L'activation des lymphocytes T est un événement fondamental de la réponse immunitaire adaptative. Elle est déclenchée par la transduction du signal médiée par le complexe TCR/CD3.Le mécanisme de déclenchement du signal via le TCR reste, mal compris. Mon projet de thèse vise à examiner la contribution des PI(4,5)P2 dans le mécanisme du déclenchement du signal TCR. L'expression ectopique d'une phosphatase spécifique de PIP2 a permis de réduire de 50% les PI(4,5)P2 membranaires. L'analyse du profil de phosphorylations spécifiques des tyrosines montre que l'expression ectopique de cette 5-phosphatase augmente les événements de phosphorylation à l'état basal comparés à des conditions analogues pour des cellules contrôles. En revanche, alors que suite à l'engagement du TCR par le complexe MHC-peptide indépendamment du co-récepteur CD4 nous observons une augmentation des phosphorylations, l'activation par le complexe MHC-peptide CD4 dépendant semble affectée. Nous avons analysé la contribution des PI(4,5)P2 dans la dynamique membranaire du TCR grâce à la technique svFCS. PIP2 peuvent jouer un rôle essentiel dans la régulation de la dynamique latérale du TCR à la membrane plasmique. Enfin, nous avons observé que l'inhibition par la néomycine (aminoglycoside qui en tant que polycation peut se lier et neutraliser le groupement anionique de PI(4,5)P2), aboutit à des changements similaires dans la dynamique membranaire du TCR et la régulation proximale dans des cellules T primaires murines CD4+. Ensemble, nos données révèlent le rôle régulateur fondamental de PI(4,5)P2 dans la dynamique membranaire du TCR et de CD4, pour le contrôle de l'initiation des voies de signalisation du TCR. / PI(4,5)P2 plays important roles in a large spectrum of membrane-based cellular activities . It is therefore surprising that it is currently not known if PI(4,5)P2 is also involved in the T cell receptor (TCR) signal transduction mechanism. We investigate here the role of PI(4,5)P2 in the regulation of the TCR membrane dynamics and signaling initiation using a combination of biophysical, biochemistry and cell biology approaches. Ectopic expression of the Inp54p, a 5-phophatase that hydrolyzes PI(4,5)P2 into PI(4)P, with a membrane targeting signal specifically decreased by 50% of the PI(4,5)P2 in a CD4+ T cell hybridoma. Interestingly, we observed that this decrease caused modified TCR (and CD4 co-receptor) dynamics in the plasma membrane. The lateral diffusion switched from a regime dominated by dynamic partitioning in the cholesterol- and sphingolipid-dependent nanodomains into one dominated by dynamic partitioning in the actin cytoskeleton-assisted nanodomains. This switch was associated with a change in activation of the TCR and proximal signaling pathways both at the basal level and upon stimulation. Upon pMHC engagement, the CD4-independent activation of the TCR signaling pathways was found significantly augmented while that of CD4-dependent was affected. We further provided evidence for the involvement of PI(4,5)P2 in the Finally, we found that inhibition of interactions between PI(4,5)P2 and endogenous proteins with neomycin resulted in the modified TCR membrane dynamics and proximal signaling in primary murine CD4+ T cells. Altogether, our data reveal that PI(4,5)P2 is crucially involved in the control of the activation of TCR early signaling pathways.

A T helper cell clone, referred to as clone 9, was derived from an allogeneic mixed lymphocyte culture. Clone 9, as well as supernatant factor(s) derived from it, could help the cytotoxic T lymphocyte (CTL) responses of H-2 Db (Db) responder cells to alloantigens, or they could help the CTL responses of non- Db responder cells to Db alloantigens. Clone 9 cells or their factor(s) were active only when added during the first 24 hours of a five-day culture period. Clone 9 or its factor(s) could also synergize with interleukin-2 (IL-2)-containing medium in mounting cytotoxic responses to alloantigens. The helper activity in clone 9 supernatant was not due to IL-2 and it was specifically absorbed out by Db -spleen cells. The characterization of the Db -specific helper factor(ASHF) was facilitated by the isolation of a T hybridoma clone (clone 25), obtained from fusion of clone 9 cells with the T cell lymphoma, BW5147, and a B cell hybridoma that produced an IgM monoclonal antibody (clone 30 IgM) which bound ASHF. An additional monoclonal antibody (F23.1), which recognizes a determinant of the Vβ8 family of the T cell receptor, was also particularly useful for the characterization of ASHF. Analysis with these reagents showed that both clone 30 IgM and F23.1 immunoadsorbents could retain ASHF activity. Preabsorption of the ASHF with Db spleen cells prior to affinity purification over a clone 30 IgM column resulted in the absorption of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band on SDS-PAGE under reducing conditions. Furthermore, affinity purification of ASHF over the F23.1 immunoadsorbent, but not an irrelevant monoclonal antibody (mAb) column, also yielded a 50,000 MW molecule. Taken together, these findings suggest that the 50,000 MW molecule is a component of the ASHF and it is intimately related to the B chain of the T-cell receptor. The mode of action of clone 9 and its products in the induction bfCTL responses was also investigated. It was found that clone 9 and ASHF could help CTL responses by inducing IL-2 production in B6-stimulated cultures. In addition to ASHF, clone 9 cells also produced an additional factor(s) which participated in the induction of CTL responses. This additional factor(s) was referred to as IL-X. IL-X synergized with excess human recombinant IL-2 in the activation of CTL precursors (CTL-P) in the absence of antigenic stimulation. A model which involves the participation of ASHF, T helper cells, IL-2 and IL-X in the induction of CTL responses is proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Antigen-specific CD8 T cells provide an important protective role in response to infection by viruses, intracellular bacteria, and parasites. Pathogen-specific CD8 T cells render this protection by undergoing robust expansion in numbers while gaining the ability to produce cytokines and cytolytic machinery. Following expansion and effector differentiation, pathogen-specific CD8 T cells will contract in number while further differentiating into a highly functional population of memory CD8 T cells. These antigen-experienced cells persist in secondary lymphoid organs and the periphery in order to rapidly respond to repeated infection. Creating optimal CD8 T cell responses to infection can be critical for raising sufficient armament to provide protection against invading intracellular pathogens. Although CD8 T cells have protective value, many vaccine strategies tend to focus on creating productive B cell antibody responses to promote immunological protection. Even though antibody responses can be highly protective, coupling optimal CD8 T cell responses with B cell responses could provide higher orders of protection than either one on their own. Therefore, a deeper understanding of the pathways that ultimately guide the magnitude of CD8 T cell responses is required to achieve this potential therapeutic benefit. My studies evaluate the role of receptor signaling events in guiding the expansion of activated CD8 T cells during primary and secondary responses. Specifically, the first portion of my studies dissect the mechanism by which direct IL-12 and Type I IFN stimulation can substantially bolster primary CD8 T cell responses in vivo. Within this context, I demonstrate that direct IL-12 and Type I IFN signaling increases CD8 T cell accumulation during primary expansion by prolonging division without altering survival. IL-12/Type I IFN signaling promoted prolonged division of activated CD8 T cells by maintaining high-affinity IL-2 receptor subunit (CD25) expression and IL-2 signaling. The other portion of my work was dedicated to understanding the expression and role of the inhibitory FcgR (FcgRIIB) during primary and secondary CD8 T cell responses. FcgRIIB expression could be detected as early as the peak of the CD8 T cell response and marked activated CD8 T cells that were highly sensitive to antigen stimulation. Although FcgRIIB did not appear to play a substantial role in regulating the magnitude of primary CD8 T cell responses, it played an important role in inhibiting the expansion and cytotoxicity of memory CD8 T cells during homologous challenge. Collectively, these data highlight potential avenues that could be exploited by future therapies that aim to achieve appropriately sized CD8 T cell responses.

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy is a new treatment for hematologic malignancies including aggressive B-cell non-Hodgkin’s lymphoma (NHL). Although it has provided an effective treatment option for patients who have few options, CAR T-cell therapy does have many associated toxicities. Prolonged cytopenias are one of the lesser understood toxicities that can affect upwards of 40% of patients. METHODS: In this retrospective study, we reviewed 106 patients who received commercial CAR T-cell therapy between November 2017 and September 2019. Prolonged cytopenias were defined as having absolute neutrophil count (ANC) <1000/mm3, platelets (PLT) <50,000/mm3, and/or hemoglobin (Hgb) <10 g/dL at least once after 30 days post-CAR T-cell infusion. Furthermore, if only one incidence of cytopenia was recorded 30 days post infusion, we required that the patient had to have received either a transfusion or granulocyte-colony stimulating factor (GCSF) after the date of the recorded cytopenic value to be considered a part of the cytopenic cohort. RESULTS: 22 patients met the criteria of having prolonged cytopenias. 64% of the cytopenic cohort had >1 type of prolonged cytopenias. Anemia was the most prevalent affecting 72% of cytopenic patients. The length of time from diagnosis of aggressive B-cell NHL to date of CAR T-cell infusion was found to be positively correlated with an increased risk of developing prolonged cytopenias following CAR T-cell therapy. Additional risk factors associated with an increased risk of delayed cytopenias by univariate analysis included neutropenia on the day of infusion (day 0), a high C-reactive protein (CRP) before lymphodepletion and on day 0, day 0 PLT count, and Hgb before lymphodepletion and on day 0. On multivariate analysis, only high CRP before lymphodepletion was associated with an increased risk of prolonged cytopenias while high ferritin and PLT values on day 0 were associated with not developing prolonged cytopenias. There was no statistical difference between the cytopenic and non-cytopenic cohorts in rates of progression free survival (PFS) and overall survival (OS). Also, no difference was seen in rates or severity of other toxicities between cohorts. 41% of the cytopenic cohort experienced infectious complications post-infusion with one patient dying from their infectious complications. However, there was no association with incidence of infection and prolonged cytopenias when compared to the incidence of infection in the non-cytopenic cohort. CONCLUSIONS: A longer time from diagnosis of aggressive B-cell NHL to time of CAR T-cell infusion was associated with prolonged cytopenias while the number of lines of prior chemotherapy and rate of prior high dose chemotherapy with an autologous stem cell transplant (HD-ASCT) were not associated. It would be valuable to confirm this association and why it is associated since the other two factors were not. We lacked bone marrow biopsies before CAR T-cell infusion and did not have bone marrow biopsies for many patients after CAR T-cell infusion. It would be beneficial to collect data regarding bone marrow biopsies from these time points to highlight any changes that could be related to CAR T-cell therapy. Cytogenetic information of individual patient’s diseases would be worth analyzing to help determine if there are biological factors associated with prolonged cytopenias in response to CAR T-cell therapy. Additional studies should investigate the laboratory values we found to have associations with either cohort to help identify possible predictive values providers could use to identify patients at higher risk of having prolonged cytopenias. There is also a need to see if specific prior chemotherapy regimens increase a patient’s risk of having prolonged cytopenias. Overall, since prolonged cytopenias after CAR T-cell infusions have not been heavily investigated, further investigation is needed to better understand the predictive factors and identify possible mechanisms of prolonged cytopenias seen in CAR T-cell patients.

abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates. / Dissertation/Thesis / Masters Thesis Industrial Engineering 2020

Acute viral infections induce CD8 memory T cells that play an important role in the protection of the host upon re-infection with the same pathogen. These virus epitope-specific memory CD8 T cells develop complex TCR repertoires that are specific for that epitope. As individuals age virus-specific immunity appears to wane. Older people have difficulty controlling infection with common viruses such as influenza A (IAV), a RNA virus which causes recurrent infections due to a high rate of genetic mutation, and Epstein Barr virus (EBV), a DNA virus which persists in B cells for life in the 95% of people that become acutely infected. Many factors may contribute to this waning immunity including changes in virus-specific TCR repertoires. We hypothesize that epitope-specific memory CD8 TCR repertoires to these two common viruses change with increasing age and that CD8 T cell cross-reactivity may be one of the mechanisms mediating these changes. To address this hypothesis in our first study, we compared epitope-specific CD8 memory TRBV repertoires directly ex vivo for these two common human viruses. In cross-sectional and longitudinal studies of EBV seropositive, HLA-A2+, young (18-22 years), middle age (25-59 years), and older (>60 years) donors, we demonstrated that CD8 memory TCR repertoires to three immunodominant epitopes, known to have cross-reactive responses, IAV-M158-66, EBV-BM280-288, and EBV-BR109-117 co-evolve as individuals age. Cross-sectional studies showed that IAV-M1-and both EBV-specific repertoires narrowed their TRBV usage by middle-age. In fact, narrowing of EBV-BM and EBV-BR-specific TRBV usage correlated with increasing age. Although narrowing of IAV-M1-specific TRBV did not directly correlate with increasing age there was clear evidence that the TRBV usage was changing with age. The dominant TRBV19 usage appeared to become bimodal in the older age group and interestingly TRBV30 usage did directly correlate with age. For the EBV epitope-specific responses there was preferential usage of particular TRBV and changes in the hierarchy of TRBV usage in the different age groups. Longitudinal studies tracking 3 donors over 10-15 years (middle age to older) showed that there were changes in the TCR repertoire of IAV-M1, EBV-BM and -BR-specific responses over time. In two of the donors who experienced acute IAV infection there was evidence these repertoire changes may be influenced by TCR cross-reactivity, which is enhanced during acute IAV infection. The results of this first ex vivo study are consistent with our hypothesis. They suggest that virus-specific TCR repertoires change over time as an individual ages leading to narrowing of the repertoire and may co-evolve in the presence of CD8 T cell cross-reactivity. To further test our hypothesis in a second study we compared CD8 memory TRAV and TRBV repertoires to the three immunodominant epitopes IAV-M1, EBV-BM, and EBV-BR in the two extreme age groups, young donors (YSP) (18-22 years) and older donors (OSP) (>60 years) using the same donors as in the first study. Since these three epitopes are known to generate cross-reactive CD8 T cell responses and humans during their lifetime are frequently infected with both viruses at the same time these studies were also designed to more closely examine if TCR cross-reactivity could contribute to changes in TCR repertoire with increasing age. We examined the differences in both TRAV and TRBV in these two age groups by monoclonal antibody (mAb) staining and by deep sequencing and single cell sequencing in tetramer positive sorted cells from short-term cultures. Our initial studies showed that there were strong correlations in TRBV usage between short-term cultured and ex vivo antigen-specific responses; functional differences as well as differences in TRBV usage and diversity as measured by mAb staining particularly for the EBV epitope-specific responses between YSP and OSP donors. The TCR deep sequencing data also showed significant differences in TRBV usage between YSP and OSP. However, there were many more differences in TRAV and TRAJ usage than TRBV between the age groups suggesting that TRAV may play a greater role in evolution of the TCR repertoire. With increasing age, there was a preferential selection or retention of TCR for all three epitopes that have features in their CDR3a and b that increase their ease of generation, such as greater usage of convergent recombinant amino acids, and increase cross-reactive potential, such as multiple glycines. YSP and OSP differed in the patterns of TRAV/AJ and TRBV/BJ pairings and usage of dominant CDR3 motifs in all three epitope responses. Both YSP and OSP had cross-reactive responses between these 3 epitopes which were unique and differed from the cognate responses. Analyses of single cell sequencing data suggested that unique combinations of TRAV and TRBV are occurring, where one chain has features consistent with interaction with antigen one and the other chain had features consistent with interaction with antigen two. Interestingly, both the deep sequencing and single cell data show an increased tendency for the classic IAV-M1 specific clone BV19-IRSS-BJ2.7/AV27-CAGGGSQGNLIF-AJ42 to appear among the cross-reactive clones, suggesting that the dominance of this highly public clone may relate to its cross-reactivity with EBV. These results suggest that although OSP and YSP retain some of the classic TCR features for each epitope the TCR repertoire is gradually changing with age retaining TCR that are cross-reactive between these two common human viruses that we are exposed to frequently, one with recurrent infections and the other a persistent virus which frequently reactivates. These results are highly supportive our hypothesis and their importance in relation to viral immune-pathogenesis and potential novel immunotherapies will be discussed. These studies further emphasize the complexity and potential importance of human virus-specific T cell responses and TCR repertoires as people age and the need for a better understanding of TCR cross-reactivity between different viruses. For instance, at the present time these studies are highly relevant to better understanding the immune-pathogenesis observed during the COVID19 pandemic.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22041号 / 医博第4526号 / 新制||医||1038(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 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 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22122号 / 医科博第107号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 濵﨑 洋子, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22830号 / 医博第4669号 / 新制||医||1047(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 江藤 浩之, 教授 武藤 学, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM