Impaired cell mediated immunity is the clinical hallmark of HIV infection yet the manner in which CD8 T-cells are disabled is not yet fully understood. IL-7 signalling is essential for normal CD8 T-cell development and function. Our lab has previously shown decreased expression of the IL-7 receptor a-chain (CD127) on circulating CD8 T-cells in HIV+ patients is mediated by the HIV Tat protein which results in poor CD8 T-cell function. Soluble Tat protein is secreted by infected CD4 T-cells and taken up by neighbouring uninfected CD8 T-cells through endocytosis. Once in the cytoplasm, Tat translocates to the inner leaflet of the cell membrane where it binds directly to the cytoplasmic tail of CD127 inducing receptor aggregation, internalization, and degradation by the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is able to reduce IL-7 signaling and impair CD8 T-cell proliferation and function. To determine which domain(s) in the cytoplamic tail of CD127 are required for interaction with Tat, a series of plasmids encoding for CD127 deletion mutants were successfully created. These series of mutant CD127 coding sequences were transfected into a eukaryotic expression system, the Jurakt cell line, where CD127 mutants were successfully expressed. Before determine which region on CD127 is required for Tat binding, an optimized Ni-NTA column system was used to successfully isolate histidine-tagged HIV-1 Tat at a high yield and purity from E. coli. This HIV Tat protein was used to treat the lysates of the Jurakt cells transfected with the panel of CD127 mutants. CD127 was then immunoprecipitated, followed by Western analysis of the immune complexes to detect Tat protein. Tat was immunoprecipitated with all CD127 mutants suggests neither tyrosine 449, box 1, the acidic region, serine region nor C-tail are specifically required for Tat binding to CD127.

Autoimmunity develops over an extended period of time as the result of an amalgamation of genetic, environmental, and immunologic events. Though the precise etiological factors leading to most autoimmune disease are awaiting consensus, a common thread of the autoimmune paradigm is the inappropriate activation of tissue-specific immune cells by one or more autoantigen, which begins the destruction of the tissue. To prohibit immunopathology and fine-tune the immune responses in healthy individuals, the stimulatory activities of effector/memory T (Teffs) cells must be counteracted by the suppressive mechanisms of regulatory T cells (Tregs). Thus, the potential to modulate the ratio between Teff and Tregs in autoimmune patients has been widely investigated with high hopes to permanently cure certain autoimmune diseases such as type 1 diabetes militus (T1D). Autoantigen therapies, which attempt to induce Tregs to suppress pathogenic effector cells in an autoantigen-specific manner, have shown efficacy in preventing T1D in mice, but have largely failed in clinical trials. One approach to improve the effectiveness of islet autoantigen vaccinations is to combine them with an additional modulator of the T cell response which favors a regulatory phenotype. In the work presented here, we asked whether the addition of anti-interleukin-7 receptor-alpha (anti-IL-7Rα) monoclonal antibodies (mAbs) to islet autoantigen immunizations would modulate the T cell response and prevent T1D in non-obese diabetic (NOD) mice. It was found that anti-IL-7Rα mAbs reduced the absolute numbers of islet antigen-specific T cells when immunized with islet peptide in conjunction with the commonly used vaccine adjuvant alum. Such treatments were also observed to increase nonspecific IL-2, IFN-𝛾, and IL-10 cytokine production, resulting in no improvement of T1D onset prevention. In another approach, we generated a conjugate vaccine by conjugating islet autoantigens to the immunogenic carrier protein, Keyhole Limpet Hemocyanin (KLH). We found that islet antigen-KLH (Ag-KLH) vaccination resulted in significant expansion of the desirable antigen-specific Tregs. Further, Ag-KLH immunization successfully delayed, and in some cases entirely prevented, T1D onset in NOD mice. Indicating that KLH-conjugated vaccine may represent a promising approach for future autoantigen therapies against autoimmunity. Interestingly, administration of anti-IL-7Rα mAbs did not improve these outcomes. To the contrary, we again observed excessive nonspecific cytokine production induced by IL-7Rα blockade that inhibited the beneficial effects of Ag-KLH vaccination. Taken together, we concluded that the addition of anti-IL-7Rα mAbs did not improve the efficacy of autoantigen vaccinations to prevent T1D. Significant work still remains to better characterize and isolate the beneficial effects of anti-IL-7Rα mAbs to treat autoimmunity.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20533号 / 生博第375号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 杉田 昌彦, 教授 米原 伸, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM