Figure 5

Host immune-derived IL-12 limits ICOS and PD1 expression by CD4+ donor T cells. Radiation chimeras (B6 or BA and IL-12p40 KO) were conditioned with 9 Gy irradiation and transplanted with 5 × 10⁶ BA.B10 TCD BM cells and 3 × 10⁵ MACS-purified B10.BR T-cells. Recipient splenocytes were harvested 10 days post transplant and analyzed via flow-cytometry. Donor T cells were gated on using congenic markers (CD45.1, CD45.2, CD90.1) as well as CD3, and were then further differentiated using CD4 and CD8. Representative histograms are shown for ICOS expression in CD4+ (A) and CD8+ (B) donor T-cells and PD1 expression in CD4+ (E) and CD8 + (F) donor T-cells (Isotype – fine dotted line, WT – black solid line, IL-12p40 KO – gray dashed line). ICOS and PD-1 expression by CD4+ and CD8+ donor T cells was analyzed; Percentages of donor T-cells expression ICOS (C) and PD1 (G) are shown as well as the median fluorescence intensity (MFI) of the signal for ICOS (D) and PD1 (H). **, p < 0.01 comparing percentages of ICOS + CD4+ donor T-cells in WT vs. IL-12p40 KO; *, p < 0.05 comparing MFI of ICOS signal in CD4+ donor T-cells in WT vs. IL-12p40 KO; *, p < 0.05 comparing percentages of PD1+ CD4+ donor T-cells in WT vs. IL-12p40 KO. Data shown for ICOS are representative from one of two independent experiments with 4 mice per group using pre-transplant irradiation of 9 Gy or 10 Gy (9 Gy is shown). Data shown for PD1 are combined from two independent experiments of 4 mice per group.