Figure 1. CXCR5⁺CD8⁺ T cells mediate in vitro cytotoxicity of alloprimed self IgG⁺ B cells.

C57BL/6 (wild-type, WT; H-2^b) mice were transplanted with FVB/N (H-2^q) hepatocytes. On day 7 post transplant, splenic CD8⁺ T cells were retrieved and purified. A) Flow cytometric analysis of purified CD8⁺ T cells shows that CXCR5⁺CXCR3⁻ (9.3±1.0%) and CXCR3⁺CXCR5⁻ (8.4±0.3%) CD8⁺ T cell subsets are detected (CD8⁺ T cells pooled from 10–15 mice) for cell sorting; data for n= 3 sorts are shown in the bar graph. B) In an in vitro cytotoxicity assay, flow-sorted alloprimed CXCR5⁺CXCR3⁻ or CXCR3⁺CXCR5⁻ CD8⁺ T cell populations and B cell targets were co-cultured at a 10:1 ratio for 4 hours and analyzed for cytotoxicity (propidium iodide (PI) uptake). Naïve CD8⁺ T cells and unsorted alloprimed CD8⁺ T cells were utilized as negative and positive controls (effector cells), respectively. Significant cytotoxicity of alloprimed, self IgG1⁺ B cells was observed in co-cultures with CXCR5⁺CXCR3⁻CD8⁺ T cells (12.7±1.8%, n=10) or with unsorted alloprimed CD8⁺ T cells (9.1±3.8, p<0.0001 for both signified by “*”; n=10) in comparison to co-cultures with naïve CD8⁺ T cells (0.9±0.4%, n=9) but no significant cytotoxicity was detected in co-cultures with CXCR3⁺CXCR5⁻CD8⁺ T cells (1.9±1.9%, p=ns, n=10). Significant cytotoxicity of allogeneic B cells was observed in co-cultures with CXCR3⁺CXCR5⁻CD8⁺ T cells (15.6±8.8%; n=6) or with unsorted alloprimed CD8⁺ T cells (12.0±5.5%; n=6, p<0.002 for both signified by “**”), but not with CXCR5⁺CXCR3⁻CD8⁺ T cells (1.8±1.8%; n=6, p=ns) in comparison to control co-cultures with naïve CD8⁺ T cells (0.8±0.7%; n=5). No significant cytotoxicity was detected against third-party (3^rd) party primed “self” IgG1⁺ B cells (H-2^b targets) or 3^rd party B cells (H-2^k targets) in any co-cultures. Error bars indicate standard error from triplicate experiments.