Figure 6. OVA-primed OT-I CXCR5⁺CD8⁺ T cells mediate suppression of anti-OVA antibody production accompanied by reduced quantity of Germinal Center B cells and IL-21⁺CD4⁺ T_FH cells.

A) Naïve OT-I CD8⁺ T cells were adoptively transferred (AT) into OVA-primed CD8 KO recipients. 7 days later, OT-I CD8⁺ T cells were isolated and analyzed by flow cytometry for expression of CXCR5 and CXCR3. A representative flow plot shows OVA-primed OT-I CD8⁺ T cells include CXCR5⁺CXCR3⁻ (11.7±1.0%) and CXCR3⁺CXCR5⁻ (17.2±0.7%) OT-I CD8⁺ T cell subsets. B) Unsorted or flow-sorted CXCR5⁺CXCR3⁻ or CXCR3⁺CXCR5⁻ OVA-primed OT-I CD8⁺ T cells (1×10⁶) were adoptively transferred into OVA-primed CD8 KO mice (day 5 post mOVA lysate stimulation). The amount of anti-OVA Ab produced in mice that received AT of CXCR5⁺CXCR3⁻ OT-I CD8⁺ T cells (5.8±0.6 μg/mL; n=6, p<0.0001 signified by “*”) or unsorted OT-I CD8⁺ T cells (10.7±0.4 μg/mL, n=4, p=0.008 signified by “**”) was significantly less than the amount produced in CD8 KO recipients without AT of CD8⁺ T cells (13.4±0.3 μg/mL, n=6). AT of CXCR3⁺CXCR5⁻ OT-I CD8⁺ T cells did not suppress anti-OVA antibody production (12.2±0.4 μg/mL; n=5, p=ns). C) The quantity of recipient spleen germinal center (GC) B cells (GL-7⁺B220⁺) and IL-21⁺CD4⁺ T_FH cells (IL-21⁺CXCR5⁺PD-1⁺CD4⁺) was analyzed in OVA-primed CD8 KO mice (day 14 following mOVA lysate stimulation). The quantity of GC B cells in mice that received AT of CXCR5⁺CXCR3⁻ OT-I CD8⁺ T cells (6,200±500 per million splenocytes; n=6, p<0.0001 signified by “*”) or with unsorted OT-I CD8⁺ T cells (8,900±700; n=4, p=0.008 signified by “**”) was significantly less than the quantity in CD8 KO recipients without AT (11,800±700 per million splenocytes; n=6). In contrast, AT of CXCR3⁺CXCR5⁻ OT-I CD8⁺ T cells was not associated with reduction in the quantity of GC B cells (10,600±400 per million splenocytes; n=6, p=ns). D) The quantity of IL-21⁺ CD4⁺ T_FH cells in mice that received AT of CXCR5⁺CXCR3⁻ OT-I CD8⁺ T cells (5,000±400 per million splenocytes; n=6, p<0.0001 signified by “*”) or unsorted OT-I CD8⁺ T cells (6,100±300, n=4, p=0.001 signified by “**”) was significantly reduced compared to the quantity in CD8 KO recipients without AT (8,800±400 per million splenocytes; n=6). In contrast, AT with CXCR3⁺CXCR5⁻ OT-I CD8⁺ T cells was not associated with a reduction in the quantity of IL-21⁺ CD4⁺ T_FH cells (7,200±400 per million splenocytes; n=6, p=ns). Error bars indicate standard error from duplicate experiments.