*Purpose: We have reported that the novel subset of antibody-suppressor CD8⁺ T cells (termed CD8⁺ T_Ab-supp cells) require the expression of CXCR5. In addition, alloprimed CXCR5⁺CD8⁺ T_Ab-supp cells inhibit production of alloantibody (but do not reject allografts) while CXCR3⁺CD8⁺ T cells reject allogeneic transplants (but do not inhibit antibody). Recent report demonstrates that there are many subsets of CXCR5⁺CD8⁺ T cells with different effector function including anti-viral, anti-tumor, anti-autoimmune, or antibody enhancers. By investigating CD8⁺ T_Ab-supp cell protein and transcript expression, we aim to determine a profile that might distinguish this novel CD8⁺ T_Ab-supp cell subset.

*Methods: C57BL/6 (wild-type; WT; H-2^b) were transplanted with FVB/N (H-2^q) hepatocytes. On day 7 (peak activation of CD8⁺ T_Ab-supp cells), splenic CXCR5⁺CD8⁺ T cells were evaluated by RNA-seq and flow cytometry. Alloprimed CXCR5⁺CD8⁺ T cells were compared to naïve CD8⁺ T cells as well as alloprimed CXCR3⁺CD8⁺ T cells. Fluorescence minus one controls were used to determine background staining (flow cytometry).

*Results: RNA-seq analysis show that 1670 transcripts were upregulated or downregulated when comparing between flow sorted alloprimed (CD62L^–CD44⁺) CXCR5⁺CD8⁺ T cells and naïve (CD62L⁺CD44^–) CD8⁺ T cells. Transcripts of note include Bcl-6 (3.3-fold upregulated), CXCR3 (8.4-fold downregulated), and S1pr3 (140-fold upregulated). The latter suggests an important role for S1PR3 on CXCR5⁺CD8⁺ T_Ab-supp cell trafficking into the circulation, perhaps from the germinal center. Flow cytometry analysis suggests that CD8⁺ T_Ab-supp cells are short-lived effectors cells based on the expression of CD44, KLRG1, and CD122 (not CD62L or CD127). An expansive flow analysis panel (not shown, and review of the literature) comparing differences in transcript and protein expression between alloprimed, antibody-suppressor CXCR5⁺CD8⁺ T_Ab-supp cells and other reported CXCR5⁺CD8⁺ T cell subsets indicates that the absence of PD-1, IL-10 and FoxP3 expression distinguishes CD8⁺ T_Ab-supp cells from all reported CXCR5⁺CD8⁺ T cell subsets (including CD8⁺ T_reg and CD8⁺ T follicular regulatory cells).

*Conclusions: The phenotype of novel CXCR5⁺CD8⁺ T_Ab-supp cells suggests cytotoxic CD8⁺ T cells with short-lived effector function.

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