Single-Cell Analysis of Transcriptome and TCR Sequencing Reveals Functional Heterogeneity of T Cell in Murine Heart Transplantation

To illustrate the functional heterogeneity of T cells in the heart allograft rejection,we established the murine heterotopic heart transplantation model and isolated CD45 positive cells from cardiac grafts and spleens for single cell transcriptome and TCR sequencing. Overall design: 8-10 weeks male C57BL/6 (H-2b) and BALB/c(H-2d) were purchased from Charles River (Beijing, China). All mice experiments were performed in a specific pathogen-free facility according to the guidelines of the animal care and use committee of Huazhong University of Science and Technology. We established the murine heterotopic heart transplantation model as previously described . Allograft group: BALB/c (H2d) hearts were transplanted into fully MHC-mismatched C57BL/6 (H2b) recipients. Isograft group: C57BL/6 (H2b) hearts were transplanted into MHC-matched C57BL/6 (H2b) recipients. Cardiac allografts normally arrest at day 7 after transplantation because of acute transplantation rejection. Therefore, we harvested spleens and cardiac allografts of allograft group recipients at day 6 before cardiac allografts arrest. At the same time, spleens and cardiac isografts of isograft group recipients were harvested. CD45+ cells were respectively isolated from cardiac allografts, allograft group recipients' spleen, cardiac isografts and isograft group recipients' spleen after digestion, centrifugation and flow sorting. In brief, cardiac grafts were cut into pieces and digested with 1 mg/mL of collagenase B (Roche 11088815001) in HEPES buffer for 45 min at 37°C. After digestion, cell suspensions were collected and filtered with 70-µm cell strainers (BIOFIL), Percoll (Beijing Solarbio Science & Technology Co., Ltd.) was used to purify mononuclear cells through density centrifugation with 600g 25min RT not-brake. Splenocytes were prepared through grinding, erythrocyte lysis, washing and centrifugation (600g, 3min). CD45+ live cells were then sorted by FACSAria flow cytometer after Cardiac graft infiltrating cells and splenocytes stained with CD45-APC (Biolegend 103112) and Zombie GreenTM Fixable Viability Kit (Biolegend 423112).