Protective Renalase-deficiency in beta cells shapes immune metabolism and function in autoimmune diabetes
Type 1 diabetes (T1D) is caused by the immune-mediated loss of pancreatic beta cells that produce insulin. The latest advances in stem cell (SC)-beta cell differentiation methods have made a cell replacement therapy for T1D feasible. However, recurring autoimmunity would rapidly destroy transplanted SC-beta cells. A promising strategy to overcome immune rejection is to genetically engineer SC-beta cells. We previously identified Renalase (Rnls) as a novel target for beta cell protection. Here we show that Rnls deletion endows beta cells with the capacity to modulate the metabolism and function of immune cells within the local graft microenvironment. We used flow cytometry and single-cell RNA sequencing to characterize beta cell graft-infiltrating immune cells in a mouse model for T1D. Loss of Rnls within transplanted beta cells affected both the composition and the transcriptional profile of infiltrating immune cells in favor of an anti-inflammatory phenotype with decreased antigen presenting capacity. We propose that changes in beta cell metabolism mediate local immune regulation and that this feature could be exploited for therapeutic goals. Overall design: WT and Rnls mutant NIT-1 cells (1.5 x 10e7 each) were injected subcutaneously (s.c.) into opposite flanks of immunodeficient NOD.scid mice. Four days later, autoreactive splenocytes from recently diabetic NOD mice were isolated and red blood cell were lysed using 5 ml of ACK lysing buffer (cat # A1049201; Thermo Fisher Scientific) for 4 min at RT. Lysis was stopped using 5 ml of PBS/ 10 % FCS followed by two washing steps using PBS only. Splenocytes were filtered two times in total through a strainer with 70 µm pore size to obtain a single cell solution for counting.  Splenocytes (1 x 10e7) were injected intravenously (i.v.) into NIT-1 cell-bearing NOD.scid mice to transfer autoimmune beta cell killing for 10 or 17 days. NIT-1 beta cell grafts were isolated and scaled on an analytical balance at indicated time points. Grafts were cut into small pieces and digested using HEPES buffered RMPI 1640 (cat # R4130-10L; Sigma-Aldrich) supplemented with 1mg/ml Collagenase D ( cat # 11088858001; Sigma-Aldrich), 20 µg/ml DNase I (cat # EN0521; Thermo Fisher Scientific), 2% FCS and 50 µg/ml Lipopolysaccharide neutralizing agent Polymyxin B sulfate (cat # 1405-20-5; Sigma-Aldrich) for 45 min at 37°C while shaking by maximum speed on a heating block. Digested grafts were further disaggregated and filtered through a strainer with 70 µm pore size two times to obtain a single cell solution. Cells were stained with anti-CD45 and PI in combination with one of four TotalSeq anti-mouse Hashtag antibodies (A0301, A0302, A0303 or A0304) (cat # 15580-1/3/5/7, clone M1/42 and 30-F11; Biolegend) to distinguish immune cells derived from individual NIT-1 beta cell grafts. Single cell RNA sequencing were then performed.