markers and can cross-present a model antigen to specific CD8 T cell responders. In vivo we demonstrate an increase in surface expression of APC makers on Vγ9Vδ2 T cells from P. falciparum infected patients.Altogether, these data outline a framework whereby P‐Ag release by iRBC into extracellular milieu can promote activation of distant Vγ9Vδ2 T cells, and opens the door to a new aspect of Vγ9Vδ2 T cell contribution to P. falciparum adaptive immune responses.

Résumé : Le carcinome hépatocellulaire (HCC) est la troisième cause commune de décès de cancer et affecte plus les hommes que les femmes. Le HCC résulte d’une dérégulation des voies de signalisation impliquées dans l’initiation de l’inflammation menant ainsi à des répercussions désastreuses. De part la complexité de ce type de cancer, les traitements qui existent à ce jour ne sont pas très prometteurs et ont un faible pourcentage de « rémission ». L’immunothérapie soulève beaucoup d’espoir quant à l’orientation vers un traitement efficace plausible. En effet, plusieurs suppresseurs de tumeur se retrouvent réprimés, parmi lesquels le SOCS1. C’est dans cette optique que nos recherches se sont orientées en mettant la lumière sur le SOCS1 «suppresseur de signalisation des cytokines 1 (SOCS1) » qui est réprimé au niveau du HCC et dont la restauration pourrait contribuer à un pronostic favorable à la rémission. La protéine SOCS1 a beaucoup attisé la curiosité des chercheurs de part son rôle suppresseur de tumeur. Pour comprendre les mécanismes d’action de SOCS1 et son implication dans la neutralisation de la tumeur, nous avons généré trois types stables de la lignée cellulaire du carcinome hépatocellulaire de souris Hepa1-6, une portant un vecteur vide, l’autre exprimant le type sauvage du gène SOCS1 (SOCS1-WT; Hepa-S) et une portant une mutation au niveau du domaine SH2 (SOCS1-R105K; Hepa-R). Le mutant ne peut plus inhiber la signalisation des cytokines. Lors de l'implantation sous-cutanée des cellules Hepa1-6 modifiées, chez des souris C57BL/6 et NOD.scid.gamma (NSG). Nous avons observé que les cellules Hepa1-6 exprimant le vecteur de contrôle (Hepa-V) formaient de grosses tumeurs tandis que les cellules Hepa-S formaient de petites tumeurs chez les deux types de souris. Les cellules Hepa-R quant à elles, formaient de grosses tumeurs seulement chez des souris immunodéficientes (NSG) mais montraient une croissance nettement retardée lorsqu’elles étaient greffées aux souris (C57BL/6) immunocompétentes. Partant de ce constat intrigant, nous avons postulé que SOCS1 favorise l'immunogénicité des cellules tumorales par son domaine SOCS Box. Par conséquent, les cellules Hepa-R offrent une occasion unique de démêler le potentiel pro-immunogène de SOCS1, et ceci dans le but d'élucider les fonctions immunogènes de SOCS1 dans le cancer du foie. Jusqu'à présent aucune précédente recherche ne s’est aventurée à chercher l’implication de SOCS1 dans l’augmentation de l’immunogénicité. / Abstract : Hepatocellular carcinoma (HCC) is the third common cause of cancer deaths and affects men more than women. HCC results from a disregulation that affects many factors including inflammation, leading to disastrous repercusions. Due to the complexity of this type of cancer, existing treatments nowadays are not promising and have a low remission percentage. Immunotherapy raises great hope regarding the direction towards a plausible effective treatment. It is in this context that our research has focused on shedding light on the SOCS1 "signaling suppressor of cytokine 1 '(SOCS1)" which was found to be suppressed in HCC and whose restoration could contribute to a favorable prognosis. The SOCS1 many fueled the curiosity of researchers due to its suppressor role in tumor. To understand the underlying mechanisms, we generated three types of stable mice cell line hepatocellular carcinoma Hepa1-6, one carrying an empty vector, the other expressing the wild-type gene SOCS1 (SOCS1-WT; hepatic S) and carrying a mutation in the SH2 domain (SOCS1-R105K; Hepa-R). These mutants cannot inhibit JAK-STAT cytokine signaling. Upon subcutaneous implantation of the modified Hepa1-6 cells in C57BL/ 6 and NOD.scid.gamma (NSG) mice, the Hepa1-6 cells expressing the control (Hepa-V) formed large tumors while the Hepa-S cells formed small tumors in both types of mice. Hepa-R cells in turn, formed large tumors only in immunodeficient mice (NSG), but showed a markedly delayed growth when transplanted into (C57BL / 6) immunocompetent mice. Based on this intriguing finding, we speculate that SOCS1 could promote immunogenicity of tumor cells, which was masked by the ability of SOCS1 to inhibit signaling of growth factors. Therefore, Hepa-R cells provide a unique opportunity to unravel the pro-immunogenic potential of SOCS1, and this in order to elucidate the immunogenic functions of SOCS1 in liver cancer. So far no previous research studied the potentiel of SOCS1 in increasing immunogenicity.

Para serem apresentados pelo MHC I em células dendríticas, os antígenos são processados pelo imunoproteassomo. O objetivo do trabalho foi examinar o efeito de pró-oxidantes em DC para avaliação da proliferação de linfócitos T CD8+, com foco no sistema ubiquitina-proteassomo. Os resultados mostram que o sistema Xantina/Xantina Oxidase aumentou a proliferação de T CD8+ isolados de camundongos imunizados com DNA-HSP65 após a co-cultura destas células com DC tratadas com XaXO. O XaXO promoveu maior maturação de DC em relação ao LPS, assim como uma queda da atividade catalítica do IP, sugerindo que o aumento da proliferação de T CD8+ não está diretamente relacionado à atividade do IP. A incubação de DC com XaXO não alterou a expressão da unidade catalítica 20S, da unidade regulatória 19S ou da subunidade <font face=\"Symbol\">&#9465i. Observou-se aumento da expressão da unidade regulatória 11S e do conteúdo de proteínas ubiquitinadas. Sugere-se que 11S se acoplaria a 20S deslocando 19S, promovendo o acúmulo de proteínas ubiquitinadas e direcionando mais fragmentos para a apresentação antigênica. / To be presented in dendritic cells MHC I, antigens are processed by immunoproteasome. The aim of the study was to examine the effect of pro-oxidants in dendritic cell-induced T CD8+ lymphocyte proliferation with focus on ubiquitin-proteasome system. The co-culture of DC incubated with Xanthine/Xanthine Oxidase system and T CD8+ isolated from mice immunized with DNA-HSP65 promoted T CD8+ proliferation. XaXO incubation was more efficient in promoting DC maturation compared to LPS. In addition, XaXO incubation decreased IP catalytic activity in relation to LPS, suggesting that increased T CD8+ proliferation is not directly related to IP activity. XaXO incubation did not alter the expression of 20S catalytic subunit, 19S regulatory unit or <font face=\"Symbol\">&#9465i subunit. XaXO incubation increased 11S regulatory unit content as well as that of ubiquitinated proteins. We suggest that the DC incubation with XaXO increased 11S content favoring its coupling to 20S in detriment of 19S. This would increase ubiquitinated protein levels and direct more peptide fragments for antigen presentation.

Este trabalho objetivou avaliar os efeitos imunomoduladores das células-tronco imaturas da polpa dentária (CTIPD) sobre a diferenciação, maturação das células dendríticas derivadas de monócitos (mo-DC), sua capacidade para ativar linfócitos T (Lin T), bem como verificar fatores solúveis liberados nos cocultivos celulares. As analises foram feitas por citometria de fluxo. Foi observado que mo-DC tiveram a diminuição de moléculas relacionadas à diferenciação para mo-DC e o aumento de moléculas relacionados ao estado precursor, o que parece ter refletido na maturação para mDC, verificado pela diminuição das moléculas de maturação. As mo-DC tiverem sua função em induzir a proliferação de Lin T reduzida, além, de favorecer o aumento da proporção de Lin T CD4+FoxP3+IL-10+ e Lin T CD4+FoxP3+IFN-<font face=\"Symbol\">g+. A mensuração dos fatores solúveis dos cocultivos mostrou que houve aumento de fatores anti-inflamatórios e redução de fatores pró-inflamatórios. Futura investigações podem suportar o uso das CTIPD em uma abordagem imunomoduladora utilizando DC em aplicações clínicas. / This study aimed to evaluate the immunomodulatory effects of immature stem cells from dental pulp on differentiation, maturation of monocyte-derived dendritic cells (mo-DC), their ability to activate T cells, as well as identify soluble factors released in cellular cocultures. The analyzes were performed by flow cytometry. It was observed a decrease of molecules related to mo-DC differentiation and an increase of molecules related to precursor state, which seems to have reflected to mDC maturation verified by reduction of maturation molecules. The mo-DC have their role in inducing T cells proliferation reduced, in addition, favored increase CD4+FoxP3+IL-10+ and CD4+FoxP3+IFN-<font face=\"Symbol\">g+ T cells population proportion. The measurement of soluble factors from coculture showed an increase of anti-inflammatory factors and a reduction of pro-inflammatory factors. Future research may support the use immature stem cells from dental pulp in an immunomodulatory approach using DC in clinical applications.

L’équipe a identifié une nouvelle GAP de RhoA, ARHGAP19, majoritairement exprimée dans le système hématopoïétique. Le projet a consisté à étudier la régulation de cette protéine dans des lymphocytes T humains. Pour cela, les analyses se sont portées sur la phosphorylation d’ARHGAP19 et sur sa localisation au cours de la division des lymphocytes T. ARHGAP19 est phosphorylée par l’effecteur de RhoA, la protéine kinase ROCK, sur la Sérine 422 et par la protéine kinase mitotique CDK1 sur les Thréonines 404 et 476. La phosphorylation par ROCK permet à ARHGAP19 d’interagir avec la famille de protéines 14-3-3 qui la protège des déphosphorylations pouvant avoir lieu au cours de la division cellulaire. L'ensemble des phosphorylations est primordial pour la régulation de la localisation cellulaire d'ARHGAP19 et contribue à une division cellulaire correcte. En effet, en absence de phosphorylation, on observe des défauts lors de la cytodiérèse entrainant la formation de cellules multinucléées. De plus, des dérégulations de RhoGTPases comme l’absence de GAP, sont aujourd’hui mises en évidence dans les cancers. C’est pourquoi nous avons généré des souris arhgap19 KO pour étudier les conséquences de l’absence du gène codant pour ARHGAP19, dans le système hématopoïétique murin. L’ensemble des cellules progénitrices et matures intervenant dans l’hématopoïèse murine a été analysé. Par ce modèle d’invalidation conditionnelle d’arhgap19, aucun rôle majeur de la protéine n'a été mis en évidence mais les résultats suggèrent une implication aux différents stades de la différenciation hématopoïétique et un impact sur l'ensemble des populations de ce système. / The team identified a new GAP of RhoA, ARHGAP19, mostly expressed in the hematopoietic system. The project consisted in studying the regulation of this protein in human T lymphocytes. For this, the analyzes focused on the phosphorylation of ARHGAP19 and on its localization during the division of the T lymphocytes. ARHGAP19 is phosphorylated by the effector of RhoA, the protein kinase ROCK, on the Serine 422 and by the protein CDK1 mitotic kinase on Threonines 404 and 476. ROCK phosphorylation allows ARHGAP19 to interact with the 14-3-3 family of proteins that protects it from dephosphorylation that occur during cell division. All phosphorylations are essential for regulating the cellular localization of ARHGAP19 and contribute to correct cell division. Indeed, in the absence of phosphorylation, defects are observed during cytodiérèse resulting in the formation of multinucleate cells. In addition, deregulation of RhoGTPases, such as the absence of GAP, are now highlighted in cancers. This is why we generated arhgap19 KO mice to study the consequences of the absence of the gene coding for ARHGAP19, in the murine hematopoietic system. All progenitor and mature cells involved in murine hematopoiesis were analyzed. By this model of conditional invalidation of arhgap19, no major role of the protein has been demonstrated but the results suggest an involvement at different stages of hematopoietic differentiation and an impact on all populations of this system.

Cancer is the second leading cause of death in the Czech Republic. Breast cancer and colorectal cancer have relatively high mortality rate. One of the areas of current clinical research in oncology is the study of prognostic biomarkers, which aims to optimize the decision-making process for a patient. Immune response and processes in the tumor microenvironment have been shown to influence to a large extent the biological nature of the tumor in terms of its aggressiveness and ability to metastasize in the host's body. There are certain tumors that could induce a strong immune response, while others do not. The ability to induce an anti-tumor cell response and to attract specific lymphocyte subpopulations directly into tumor tissue has been shown to be very closely related to the prognosis of cancer patients. There is evidence and correlation of the presence of so-called tumor infiltrating lymphocytes in tumor tissue and overall patient survival. Stratification of cancer patients based on immuno-predictors both in the plasma and directly in the tumor microenvironment makes it possible to identify suitable candidates for rediscovered modern anti-tumor immunotherapy, which can already be considered a standard therapeutic modality. In our projects, we focused on the identification of biomarkers that...

Cancer development and progression vary depending on tumor type, localization, invasion, immunogenicity and the ability of immune system to become activated. There are frequent interactions between tumor cells and immune cells, occuring locally at the site of primary tumor or distally through paracrine signalling of various mediators and cytokines. The main subject of this PhD thesis is to study key factors and aspects of immune response in cancer patients. In the first part, we analyzed immune cells infiltrating tumor tissues of ovarian cancer patients at different stages of disease. We focused on the dynamics of immune response, primarily on frequency of individual T lymphocyte populations in peripheral blood and tumor infiltrating T lymphocytes in tumors of early and advanced stages of ovarian cancer. We found that during disease progression there is a gradual decrease of proinflammatory Th17 and Th1 immune responses and a specific recruitment of regulatory T cells to the tumor site, which results in a significant immune suppression in the tumor microenvironment. In the second part, we demonstrated that the character of immune response in HPV-positive head and neck cancer patients is very different from the patients with tumors not associated with HPV infection. In HPV-positive patients, significantly...

The discovery of novel genes involved in hematopoietic development and lymphoid function is necessary for the understanding of these systems. To this end, we utilized transmembrane protein-specific gene trapping in embryonic stem (ES) cells, a method of forward genetics, to identify a novel, complex locus from which several splice variants arise. The trapped locus identified in the KST30 ES cell clone encodes several genes including outer membrane protein 25 (OMP25) and activin receptor interacting protein (ARIP2) and two novel genes, AK74 and AK88. AK74 is highly conserved between human and mouse with 85% identity at the amino acid level. The human homolog was cloned from CD34+ cord blood hematopoietic stem cell progenitors (HSCPs) implying that it may have a role in the hematopoietic system. We generated mice from the gene trapped ES cells, called KST30 mice, to analyze the expression pattern of transcripts from the trapped locus in the hematopoietic system. Utilizing the gene trap LacZ reporter and RT-PCR, we found that AK88 and AK74 are expressed in hematopoietic stem cells and thymocytes and that AK88 and ARIP2 are dramatically up-regulated in activated Band T lymphocytes. In addition, we found restricted expression of the gene trap in most non-lymphoid tissues. Interestingly, the expression pattern of the gene trap coincides with the expression of activin signaling components in many cell types including thymocytes, activated B cells, hematopoietic stem cells and the ductal cells of the pancreas. AK74, AK88 and ARIP2 share two exons that encode a 44 amino acid region. ARIP2 negatively regulates activin signaling through endocytosis of Activin type II receptors. The N-terminal PDZ domain associates with ActRII and mediates endocytosis via association with RalBP1. The region of ARIP2 that associates with RalBP1 encompasses the 44 amino acid region also found in AK74 and AK88, suggesting that these proteins may also associate with RalBP1, perhaps sequestering it from ARIP2. This possibility combined with the similarities between gene trap expression and expression of the components of activin signaling indicates a role of the trapped genes in activin signaling. AK74 and AK88 have a signal sequence and transmembrane domain that are predicted to direct them to mitochondria. To confirm this prediction, we examined the subcellular localization of AK74 and found that it localizes to a punctate, perinuclear structure identified as mitochondria using a mitochondria specific dye. AK74 was not seen in the cytoplasm, nucleus or at the plasma membrane of cells. To determine the function of these novel genes, AK74 was retrovirally over-expressed in a double positive thymoma cell line and examined the global expression profile using Affymetrix gene chip. AK74 changed the expression levels of 36 genes greater than 3-fold compared to vector alone. Of these genes, several are involved in cytoskeletal rearrangement, apoptosis or are regulated by calcium signaling. Using yeast two-hybrid, several candidate binding partners for AK74 were identified, one of which is the receptor for activated protein kinase C (RACK1). RACK1 was also identified as a potential binding partner for AK88. RACK1 is a WD40 domain-containing scaffolding protein that has been implicated in many pathways but most prominently in the protein kinase C signaling pathway. Association with RACK1 by either AK74 or AK88 suggests that they may be involved in RACK1 function. Both RACK1 and PKC are involved with Ca2+ signaling through different mechanisms. This, combined with global gene expression changes in AK74 over-expressing cells suggests a role for AK74, AK88 or ARIP2 in Ca2+ signaling. When we examined the expression of the trapped genes in mice homozygous for the gene-trapped allele (KST30tr/tr) we found that insertion of the gene trap caused a severe decrease in AK88 and ARIP2 but not AK74 transcripts. Analysis of KST30tr/tr mice showed no abnormalities in conventional lymphoid populations and precursors, however, intraepithelial lymphocyte (IEL) populations were altered by the loss of AK88 and/or ARIP2. There was an approximate 2-fold decrease CD8αα+ T cells in the small intestine while CD8αβ+ T cells were largely unaltered. Using gene trap technology, we have identified two novel, mitochondria-localized proteins. The cumulative findings described in this thesis, including the homology between AK74, AK88 and ARIP2, their expression pattern and the phenotype of KST30tr/tr mice, suggest possible roles of AK74 and AK88 in diverse pathways.

During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation. In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks). Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance. Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function. This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation. Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.