Distinct phenotype of antibody suppressor CXCR5(+)CD8(+) T cells

Abstract The novel CXCR5+CD8+ T cell subset has been shown to inhibit alloantibody production (termed CD8+ TAb-supp cells) but do not reject allografts. In contrast, CXCR3+CD8+ T cells reject allogeneic transplants (but do not inhibit antibody). Recent literature indicates there are many CXCR5+CD8+ T cell subsets with different effector functions (anti-viral, anti-tumor, anti-autoimmune, or antibody enhancement). By investigating CD8+ TAb-supp cell by RNA-seq and flow cytometry, we aimed to determine a profile that distinguishes this novel subset. To investigate this, C57BL/6 (H-2b) were transplanted with FVB/N (H-2q) hepatocytes and CD8+ T cell subsets were analyzed on day 7. Alloprimed CXCR5+CD8+ T cells were compared to naïve CD8+ T cells and alloprimed CXCR3+CD8+ T cells. Fluorescence minus one controls were used to determine background staining (flow cytometry). RNA-seq analysis show that 1670 transcripts were upregulated or downregulated when comparing between flow-sorted alloprimed (CD62L−CD44+) CXCR5+CD8+ T cells or 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). Flow cytometry analysis suggests that CD8+ TAb-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 comparing differences in transcript and protein expression between alloprimed CXCR5+CD8+ TAb-supp cells and other reported CXCR5+CD8+ T cell subsets indicates that the absence of PD-1, IL-10, and FoxP3 expression distinguishes CD8+ TAb-supp cells from all reported CXCR5+CD8+ T cell subsets (including CD8+ T follicular regulatory cells).