Quantitative Assessment of TCR Repertoire and Allo-Reactivity of Human Acute Gvhd Tissue-Infiltrating T Cells

Abstract Introduction: Due to difficulty in isolating T cells from human tissues, the diversity of allo-reactive T cells that are infiltrating human acute GVHD (aGVHD) tissues remains largely uninvestigated. Here, we report the analysis of comprehensive T cell repertoire and allo-reactivity of human aGVHD tissue-infiltrating T cells. Patients and Methods: Skin (n = 7), stomach (n = 3) and colon (n = 2) biopsy samples and blood samples (n = 8) were obtained from eight post-transplant patients when the patients were supposed to develop aGVHD and prior to the fist-line treatment of aGVHD. Total RNA was extracted from tissue and blood samples, and T cell receptor (TCR)-β gene deep sequencing was performed (~100000 reads for each sample). To estimate the TCR-β repertoire diversity, inverse Simpson's diversity index (1/Ds) were calculated. T cells were isolated from tissues and cloned by limiting dilution. This study was approved by the ethics committees of Nagoya University. Results: In the skin of patient #1, the relative abundance of the most and second most frequent clonotypes were 74.9% and 22.8%, respectively, suggesting that only two T cell clones accounted for approximately 98% of GVHD skin-infiltrating T cells. Two T cell clones, termed clones A and B, were isolated from the skin of patient #1. Both clones lysed patient peripheral blood mononuclear cells, but failed to lyse donor cells. Thus, clones A and B were allo-reactive cytotoxic T lymphocytes (CTLs). The IFN-γ production of clone B was significantly increased when stimulated by COS cells transfected with HLA-DRB1*14:54 cDNA, suggesting that the clone B recognized the mismatched patient's HLA-DRB1*14:54 molecule. To confirm the relative abundance of clones A and B in his skin, the TCR-β usage and CDR3 were determined. Surprisingly, clones A and B were the most and second most frequent skin-infiltrating T cell clones. Taken together, these data suggested that a limited allo-reactive CTL clones were involved in GVHD development of patient #1. In all samples, the mean 1/Ds of skin, stomach, colon and all tissues were 9.4 (range, 1.2-21.3), 7.5 (1.9-14.9), 3.3 (1.0-5.5) and 7.9 (1.0-21.3), respectively, while that of all blood samples was 247 (18.8-888.7) (P 10% frequency) clonotypes in a certain tissue were detected in other tissue(s) of the same patient at an extremely low frequency ( 10% frequency) clonotypes in the skin of a certain patient were detected in those of other patients with an extremely low frequency ( Conclusion: In humans, a limited allo-reactive CTL clones, which differ from tissue to tissue and from patient to patient, may induce aGVHD. The degree of skewing of tissue-infiltrating T cell repertoire may be predictive of GVHD outcome. Download : Download high-res image (62KB) Download : Download full-size image Disclosures Kiyoi: Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; MSD K.K.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding.