*Purpose: Antibody-mediated rejection (AMR) is a significant contributor to transplant rejection. Our prior work identified a novel subset of CXCR5⁺CD8⁺ T cells (CD8⁺ T_Ab-supp cells) that suppresses alloantibody production in part by mediating cytotoxic of killing IgG⁺ alloantibody-producing B cells. The purpose of these studies was to investigate which B cell subsets are susceptible to CD8⁺ T_Ab-supp cell-mediated cytotoxicity.

*Methods: C57BL/6 (wild-type, WT) and CD8 KO (H-2^b) mice were stimulated with FVB/N (H-2^q) allogeneic lysate (2mg bulk protein). Alloprimed CXCR5⁺CD8⁺ T cells were isolated from splenocytes of alloprimed C57BL/6 mice on day 7 following allo-lysate stimulation using CD8 StemCell isolation and flow sorting. Various B cell subsets (IgG⁺ StemCell isolation kits and/or flow sort of splenocytes), macrophages (peritoneal lavage)), and dendritic cells (density gradient centrifugation and culture plate non-adherence of splenocytes) were isolated from alloprimed CD8 KO on day 7 following allo-lysate stimulation. Alloprimed CD8 KO mice are known to be high alloantibody producers from our prior studies. Cells were tested as targets in co-cultures to evaluate their susceptibility to CXCR5⁺CD8⁺ T -mediated in vitro cytotoxicity. Cell purity was greater than 90% for all subsets.

*Results: CXCR5⁺CD8⁺ T cells exhibited significantly higher in vitro cytotoxicity towards alloprimed GC B cells (IgG⁺GL-7⁺Fas⁺; 11.4±1.0%; n=21) than towards other antibody producing B cell subsets, including alloprimed extrafollicular plasmablasts (IgG⁺CD138⁺; 0.9±0.3%; n=13) and plasma cells (IgG^–CD138⁺; 1.6±0.5%; n=10; p<0.0001 for both). In addition, CXCR5⁺CD8⁺ T cells did not kill alloprimed dendritic cells (MHCII⁺CD11c⁺; 1.5±0.7; n=6) or macrophages (F4/80⁺; 1.7±0.4; n=6) in co-culture. CXCR5⁺CD8-mediated cytotoxic killing of GC B cells is allospecific as alloprimed CXCR5⁺CD8⁺ T cells mediated in vitro cytotoxicity towards alloprimed GC B cells (11.4±1.0%; n=21) whereas third party primed CXCR5⁺CD8⁺ T cells did not (1.1±0.2; n=6, p<0.0001). No significant cytotoxicity was observed in control co-cultures using naïve CD8⁺ T cells (1.2±0.3%; n=15) or alloprimed CXCR5-negative CD8⁺ T cells (0.6±0.3%; n=15).

*Conclusions: Antibody-suppressor CXCR5⁺CD8⁺ T cells preferentially mediate cytotoxic killing of allogeneic germinal center B cells.

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