The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-induced proteins. The TCR proximal tyrosine kinase ITK simultaneously influences many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, I examined NFAT, NF-κB and MAP kinase pathways during early activation of individual naïve OT-I CD8+ T cells using peptide-loaded antigen presenting cells. Utilizing both measurement of transcription factor translocation in single T cell nuclei and conventional phospho-flow cytometry, I observed digital activation of Erk-MAPK and NFAT1 at all peptide doses and avidities. However, NF-κB activation showed a graded response to variation in TCR signal strength and was more sensitive to treatment with an ITK inhibitor. Inhibitor-treated cells showed poor induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis revealed genomic regions most sensitive to ITK inhibition are enriched for NF-κB and AP-1 motifs. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, I describe a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.

The T cell receptor (TCR) activation induced signaling cascade is a major driver of T cell effector responses such as cytokine production and actin cytoskeletal rearrangement. Characterizing chemical modulators of this pathway has the benefits of both revealing basic science knowledge about these signaling processes and providing foundation for development of novel therapeutics. Glycerol Monolaurate (GML) is a naturally occurring fatty acid monoester that is found as a monoglyceride in human breast milk and coconut oil. It is widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a wide range of bacteria, fungi, and enveloped viruses. Because of this, GML has been developed as a preventative for menstrual associated Toxic Shock Syndrome, and is being tested to prevent HIV transmission and superficial skin infections. Interestingly, GML suppresses mitogen induced lymphocyte proliferation and inositol triphosphate production, suggesting that GML has immunomodulatory functions. This thesis mechanistically examined how GML affects human primary T cells. Chapter III describes how GML potently altered order and disorder dynamics in the plasma membrane that resulted in reduced membrane-localized clustering of the proteins LAT, PLC-γ, and AKT, events integral for proper TCR signal propagation. Altered membrane signaling events induced selective inhibition of TCR-induced signaling events. Specifically GML reduced the phosphorylation of the regulatory P85 subunit of PI3K, and AKT and abrogated calcium influx. Functionally, GML treatment potently reduced TCR-induced production of the cytokines IL-2, IFN-γ, TNF-α, and IL-10. Chapter V shows that GML causes the mis-localization of the ARPC3 subunit of the Arp2/3 complex that leads to the formation of abnormal filopodia structures, and reduced cellular adhesion. Chapter V shows that human serum albumin binds directly to GML on the 12 carbon acyl chain. This interaction reverses GML induced suppression of TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane, AKT phosphorylation, and cytokine production. These findings establish GML as a T cell suppressive agent in addition to an antimicrobial agent. This observation reveals the potential role of naturally occurring GML in human breast milk in the formation of microbiota and immune tolerance in the infant gastrointestinal tract. It also allows for optimization of the current applications of GML in various commercial products and therapeutic strategies. Finally this information provides the rationale to investigate GML in new remedial avenues as a topical agent to treat excessive inflammation in the skin, and vaginal and gut mucosal regions.

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18463号 / 医博第3918号 / 新制||医||1005(附属図書館) / 31341 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 生田 宏一, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

abstract: Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies. / Dissertation/Thesis / Masters Thesis Biology 2020

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19405号 / 医科博第65号 / 新制||医科||5(附属図書館) / 32430 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 斎藤 通紀, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Vγ9Vδ2 T cells are the main circulating γδ T cells in human adult blood. They are known for their T cell receptor (TCR)-dependent recognition of microbe and endogenous-derived non-peptide pyrophosphate antigens (phosphoantigens, PAg). With the intrinsically biased type 1 immune responses, Vγ9Vδ2 T cells are an important force in the defense of infections and tumors. However, the immune responses of Vγ9Vδ2 T cells in early life infections and in immunotherapies are not clear yet. In this thesis, we explored Vγ9Vδ2 T cell immune responses in both aspects. Vγ9Vδ2 T cells are abundant in human fetal peripheral blood, but compared to their adult counterparts they have a distinct developmental origin, are hyporesponsive towards in vitro phosphoantigen exposure and they do not possess a cytotoxic effector phenotype. In order to obtain insight into the role of Vγ9Vδ2 T cells in the human fetus, we investigated in the first part of this thesis their responses upon in utero infection with the phosphoantigen-producing parasite Toxoplasma gondii (T. gondii). Most congenital infections are caused by viruses, T. gondii is one of the exceptions. The organelle apicoplast present in T. gondii can generate the most potent Vγ9Vδ2 T cell activator. Thus infection in utero with T. gondii makes it a good model to observe Vγ9Vδ2 T cell immune responses in early life. By comparing to age-matched controls, we found that fetal Vγ9Vδ2 T cells were highly expanded in congenital T. gondii infected newborns, and these expanded cells were highly differentiated towards potent cytotoxic effector cells. While the impact of congenital infection on Vγ9Vδ2 T cell expansion and function waned after birth, the Vγ9Vδ2 TCR repertoire of infected infants possessed a clear fetal footprint with public clonotypes, reflecting the Vγ9Vδ2 T cell response in utero. Indeed, verification of the antigen recognition related complementarity-determining region 3 (CDR3) of the TCR for γ and δ chain by high-throughput sequencing revealed the enrichment of three Vδ2 sequences in congenitally-infected infants that are already generated at 8 weeks of gestation. Vγ9Vδ2 T cells possess several characteristics, including MHC-independent recognition of tumor cells and potent killing potential, that make them attractive candidates for cancer immunotherapeutic approaches. In the second part of this thesis we investigated Vγ9Vδ2 T cell responses towards two kinds of hemiparasite plant Viscum album L. (European mistletoe) extract drugs in vitro. Mistletoe therapy is the most used complementary cancer therapy in European countries. Mistletoe extract drugs are considered to benefit for increasing the quality of life of cancer patients and modulate immune cells, but the mechanism of action is not clear. Here, we investigated in-depth the in vitro response of human T cells towards mistletoe extract drugs by analyzing their functional and TCR responses using flow cytometry and high-throughput sequencing respectively. Non-fermented mistletoe-extract drugs (AbnobaViscum), but not their fermented counterparts (Iscador), induced specific expansion of Vγ9Vδ2 T cells among T cells. Furthermore, AbnobaViscum rapidly induced the release of cytotoxic granules and the production of the cytokines IFNγ and TNFα in Vγ9Vδ2 T cells. This stimulation of anti-cancer Vγ9Vδ2 T cells was mediated by the butyrophilin BTN3A, did not depend on the accumulation of endogenous phosphoantigens and involved the same Vγ9Vδ2 TCR repertoire as those of phosphoantigen-reactive Vγ9Vδ2 T cells.In summary, in the first part of this thesis we showed that the human fetus intrinsically possesses a group of Vγ9Vδ2 T cells that are responding to congenital parasite infections that provide potential protective effects to the fetus. In the second part, we provided insight into the in vitro responses of Vγ9Vδ2 T cells towards mistletoe extract drugs, indicating that Vγ9Vδ2 T cells can be an important target in mistletoe therapy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24803号 / 医博第4995号 / 新制||医||1067(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 金子 新, 教授 伊藤 能永, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Mature αβ and γδ T cells arise from a common precursor population in the thymus. Much debate has focused on the mechanism of T cell lineage choice made by these multi-potential precursor cells. It is widely believed that the decision of these precursor cells to commit to the γδ or αβ T cell lineages is regulated primarily by a specific instructive signal relayed through the appropriate T cell receptor. Contrary to this model, we present evidence for a TCR-independent lineage commitment process. Comparison of global gene expression profiles from immature αβ and γδ lineage thymocytes identified Sox13, an HMG-box transcription factor, as a γδ T cell-specific gene. Unlike other HMG-box transcription factors such as TCF1, LEF1 and SOX4, that are critical for proper αβ T cell development, Sox13 expression is restricted to early precursor subsets and γδ lineage cells. Importantly, SOX13 appears to influence the developmental fate of T cell precursors prior to T cell receptor expression on the cell surface. Transgenic over-expression of Sox13 in early T cell precursors strongly inhibits αβ lineage development, in part, by inhibiting precursor cell proliferation and concomitantly, leading to increased cell death among αβ lineage subsets. Steady-state γδ T cell numbers, however, appear unaffected. Strikingly, the DP αβ lineage cells that do develop in Sox13 transgenic mice are imprinted with a γδ- or precursor-like molecular profile, suggesting that SOX13 plays an active role in the lineage fate decision process or maintenance. Sox13-deficient mice, on the other hand, have selectively reduced numbers of γδ thymocytes, indicating that SOX13 is essential for proper development of γδ T cells. We present additional data demonstrating that SOX13 is a canonical WNT signaling antagonist modulating TCF1 activity, raising a strong possibility that WNT signals, and their modulators, are at the nexus of γδ versus αβ T cell lineage commitment.