*Purpose: Antibody-mediated rejection (AMR) contributes to both early and late kidney allograft loss. Unfortunately, conventional immunosuppressants do not effectively control development of humoral alloimmunity in a substantial proportion of recipients, underscoring the importance of animal models that enable investigation of AMR immunobiology. CCR5 KO kidney transplant (KTx) recipients have proven to be a useful model to investigate immune mechanisms impacting AMR. CCR5 KO (H-2^b) recipients of A/J (H-2^a) KTx produce alloantibody titer that is 10-fold higher than in wild-type (WT) mice. A proposed mechanism for this heightened humoral response is impaired CD4⁺ T regulatory cell trafficking to the allograft and secondary lymphoid organs. We previously reported the discovery of a novel subset of CXCR5⁺IFN-γ⁺CD8⁺ T cells capable of killing alloprimed IgG⁺ B cells and mediating significant suppression of in vivo alloantibody production in an allogeneic hepatocellular Tx model. We hypothesized that high alloantibody producing CCR5 KO KTx recipients have reduced quantities of this novel regulatory CD8⁺ T cell subset.

*Methods: We investigated the quantity of CXCR5⁺IFN-γ⁺CD8⁺ T cells in peripheral blood and splenocytes of CCR5 KO compared to WT (C57BL/6, H-2^b) hepatocyte and KTx recipients using flow cytometry.

*Results: CCR5 KO hepatocyte recipients had significantly fewer CXCR5⁺IFN-γ⁺CD8⁺ T cells in the spleen compared to WT recipients (8.75±2.73 x10³ vs. 19.1±3.07 x10³ cells; p=0.03). As expected, we found that CCR5 KO KTx recipients produce much higher quantity of alloantibody by day 14 postTx than WT recipients (5,800±700 vs. 1,200±100; p=0.0003) and have significantly reduced quantity of peripheral CXCR5⁺IFN-γ⁺CD8⁺ T cells (650±400 vs. 11,500±450 cells per million PBMCs; p=0.002). CXCR5 expression suggests that this peripheral CD8⁺ T cell subset traffics through lymphoid depots. RNA-seq analysis of mouse CXCR5⁺IFN-γ⁺CD8⁺ T cells show upregulated S1PR3 expression (140-fold vs. naïve CD8⁺ T cells), encoding a protein that facilitates temporary egress from lymphatics. Preliminary studies using GFP-fluorescent, alloprimed CXCR5⁺IFN-γ⁺CD8⁺ T cells injected into CCR5 KO KTx mice show that these cells are detected in the peripheral blood, spleen, and lymph nodes, but not the allograft. CCR5 KO KTx recipients that receive adoptive transfer of CXCR5⁺IFN-γ⁺CD8⁺ T cells (n=9) on day 5 postTx produce significantly less alloantibody than WT recipients (n=7) assayed on day 14 postTx (1,200±200 vs. 5,800±700; p<0.0001).

*Conclusions: Deficiency of these antibody-suppressor CD8⁺ T cells are likely an important immune mechanism driving the high alloantibody production in CCR5 KO KTx recipients. This subset exerts antibody suppressor function in lymphoid depots rather than in the kidney allograft itself.

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