Single-Cell Analysis of Transcriptome and TCR Sequencing Reveals Immune Cell Atlas and Functional Heterogeneity of T Cell Repertoire in Murine Heart Transplantation
To illustrate the  immune cell atlas and functional heterogeneity of T cell repertoire in murine heart transplantation,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. CD45+ cells were sorted from recipients' heart grafts and spleens on Day 6 after transplantation. Each group (allograft, allo-spleen, and iso-spleen) was pooled from three mice. 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).