*Purpose: Evaluating kidney allografts using single cell RNA sequencing (scRNAseq) can provide a comprehensive qualitative and quantitative gene expression data on thousands of individual cells. These data can be helpful in identifying novel therapeutic targets and tailoring anti-rejection immunosuppressive strategies for individual transplant recipients. In the present, we analyzed rejecting and tolerized murine kidney allografts employing scRNAseq technique.

*Methods: Bilaterally nephrectomized C57BL/6 mice were transplanted with BALB/c kidneys. Rejecting allografts were retrieved from untreated recipients. Non-rejecting allografts were retrieved from tolerized recipients intravenously infused with donor apoptotic splenocytes (treated with a chemical cross-linker called ethylenecarbodiimide) on day -7 and 1. On day 15 post-transplant, the allografts were retrieved, single cell preparation was subjected to 10x Chromium Platform for synthesis and construction of cDNA libraries. Libraries were sequenced on Illumina NextGen Sequencer and analyzed using SEURAT.

*Results: Untreated transplant recipients showed significantly elevated levels of serum creatinine demonstrating kidney allograft injury and rejection in comparison to tolerized recipients. scRNAseq analysis of ~20,000 cells from both rejecting and tolerized kidney allografts showed 30 cell clusters including parenchymal and non-parenchymal immune cells. Among these, we were able to identify 4 unique myeloid cell populations. Interestingly, one of these myeloid cell populations was highly enriched in rejecting kidney allografts. This was a macrophage population (Apoe, C1qa, C1qb, C1qc, Lyz2 and Ccl8) and expressed significantly high levels of major histocompatibility complex II (H2-Eb, H2-Aa and H2-Ab), T cell chemokine (Cxcl9) and allograft inflammatory factor (Aif-1). Differential gene expression analysis revealed significantly elevated expression of several ribosomal genes in this population from rejecting kidney allografts.

*Conclusions: Our data show that among various myeloid populations, a highly immunogenic macrophage population with elevated expression of ribosomal genes is associated with acute kidney allograft rejection. Further analysis of this population will elucidate its role in acute rejection.

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