TCR affinity determines S1P1, CD69, and CXCR3 expression levels on OT-I T cells. (A and B) mRNA levels of S1pr1 and CD69 expression in e670-labeled GFP+ OT-I T cells 60 h after transfer into recipient C57BL/6 mice that contained N4-, Q4-, or T4-pulsed DCs. Divided OT-I T cells (e670low) were sorted by flow cytometry and analyzed for relative mRNA levels of S1pr1 by RT-qPCR as in Fig. 2 A (A) or stained for CD69 expression on divided and undivided OT-I T cells (B; mean ± SD). (A) Data are representative of two independent experiments with RNA from LNs pooled from two to three mice per condition. (B) Data are pooled from three independent experiments with total of five to six mice per condition. (C–E) OT-I T cells were analyzed for CXCR3 expression by flow cytometry 72 and 96 h after transfer in C57BL/6 mice that contained N4-, Q4-, or T4-pulsed DCs. (C) Representative flow cytometry plots of CXCR3 expression. Data are representative of at least five independent experiments. (D) Percentage of CXCR3+ OT-I T cells. Bars indicate the mean. (E) CXCR3 MFI normalized to CXCR3+ N4-primed OT-I T cells. (D and E) Data are pooled from five to six independent experiments with a total of 10–12 mice. (F) In vitro OT-I T cell migration to CXCL9 and CXCL10 at 72 and 96 h after transfer (mean ± SEM). Data are pooled from two independent experiments with total of four mice per condition. Statistical significance was analyzed by Kruskal-Wallis tests with Dunn’s posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
