*Purpose: We reported the discovery of a novel subset of CXCR5⁺IFNγ⁺CD8⁺ T cells that suppresses antibody production (CD8⁺ T_Ab-supp cells). Alloprimed CD8⁺ T_Ab-supp cells significantly inhibit posttransplant alloantibody production and prolong allograft survival. The requirements for CD8⁺ T_Ab-supp cell development are not understood. We hypothesized that CD4⁺ T cells and IFN-γ are required for the development of CD8⁺ T_Ab-supp cells following antigen stimulation.

*Methods: Wild-type (WT; C57BL/6) and IFN-γ KO knockout (KO) mice were alloprimed (FVB/N, H-2^q, allogeneic lysate). Cohorts of mice were CD4-depleted and/or adoptively transferred (AT; day 0) with Violet CellTrace stained naïve CD8⁺ T cells (5 million) from GFP Tg mice. On day 7 post-stimulation, splenic CD8⁺ T cells were analyzed for the development of the CXCR5⁺CD8⁺ T cell phenotype, activation and in vivo proliferation (violet stain dilution). Alloprimed CXCR5⁺CD8⁺ T cells from WT and IFN-γ KO recipients were also evaluated for in vitro killing of alloprimed IgG⁺ B cells.

*Results: The proportion of CXCR5⁺CD8⁺ T cells increased significantly following allostimulation and was maximal on day 7 in WT control recipients (11.1±1.0%) compared to proportions on day 2 (3.7±1.0%), and day 5 after allostimulation (6.8±1.0%; p<0.01 for all time points) and in naïve mice (1.4±1.0%). CXCR5⁺CD8⁺ T cells express an activated phenotype (CD44⁺CD62L^–) that peaks on day 7 (49.6±3.8%) compared to baseline before allopriming (2.3±0.7%; p<0.0001). To determine the role for host IFN-γ signaling and CD4⁺ T cells for the development of CXCR5⁺CD8⁺ T cells, we tracked the expression of CXCR5, activation markers, and in vivo proliferation. We found that the development of CXCR5⁺CD8⁺ T cells was significantly impaired in IFN-γ KO (6.3±1.0% of CD8⁺ T cells), CD4-depleted WT recipients (5.8±0.6%), and CD4-depleted IFN-γ KO (4.1±0.5%), compared to WT recipients (11.1±1.7%; p<0.02 for all), suggesting that host CD4⁺ T cells and host IFN-γ are critical for the development of alloprimed CXCR5⁺CD8⁺ T cells. Expression of activation marker CD44 and proliferation were similar for all experimental groups. Similar results for the development of endogenous CXCR5⁺CD8⁺ T cells were observed. IgG⁺ B cells were harvested from CD8 KO alloprimed hosts (day 7) and co-cultured with CXCR5⁺CD8⁺ T cells from different groups of alloprimed hosts to test for CD8-mediated in vitro cytotoxicity. WT CXCR5⁺CD8⁺ T cells exhibited significantly greater cytotoxicity (15.6±1.2%) compared to CXCR5⁺CD8⁺ T cells from CD4-deficient hosts (11.3±0.7%) or IFN-γ KO hosts (12.1±0.3%; p<0.04 for both).

*Conclusions: Host IFN-γ expression and CD4⁺ T cells are critical for optimal development and cytotoxic effector function of antibody-suppressor CXCR5⁺CD8⁺ T cells.

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