Fig. 7. Tim-3 binding to HMGB-1 controls CD8⁺ T_reg cell suppression independently of Gal-9.

(A) The number of dividing OT-I T cells at D3 was determined when rGal-9 was included in the media of CFSE-labeled naïve Thy1.1⁺CD8⁺ OT-I T cells cultured with SIINFEKL-pulsed BMDCs and when these cells were co-cultured with D7 Ad-Ova liver-primed CD8⁺ T_reg cells in the presence of anti-Gal-9 Ab (n = 3 per group). (B) D7 Ad-Ova primed CD8⁺ T_reg cells were isolated from livers, left alone or pre-coated with anti-Tim-3 Ab, and co-cultured with CFSE-labeled naïve Thy1.1⁺CD8⁺ OT-I T cells at a 1:1 ratio and SIINKFEKL-pulsed BMDCs. Select wells also contained anti-HMGB-1 Ab in the media during culture. D3 CFSE dilution and number of dividing OT-I T cells and OT-I T cell division index were evaluated (one-way ANOVA/Tukey’s post test; n = 6 per group). (C) Supernatants from media obtained from BMDCs maturing in the presence of rIL-4 and rGM-CSF and from D1 BMDC co-culture with OT-I T cells were incubated with rTim-3Fc chimeric protein for 1 hr, followed by immunoprecipitation with protein A/G and immunoblot analysis with anti-HMGB-1 Ab or anti-human Fc (n = 3 per group). (D) 5×10⁵ naïve Thy1.1⁺CD8⁺ OT-I T cells were transferred into D7 Ad-Ova infected mice, which were left untreated or received 300 μg anti-HMGB-1 Ab or anti-Tim-3 Ab IP at D5 and D6 prior to a D7 MCMV-Ova infection. The number of Thy1.1⁺CD8⁺ OT-I T cells was assessed in the livers of infected animals at D14 (one-way ANOVA/Tukey’s post test; n = 3–6 per group). (E) PLP-fixed/OCT-frozen liver cross sections from D7 livers were stained with anti-HMGB-1 (red), anti-CD4 (blue), and anti-CD8 (green). Central vein (CV) is indicated (n = 6 per group). Scale bar, 100 μm. (F) 3D orthogonal view representative of PLP-fixed/OCT-frozen Ig LN cross sections from C57BL/6 mice that received CM-DiI-labeled CD8⁺ T_reg cells from D7 infected liver (red) and CFSE-labeled naïve Thy1.1⁺CD8⁺ OT-I T cells stained with anti-HMGB-1 (blue) and anti-Tim-3 (green) is shown. Scale bar, 5 μm. Quantification of intracellular HMGB-1⁺ perinuclear vesicles within uncoated, anti-PD-L1 Ab pre-coated, and anti-Tim-3 Ab pre-coated CD8⁺ T_reg cells was assessed per cell in images (one-way ANOVA/Tukey’s post test; n = 3 per group). Numbers in the histograms represent percentages. Mean ± s.e.m.; *P < 0.05, **P < 0.01, and ***P < 0.001.