Fig 6.

In vivo CD8⁺ T_reg cell suppression of SC-primed OT-I cells entering draining lymph nodes is regulated by Tim-3. C57BL/6 mice were SC or IV infected with 2.5 × 10⁷ IU of Ad-Ova, and bulk CD8⁺ T cells were isolated from day 7 SC spleens and IV livers. D7 CD8⁺ T_reg cells from IV-infected livers were left alone or precoated with anti-PD-L1 Ab or anti-Tim-3 Ab. CFSE-labeled naïve Thy1.1⁺CD8⁺ OT-I T cells were adoptively transferred alone or in combination with CD8⁺ T cells from infected spleens and livers at a 1:2 ratio, respectively, into naïve C57BL/6 mice. Shortly thereafter, these recipient mice were SC infected. At day 3 postinfection, spleens, C LN, and (A and B) Ig LN were harvested, and (C) the number of dividing OT-I T cells was determined in each organ. The division index and percent suppression of OT-I T cells in the Ig LN is displayed (one-way ANOVA/Tukey's posttest; n = 3-6 per group). (D) CM-DiI-labeled CD8⁺ T_reg cells from day 7 IV-infected liver (red) and CFSE-labeled naïve Thy1.1⁺CD8⁺ OT-I T cells (green) were transferred in a similar experiment, and Ig LNs were harvested at day 2 SC postinfection. PLP-fixed/OCT-frozen Ig LN cross-sections were stained with anti-CD4 (blue, upper panel), anti-CD31 (blue, lower panel), anti-B220 (magenta, upper panel), and CD105 (magenta, lower panel; n = 3 per group). Scale bar, 100 μm. Numbers in histograms represent percentages (mean ± standard error of the mean). *P < 0.05.