Die Herstellung von T-Zellen mit definierten Spezifitäten durch den Transfer von T-Zellrezeptor (TCR) Genen ist eine effiziente Methode, um Zellen für eine Immuntherapie bereitzustellen. Eine besondere Herausforderung ist dabei, ein ausreichend hohes Expressionsniveau des therapeutischen TCR zu erreichen. Da T-Zellen mit einem zusätzlichen TCR ausgestattet werden, entsteht eine Konkurrenzsituation zwischen dem therapeutischen und dem endogenen TCR. Bevor diese Arbeit begonnen wurde war nicht bekannt, welche TCR nach einem Gen-Transfer exprimiert werden. Daher haben wir Modelle etabliert, in denen TCR Gene in Maus und humane T-Zellen mit definierten endogenen TCR transferiert wurden. Die Expression beider TCR wurde mithilfe von Antikörpern und MHC-Multimeren analysiert. Diese Modelle haben gezeigt, dass bestimmte TCR andere TCR von der Zelloberfläche verdrängen können. Dies führte in einem Fall zu einer vollständigen Umkehr der Antigenspezifität. Aufgrund dieser Ergebnisse haben wir das Konzept von „starken“ (gut exprimierten) und „schwachen“ (schlecht exprimierten) TCR vorgeschlagen. Zusätzlich wurde die Verdrängung „schwacher“ und „starker“ humaner TCR durch Maus TCR beobachtet. Parallel dazu wurde berichtet, dass die konstanten (C) Regionen von Maus TCR für die erhöhte Expression auf humanen Zellen verantwortlich sind. Dies führte zu einer Strategie zur Verbesserung der Expression humaner TCR, die auf dem Austausch der humanen C-Regionen durch die von Maus TCR basiert (Murinisierung). Ein Problem ist dabei die mögliche Immunogenität dieser hybriden Konstrukte. Deshalb haben wir jene Bereiche der Maus C-Regionen identifiziert, die für die erhöhte Expression verantwortlich sind. In der TCRalpha Kette wurden vier und in der TCRbeta Kette fünf Aminosäuren gefunden, die ausreichend für diesen Effekt waren. Primäre humane T-Zellen mit TCR, die diese neun „Maus“ Aminosäuren enthielten, zeigten eine bessere Funktionalität als T-Zellen mit Wildtyp TCR. / The in vitro generation of T cells with a defined antigen specificity by T cell receptor (TCR) gene transfer is an efficient method to create cells for immunotherapy. One major challenge of this strategy is to achieve sufficiently high expression levels of the therapeutic TCR. As T cells expressing an endogenous TCR are equipped with an additional TCR, there is a competition between therapeutic and endogenous TCR. Before this work was started, it was not known which TCR is present on the cell surface after TCR gene transfer. Therefore, we transferred TCR genes into murine and human T cells and analyzed TCR expression of endogenous and transferred TCR by staining with antibodies and MHC-multimers. We found that some TCR have the capability to replace other TCR on the cell surface, which led to a complete conversion of antigen specificity in one model. Based on these findings we proposed the concept of ‘‘strong’’ (well expressed) and “weak” (poorly expressed) TCR. In addition, we found that a mouse TCR is able to replace both “weak” and “strong” human TCR on human cells. In parallel to this result, it was reported that the constant (C)-regions of mouse TCR were responsible for the improved expression of murine TCR on human cells. This led to a strategy to improve human TCR by exchanging the C-regions by their murine counterparts (murinization). However, a problem of these hybrid constructs is the probable immunogenicity. Therefore, we identified the specific parts of the mouse C-regions which are essential to improve human TCR. In the TCRalpha C-region four and in the TCRbeta C-region five amino acids were identified. Primary human T cells modified with TCR containing these nine “murine” amino acids showed an increased function compared to cells modified with wild type TCR. For TCR gene therapy the utilization of these new C-regions will reduce the amount of foreign sequences and thus the risk of immunogenicity of the therapeutic TCR.

Les contrôleurs du VIH (HICs) sont de rares patients qui contrôlent spontanément la réplication du VIH en absence de thérapie antirétrovirale. Ces patients sont caractérisés par un taux normal de cellules T CD4+, le maintien d’une charge virale indétectable (<50 copies d’ARN viral/mL de plasma), et présentent un très faible risque de progression vers le SIDA. De nombreux travaux suggèrent que le contrôle de la réplication virale chez ces patients est dû à une réponse cellulaire antivirale particulièrement efficace. Notre équipe a récemment montré que les HICs inclus dans la Cohorte ANRS CO21 CODEX maintiennent des réponses T CD4+ très sensibles, associées à l'expression de TCRs ayant une forte avidité pour certains peptides Gag du VIH. Nous avons montré en particulier que les cellules T CD4+ de HICs répondent à de très faibles concentrations de l’épitope immunodominant Gag293. Dans une première étude, nous avons pu mettre en évidence que la réponse de haute avidité observée pour les cellules T CD4+ des HICs semble s'expliquer par une propriété intrinsèque de leur TCR. Pour identifier les déterminants moléculaires qui sous-tendent cette réponse de haute avidité, nous avons caractérisé le répertoire TCR des cellules T CD4+ spécifiques de l’épitope immunodominant Gag293. Les HICs présentent un répertoire TCR hautement biaisé, caractérisé par une expression préférentielle des chaînes variables TRAV24 et TRBV2, la présence de motifs conservés au sein des régions CDR3 de ces deux chaînes, et une prévalence élevée de clonotypes publics. Le clonotype public le plus représenté est capable de conférer une réponse spécifique à Gag293 très sensible et polyfonctionnelle aux cellules T CD4+ primaires, ainsi qu'une reconnaissance de multiples allèles HLA-DR. Nous cherchons à présent à déterminer si les candidats vaccins peuvent induire des réponses T CD4+ similaires à celles observées chez des patients HIC. Pour cela, nous avons analysé les réponses spécifiques de Gag293 chez des volontaires sains ayant reçu le candidat vaccin ADN ADVAX administré par soit par électroporation (EP) ou soit par injection intramusculaire (IM). La comparaison des réponses spécifiques de Gag293 dans des lignées T CD4+ primaires par test ELISpot IFNγ a montré que la sensibilité à l'antigène chez les volontaires vaccinés était proche de celle des patients contrôleurs et significativement plus élevée que celle mesurée chez les patients traités (p<0.05). Par contre, les volontaires du groupe IM n'ont pas montré de réponse détectable contre Gag293. L'analyse du répertoire TCR des cellules spécifiques de Gag293 chez les volontaires vaccinés a révélé une amplification préférentielle de la chaîne variable TRBV2, chaîne qui prédomine également chez les patients HIC. Cependant, l'utilisation des chaînes variables TRAV est apparue plus diverse chez les sujets vaccinés, avec une amplification préférentielle des chaînes TRAV29 et TRAV24. L'analyse des clonotypes TCR amplifiés chez les vaccinés a mis en évidence un recoupement inattendu avec le répertoire des HIC, avec une fréquence élevée de motifs partagés (30%). Les clonotypes publiques TRAV24 et TRBV2 les plus fréquemment retrouvés dans le répertoire des Contrôleurs ont également été induits par la vaccination ADN EP, indiquant le potentiel pour une réponse de forte avidité. Par contre, le clonotype TRAV29 le plus exprimé, chez les vaccinés a induit des réponses de plus faible avidité, même si ces réponses étaient restreintes par de multiples allèles HLA-DR. L'ensemble de ces résultats suggère que les clonotypes TRAV24, plutôt que les clonotypes TRAV29, sont responsables de la réponse de forte avidité contre Gag293. En conclusion, un candidat vaccin ADN administré par EP possède la capacité d'induire des clonotypes TCR associés au contrôle du VIH. Cette étude suggère que les clonotypes TCR publics pourraient servir de nouveaux biomarqueurs permettant d'évaluer la qualité des réponses vaccinales induites contre le VIH / HIV controllers are rare patients who spontaneously control HIV replication to levels below 50 copies viral RNA/mL in the absence of antiretroviral therapy. Converging evidence indicate that HIV controllers mount a cellular antiviral response that is highly efficient at detecting and eliminating infected cells. In particular, we previously reported that HIV controllers from the ANRS CO21 CODEX cohort harbour a pool of memory CD4+ T cells able to respond to minimal amounts of viral antigens, due to the expression of TCRs with high avidity for immunodominant Gag epitopes. In a first study we show that the high functional avidity observed in Controller CD4+ T cells could be explained by the expression of high avidity T cell receptors (TCRs) which efficiently bound Gag peptide-loaded MHC class II tetramers. Repertoire analysis of CD4+ T cells specific for the immunodominant Gag293 epitope showed a highly skewed repertoire in HIV controllers, with a predominance of the TRAV24 and TRBV2 variable gene families, the presence of conserved motifs in both CDR3 regions, and a high prevalence of public clonotypes. When transferred to primary CD4+ T cells, the most frequent public TCR clonotypes could confer properties associated with an efficient T cell response, such as polyfunctional cytokine secretion, high affinity antigen recognition, and broad HLA-DR crossrestriction. To determine whether candidate HIV vaccines could induce the high sensitivity responses seen in controllers, we analysed Gag293-specific responses in healthy volunteers who received the ADVAX DNA vaccine administered by electroporation (EP) or intramuscular injection (IM). Comparison of Gag293-specific responses in primary CD4+ T cell lines via IFNγ ELISpot revealed that the median antigen sensitivity in vaccinees was close to that observed for controllers but significantly higher than that in treated patients (p <0.05). In contrast, volunteers from the IM group did not show a detectable Gag293-specific response. TCR repertoire analysis of Gag293-specific CD4+ T cells from vaccinees revealed a preferential amplification of TCRβ family chain TRBV2, which also predominates in controllers. However, TRAV family gene usage appeared more diverse in vaccinees compared to controllers, with the preferential amplification of TRAV29 and TRAV24. Sequence analysis revealed an unexpected degree of overlap between the specific repertoires of vaccinees and HIV controllers, as evidenced by a high frequency of shared TCR motifs (>30%). The most frequent TRAV24 and TRBV2 public clonotypes found in the controller TCR repertoire were also induced by EP DNA vaccination, suggesting the potential for high avidity responses. In contrast, an abundant TRAV29 clonotype from vaccinees induced Gag293-specific responses of lower avidity, even though these responses were broadly HLA-DR cross-restricted. Thus, our results suggest that TRAV24 rather than TRAV29 clonotypes may be responsible for the high avidity Gag293-response. In conclusion, DNA vaccination by electroporation has the potential to inducer TCR clonotypes associated with HIV control. Monitoring the amplification of public TCR clonotypes could provide a novel approach to evaluate the quality of HIV vaccine responses

A small percentage (1-5%) of the blood lymphocytes expresses alternative T-cell antigen receptor that uses g and d TCR rearranging genes. A subset of them expresses the Vg9Vd2 TCR. Those cells respond to self-nonpeptide and foreign antigens presented by unknown antigen-presenting molecules. Vg9Vd2 T cells also express Toll-like receptors and natural killer receptors that allow them to respond to other nonpeptide microbial components or to alterations in the expression of stress cell surface ligands such as NKG2D ligands. Vg9Vd2 T cells frequently are regulated by the expression of activating and/or inhibitory NKRs (iNKRs) that can fine-tune their activation threshold and the activating NKG2D receptor is one of the most studied until now. NKG2D, a C-type lectin receptor directed against MICA/MICB and UL16-binding protein (ULBP) molecules, have been reported a powerful co-stimulus for Ag-mediated activation of CD8 and Vg9Vd2 T cells. Indeed, NKG2D is recruited within the Vg9Vd2 TCR immunological synapse and enhances recognition by Vg9Vd2 T cells of Mycobacteria-infected DCs and various MICA/MICB or ULBP hemopoietic and non-hemopoietic tumors. The level of NKG2D is upregulated by inflammatory cytokines (e.g. IL-15), and NKG2D ligands are induced after a physical or genotoxic stress and/or along infection by intracellular pathogens. Therefore, NKG2D is a key stress sensor that strongly enhances recognition of altered or infected self by human gd T cells. Recent progress in the field supports the idea that gd T cells fulfill a role in the innate and adaptative immune response in different way of the conventional ab T cells. We demonstrated direct activation of Vg9Vd2 T cells by NKG2D ligation through the association with DAP10 adapter molecules and independently of TCR-Ag recognition, similar to the NKG2D-mediated activation of NK cells. Culture of peripherical blood mononuclear cells with immobilized NKG2D mAb or NKG2D ligand MICA induces up-regulation of CD69 and CD25 in NK and Vg9Vd2 T cells but not in CD8 T cells. Additionally, the ligation of NKG2D induces in Vg9Vd2 T cells the up-regulation of molecules typical for antigenpresenting cells, such as co-stimulator molecules (CD86) antigen presenting molecules (CD1a, HLA-DR), adhesion molecules (CD54), and activation molecules (CD69). Furthermore, NKG2D ligation in Vg9Vd2 T cells induces the production of cytokines such as TNF-a and chemokines such as, MIP-1a, but cannot induce the production of cytokines such as IL-6 or IFN-g and chemokines such as RANTES, MCP-1 and GM-CSF. In addition, NKG2D triggers the activation of the cytolytic machinery as efficient as CD3 stimulation as shown by measurement of the release of granules with esterase activity (BLT assay), perforin and the up-regulation of CD107a on the surface of Vg9Vd2 T cells. This NKG2D dependent cytolysis has been confirmed using purified Vg9Vd2 T cells, which kill MICA-transduced RMA cells but not the control cells. The TCR independence and NKG2D dependence of this killing is supported by mAb inhibition experiment. Finally, DAP 10, which mediates NKG2D signaling of human NK cells, is found in resting and activated Vg9Vd2 T cells. Moreover, data of intracellular signaling studies suggest an important role of Scr kinases in the NKG2D mediated killing and involvement of DAP-10-PI3K and PLCg 1 pathways as mayor proteins implicated in target cell lysis, and shows remarkable difference with the TCR signaling. The identification of these similarities in NKG2D function between NK and Vg9Vd2 T cells may be of interest for development of new strategies for Vg9Vd2 T cell-based immunotherapy in certain types of cancer and help to understand Vg9Vd2 T cell function in general. / Ein geringer Prozentsatz (1-5%) der T-Lymphozyten (T-Zellen) besitzt einen alternativen TZellrezeptor (TCR), der aus der g und d Kette der rearrangierten Gene aufgebaut ist. Eine geringe Population dieser T-Zellen exprimiert den Vg9Vd2 TCR. Diese Zellen werden durch körpereigene nicht-Peptide und fremde Antigene, die von bisher unbekannten antigenpräsentierenden Molekülen präsentiert werden, aktiviert. Vg9Vd2 T-Zellen exprimieren zudem Toll-like Rezeptoren und NK-Rezeptoren die es ihnen ermöglichen auf weitere, mikrobielle nicht-Peptid Moleküle oder die veränderte Expression von stressspezifischen Zelloberflächenmolekülen, wie dem NKG2D Liganden zu reagieren. Vg9Vd2 T-Zellen werden häufig über die Expression von aktivierenden und/oder hemmenden NKRs (iNKRs) reguliert, die deren Aktivierungsschwelle fein einstellen können. Der bisher am Besten untersuchte NKR ist der aktivierende NKG2D Rezeptor. Es wurde gezeigt, dass NKG2D, ein C-Typ Lektinrezeptor, der sich gegen MICA/MICB und UL16- bindende Proteine (ULBP)-Moleküle richtet, als starker Kostimulus für die antigenvermittelte Aktivierung von CD8 und Vg9Vd2 T-Zellen dient. In der Tat wird NKG2D zu der Vg9Vd2 TCR immunologischen Synapse rekrutiert und stimuliert dort die Erkennung von mycobakteriell infizierten DCs und verschiedenen MICA/MICB oder ULBP hämopoetischen und nicht hämopoetischen Tumoren durch Vg9Vd2 T-Zellen. Die Expression von NKG2D wird durch inflammatorische Zytokine (wie z.B. IL-15) stimuliert, sowie nach physikalischem oder genotoxischem Stress und/oder während einer Infektion mit intrazellulären Pathogenen induziert. Daher gilt NKG2D als entscheidender Stress-Sensor, der eine verstärkte Identifikation von veränderten oder infizierten körpereigenen Antigenen durch menschliche gd -Zellen bewirkt. Jüngste Fortschritte auf dem Gebiet stützen die Hypothese, dass gd TZellen eine Rolle in der angeborenen, sowie der adaptiven Immunantwort spielen, allerdings auf andere Weise wirken wie die konventionellen ab T Zellen. Wir haben gezeigt, dass die direkte Aktivierung von Vg9Vd2 T-Zellen durch die Bindung von NKG2D mittels Interaktion mit DAP10 Adaptermolekülen und unabhängig von TCR/Antigen Erkennung erfolgt, ähnlich der NKG2D vermittelten Aktivierung von NKZellen. Kulturen aus peripheren Blutzellen, die mit immobilisierten NKG2D monoklonalem Antikörper (mAb) oder dem NKG2D Liganden MICA behandelt wurden zeigten vermehrte Expression von CD69 und CD25 in NK und Vg9Vd2 T-Zellen, jedoch nicht in CD8-Zellen. Desweiteren führte die Bindung von NKG2D in Vg9Vd2 T-Zellen zur Regulation von antigenpräsentierenden Molekülen nach NKG2D Stimulation, wie z.B. kostimulatorische Moleküle (CD80, CD68), antigenpräsentierende Moleküle (CD1a, HLA-DR), Adhäsionsmoleküle (CD54) und Aktivierungsmoleküle (CD69, CD95). Die Interaktion von NKG2D und Vg9Vd2 T-Zellen induzierte zudem die Produktion von Zytokinen, wie TNF-a, sowie Chemokinen wie MIP-1a, jedoch nicht von IL-6, IFN-g oder RANTES , MCP-1 und GM-CSF. Desweiteren aktivierte NKG2D die zytolytischen Maschinerie ebenso effizient, wie CD3. Dies konnte durch Messung der Freisetzung von Granula mit Esterase-Aktivität (BLTAssay) und von Perforin, sowie die verstärkte Expression von CD107a an der Zelloberfläche von Vg9Vd2 T-Zellen nachgewiesen werden. Die zytologische Aktivierung durch NKG2D konnte durch Vg9Vd2 T-Zellen, die MICA transduzierte RMA Zellen jedoch nicht die Kontrolzellen töten konnten, bestätigt werden. Die Tatsache, dass das Töten dieser Zellen unabhängig von TCR und abhängig von NKG2D erfolgt, wurde durch mAb Hemm- Experimente unterstützt. Schließlich wurde DAP10, das die Signalweiterleitung von NKG2D in menschlichen NK Zellen überträgt, sowohl in nicht aktivierten und activierten Vg9Vd2 Zellen nachgewiesen werden. Zudem lassen Daten von intrazellulären Signalstudien vermuten, dass Scr Kinase, wie auch DAP-10-PI3K und der PLCg 1 eine wichtige Rolle beim Töten durch den NKG2D Signalweg spielen, der beachtliche Unterschiede zum TCR Signalweg aufweisen. Die Entdeckung, dass NK-Zellen und Vg9Vd2 T-Zellen ähnlich auf die Bindung von NKG2D reagieren, könnte für die Entwicklung von einer Vg9Vd2 T-Zell-basierten Immuntherapie für die Behandlung bestimmter Krebsarten von Bedeutung sein und im Allgemeinen helfen die Vg9Vd2 T-Zell Funktion zu verstehen.

CD4⁺ T cells are a critical component of the adaptive immune compartment. Each T cell expresses a clonotypic T cell receptor (TCR) that must discriminate between self and foreign peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells to direct T cell fate decisions. Information regarding TCR-pMHC interactions must then be transmitted to the TCR-associated CD3 signaling modules, which contain ITAMs that serve as signaling substrates for Src kinases. The Src kinase, Lck, is recruited to the pMHC-bound TCR-CD3 complex via association with the CD4 coreceptor that binds MHCII. It is therefore through the coordinated interactions within the TCR-CD3-pMHC-CD4 macro-complex that productive TCR signaling can occur to inform T cell activation and fate decisions. While much is known regarding the structure of the individual subunits that make up the TCR-CD3-pMHC-CD4 macro-complex, there is little information regarding how these components come together to initiate TCR signaling and determine functional outcomes. Here, we have interrogated how interaction of these individual components leads to productive T cell activation. Specifically, we interrogated the nature of TCR-MHC interactions and provide evidence that there is intrinsic specificity of the TCR for MHCII. We have also built mouse models to determine the role of TCR-CD3 interactions and TCR dimerization in the transmission of information from the TCR to the CD3 subunits following TCR-pMHC engagement. Finally, we show that both the CD4 transmembrane and extracellular domains contribute to T cell activation in vitro. Overall, this work provides insight into how the constituents of the TCR-CD3-pMHC-CD4 macro-complex interact to initiate T cell fate and function.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

Els limfòcits T autorreactius, responsables de la destrucció de les cèl.lules beta pancreàtiques en la DT1, presenten un repertori TCR esbiaixat segons s’ha demostrat al model experimental del ratolí NOD. Per a definir el repertori TCR autorreactiu en la diabetis humana, hem estudiat les poblacions i expansions monoclonals intra-illot d’un pàncrees de debut i les hem comparat amb les de la sang perifèrica i a la melsa del mateix individu. L’estudi de les cèl.lules T intra-pancreàtiques ha mostrat un repertori divers però amb cinc expansions monoclonals per a les famílies gèniques Vβ1, Vβ7, Vβ11, Vβ17, i Vβ22. Mitjançant el -CDR3, s’ha identificant la mateixa expansió monoclonal Vβ22 als illots i a la melsa, i també se n’ha detectat la seqüència en PBMCs. Per a valorar el biaix als TCR, s’ha analitzat 139 seqüències CDR3 de cadena beta de diferents clons infiltrants i s’ha observat una prevalença d’alguns aminoàcids a l’ NDN, que indica una restricció allunyada de l’atzar. Les seqüències CDR3 de les cinc expansions monoclonals pancreàtiques (incloent la comú a la melsa) s’ajusten a la restricció en NDN definida a partir dels limfòcits del pàncrees. El sub-clonatge i la expansió in–vitro (amb extracte d’illots i anti-CD3) de l’infiltrat pancreàtic, ha mostrat una alteració del repertori original però ha permès aïllar un clon present a l’infiltrat que expressa un doble TCR i que manté també els paràmetres de restricció definits a l’infiltrat original. El clon V22 s’ajusta a la restricció de -CDR3 establerta a l’infiltrat, i representa la única expansió monoclonal del pàncrees observada a la sang perifèrica i la melsa de l’individu. Aquest clon, es podria haver expandit o acumulat in situ per la presència d'un autoantigen comú en ambdós òrgans i d’aquesta manera es postula que la melsa de l’individu podria contribuir a la perpetuació de la malaltia per expansió o retenció d’alguns clons T autorreactius. / Autoreactive T cells, responsible for the destruction of pancreatic beta cells in type 1 diabetes (T1D), have a biased TCR repertoire as has been demonstrated by the NOD mouse experimental model. To define the TCR repertoire of autoreactive cells in human diabetes, we have studied intra-islet populations and monoclonal expansions of a pancreas in diabetic onset and compared them with peripheral blood and spleen populations from the same patient. The study of intra-pancreatic T cells, showed a diverse repertoire but with five monoclonal expansions in the gene families Vβ1, Vβ7, Vβ11, Vβ17, and Vβ22. By TCR’s  chain CDR3 (-CDR3) analysis, the same Vβ22 monoclonal expansion could be identified in the spleen and islets, and the same sequence was detected in PBMCs. To evaluate the existance of a bias in the pancreatic TCR repertoire, we analyzed 139 -CDR3 sequences from different infiltrating clones and prevalence of certain amino acids in the NDN was observed, thus indicating the existance of non-random restriction. CDR3 sequences from that five pancreatic monoclonal expansions (including the one equal in spleen) adjusted to the restrictions defined in NDN from the pancreas lymphocytes. The sub-cloning and in vitro expansion of pancreas infiltrating cells (with islets’ extract and anti-CD3) showed an alteration of the original repertoire, but allowed to isolate a clone expressing two TCRs that did also maintain the restriction parameters defined in the original infiltrate. The clone V22, fits that -CDR3 restriction established for infiltrating cells, and represents the only monoclonal expansion in the pancreas that is also observed in the peripheral blood and spleen of the patient. That clone should have been expanded, or accumulated, in situ in the presence of an autoantigen common to both organs, so is postulated that the spleen could contribute to the perpetuation of the disease by expansion or retention of some T autoreactive T cell clones.

Nuclear factor kappa B (NF-kB) is a widely expressed family of transcription factors that are involved in a diverse number of processes. These include inflammation or differentiation, survival or apoptosis, and proliferation or cell cycle arrest. NF-kB is usually associated with inhibitory kB proteins (IkB), which mask the nuclear localisation sequence (NLS) of NF-kB and renders it in the cytoplasm. Various stimuli result in the activation of the I kappa B kinase (IKK) protein complex, which phosphorylates IκB proteins and thereby marks them for degradation by the ubiquitin-proteasome pathway. Thereby NF-kB enters the nucleus and acts on its target genes. The study of T- and B-lymphocyte antigen receptor signalling to NF-kB is a field of intense investigation, with much attention being focused on the molecular scaffolding proteins Carma1, Bcl10 and MALT1 and their post-translational modifications. These have been shown to be crucial for the organization of the immunological synapse structure under the activated receptor, to which IKK is recruited and becomes activated, which subsequently leads to the activation of NF-kB. T cell receptor (TCR) activation results in a rapid increase in the intracellular Ca2+ level and NF-kB activation is known to be regulated by those increases, but the mechanisms have remained unclear. Calmodulin (CaM) is a calcium sensory protein that responds to increases in intracellular Ca2+ levels. When CaM binds Ca2+ ions, it leads to structural changes that directly as well as indirectly, through CaM dependent kinases (CaMKs), phosphatases and other enzymes, alters a variety of cellular processes, among them transcriptional regulation. Here CaM is shown to interact directly with Bcl10 in a Ca2+ dependent manner. Increases in the intracellular Ca2+ level are shown to induce the proximity of Bcl10 and CaM in vivo. Carma1 associates with Bcl10 through a CARD-CARD domain interaction that is known to be crucial for TCR signalling to NF-kB. The interaction of CaM with Bcl10 was mapped to the CARD domain and was shown to be a negative regulator for the Bcl10-Carma1 interaction. Inhibition of the CaM interaction by a point mutation within the CaM binding site of Bcl10 results in decreased binding of CaM to Bcl10 in vivo, as well as an increased ability of Bcl10 to induce NF-kB transcriptional activity, which is further enhanced by TCR activating stimuli. NF-kB activation is also shown here to be regulated by CaM indirectly through actions of CaMKII. The CaMKII is recruited to the immunological synapse where it interacts with Bcl10 in an inducible fashion and phosphorylates Bcl10. Phosphorylations of Bcl10 by CaMKII are shown to be important for the ability of Bcl10 to induce NF-κB transcriptional activity. Upon mutation of its most important CaMKII site, Bcl10 fails to activate an NF-kB reporter and an NF-kB target gene (IL-2). This mutated Bcl10 also fails to induce activating phosphorylations of IKKa/b and the kinase JNK2 but not JNK1. Furthermore, phosphorylation of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signaling complex and the essential signal induced ubiquitinations of Bcl10 and IKKg. Phosphorylation of IKK by TAK1 is also regulated by CaMKII, and serine 82 is a putative CaMKII target site of TAK1 that appears to be important for IκBα degradation. In summary, this thesis explores that not only NF-kB but also CaM is a double-edged sword, since the multi-functional NF-kB family of transcription factors is regulated by CaM both negatively and positively.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13277号 / 論医博第2185号 / 新制||医||1039(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 河本 宏, 教授 杉田 昌彦 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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