Fig. 4: ALK5^ΔCD8 T cells upregulate CXCR3 and preferentially migrate to the tumor.

a Experimental schema for adoptive co-transfer of congenic C57BL/6 WT and ALK5^ΔCD8 CD8⁺ T cells into MC38 tumor-bearing mice. b The frequency of transferred CD45.1 WT and CD45.2 ALK5^ΔCD8 CD8⁺ T cells (n = 9 recipients) analyzed in the spleen, lymph node, and tumor; each symbol represents one mouse, measure of center = mean, CD45.1 WT transferred T cells (black circles) and CD45.2 ALK5^ΔCD8 transferred T cells (white boxes). Shown is a compilation of two independent experiments. ****p < 0.000001. c The percent of CFSE^low cells gated on CD45.1 WT or CD45.2 ALK5^ΔCD8 transferred T cells in spleen, lymph node, and MC38 tumors. *p = 0.0155 (spleen), 0.04393 (lymph node). N = 5 recipients, reflective of two independent experiments. d In vitro proliferation of naïve splenocyte-derived CD8⁺ T cells from C57BL/6 or ALK5^ΔCD8 mice stimulated with plate-bound αCD3/αCD28 +/− TGFβ1 (1 or 10 ng/ml) for 68 h; n = 3 biologic replicates/group, displayed as mean + SD. One representative experiment is shown reflective of 3 independent experiments. P-values as follows: WT vs. ALK5^ΔCD8 = 0.000016 (1 ng/ml), 0.00086 (10 ng/ml); WT vs. WT = 0.0002 (0 vs. 1 ng/ml), 0.0201 (0 vs. 10 ng/ml). e The percent of CXCR3⁺ transferred CD8⁺ T cells assessed by FACS analysis 7 days post transfer in tumor bearing mice. Symbols represent one mouse, measure of center=mean, N = 5 recipients, reflective of two independent experiments. *p = 0.011, ***p = 0.0002, ****p = 0.000083. f Frequency of CXCR3⁺CD8⁺ T cells in vitro following treatment as in d. n = 3 biologic replicates/group. One experiment is shown reflective of 3 independent experiments, displayed as mean +/− SD. For WT vs. ALK5^ΔCD8, ****p < 0.000001 (unstimulated); ***p = 0.000173 (0 ng/ml TGFβ); ***p = 0.000175 (1 ng/ml); ***p = 0.00042 (10 ng/ml). For WT vs. WT, ***p = 0.0003 (unstimulated vs. 0 ng/mL); **p = 0.0023 (0 vs. 1 ng/mL). g In vitro migration of WT and ALK5^ΔCD8 CD8⁺ T cells to CXCL10 in the bottom chamber, displayed as mean +/−SD, n = 3 biologic replicates/group, reflective of 2 independent experiments. WT vs. ALK5^ΔCD8: p = 0.00067 (10 ng^/mL); p = 0.0489 (50 ng/mL). For panels d, f, and g, p-values were derived using 1-way ANOVA with multiple comparisons, all other panels used unpaired, two-tailed t-test.