IFNγR is required for trafficking of Tconvs to GVHD target organs and CXCR3 expression. (A) In vivo BLI was performed to specifically track CBRluc-transduced Tconvs (2 or 4 × 10⁶ cells) after allo-HSCT. BLI images of 1 dissected representative mouse from WT (left) and IFNγR^−/− (right) T cell recipients at day 31 after allo-HSCT (top panels). Spleens and GI tracts were separated from the body cavities. White arrows indicate LNs. Ratio of signal intensities (photons/s/cm²/sr) from spleen and GI tract and the rest of body were compared in the bottom panel. Data represent the pool of 2 independent experiments. (B) WT Tconvs express IFNγR (CD119) and CXCR3 before (top panels) and after (bottom panels) activation by anti-CD3/CD28 antibody-coated beads. The expressions of IFNγR (CD119) and CXCR3 are correlated in WT Tconv. (C) IFNγR^−/− Tconv (both CD4⁺ and CD8⁺ T cells; CD4− T cells are CD8⁺ T cells right panels) express CXCR3 significantly less than WT Tconvs (left panels) after activation (bottom panels). Mean and SD of activated WT Tconvs are as follows (n = 8). CD8⁺CXCR3⁺: 42.7% ± 5.5%, CD8⁺CXCR3⁻: 5.2% ± 1.3%, CD4⁺CXCR3⁺: 41.2% ± 5.0%, CD4⁺CXCR3⁻: 11.0% ± 4.7%. Mean and standard deviation of activated IFNγR^−/− Tconv are as follows (n = 8). CD8⁺CXCR3⁺: 24.1% ± 8.2%, CD8⁺CXCR3⁻: 21.0% ± 3.3%, CD4⁺CXCR3⁺: 5.5% ± 1.7%, and CD4⁺CXCR3⁻: 49.3% ± 4.8%. (D) IFNγ^−/− Tconv (both CD4⁺ and CD8⁺ T cells; CD4⁻ T cells are CD8⁺ T cells) express CXCR3 significantly less than WT Tconvs (C) after activation (bottom panels) but up-regulate IFNγR expression similar to activated WT Tconvs (left panels). Mean and standard deviation of activated IFNγ^−/− Tconv are as follows (n = 9). CD8+CXCR3⁺: 23.4% ± 5.3%, CD8⁺CXCR3⁻: 24.6% ± 4.9%, CD4⁺CXCR3⁺: 5.6% ± 2.6%, and CD4⁺CXCR3⁻: 46.4% ± 6.9%. (E) IFNγR^−/− Tconvs were retrovirally transduced with either the CBRluc-GFP gene (IFNγR^−/−) or the CXCR3-ires-CBRluc-GFP gene (CXCR3⁺ IFNγR^−/−). WT Tconvs were also transduced with the CBRluc-GFP gene. Transduced cells were purified using the Reflection cell sorter (iCyt; GFP+ WT Tconvs and GFP+ IFNγR^−/− Tconvs) or AutoMACS (CXCR3⁺ IFNγR^−/− Tconvs; all cells > 96% pure) and transplanted (2 × 10⁶) into recipient mice along with TCD BM (5 × 10⁶). Shown is percent survival after allo-HSCT (5 × 10⁶ TCD BM + 2 × 10⁶ Tconv). Data represent a pool of 3 independent experiments.