*Purpose: We previously reported the novel activity of alloprimed CD8⁺ T cells that suppress post-transplant alloantibody production. The purpose of the current study was to investigate the expression and role of CXCR5 on antibody-suppressor CD8⁺ T cell (CD8⁺ T_Ab-supp cell) function.

*Methods: C57BL/6 mice were transplanted (Tx) with FVB/N hepatocytes (Hc). Alloprimed CD8⁺ T cells were retrieved on day 7 from hepatocyte Tx recipients and CD8⁺ T cell subsets were sorted into CXCR5⁺CXCR3^– (9.3±1.0%) or CXCR3⁺CXCR5^– (8.4±0.3%) subsets by flow cytometry (both subsets were CD44⁺IFN-γ⁺).

*Results: Flow-sorted (CXCR5⁺CXCR3^– and CXCR3⁺CXCR5^‑) alloprimed CD8⁺ T cell subsets were analyzed for in vitro cytotoxicity and capacity to inhibit in vivo alloantibody production following adoptive transfer (AT) into C57BL/6 or high alloantibody-producing CD8 KO hepatocyte Tx recipients. Alloprimed CXCR5⁺CXCR3^–CD8⁺ T cells mediated in vitro cytotoxicity of alloprimed IgG⁺ (antibody-producing) B cells (12.7±1.8%, n=10, p<0.0001) while CXCR3⁺CXCR5^–CD8⁺ T cells did not (1.9±1.9%, n=10, p=ns). Only flow-sorted alloprimed CXCR5⁺CXCR3^–CD8⁺ T cells (not flow-sorted alloprimed CXCR3⁺CXCR5^–CD8⁺ T cells) suppressed alloantibody production after AT into C57BL/6 HcTx recipients (titer=60±10 compared to 120±20 in untreated control HcTx mice, N≥4 in each group, p=0.02). The lower alloantibody titer following AT of CXCR5⁺CXCR3^–CD8⁺ T cells into C57BL/6 recipients also correlated with a 2-fold reduction in the quantity of germinal center B cells (p=0.001) and IL-4⁺IL-21⁺ CD4⁺ T_FH cells (p=0.003) compared to control recipients without AT. Adoptive transfer of CXCR5⁺CXCR3^–CD8⁺ T cells also suppressed alloantibody production in high alloantibody-producing CD8 KO HcTx recipients (titer= 90±40 vs.1,300±500 in control mice, N≥5 in each group, p<0.0001). CD8 KO hepatocyte recipients that received AT of CXCR5⁺CXCR3^–CD8⁺ T cells had prolonged allograft survival (MST=day 32; N=5, p=0.002) compared to those that received AT of CXCR3⁺CXCR5^–CD8⁺ T cells (MST= day 14, N=5) or those with no AT (MST= day 14, N=7). Adoptive transfer of CD8⁺ T cells retrieved from wild-type (but not CXCR5 KO) mice into CD8 KO HcTx recipients suppresses alloantibody production (titer= 300±70 vs. 1,250±250 in control mice p=0.004).

*Conclusions: These data support the conclusion that expression of CXCR5 by antigen-primed CD8⁺ T cells is critical for the function of CD8⁺ T_Ab-supp cells.

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