Fig. 3: ALK5 loss lowers the TCR activation threshold and enhances effector function of CD8+ T cells.
From: TGFβ suppresses CD8+ T cell expression of CXCR3 and tumor trafficking
For a–e: On day 14 following MC38 implantation into WT (n = 6) and ALK5ΔCD8 (n = 5) mice, tumors were harvested, digested into a single cell suspension, and analyzed by flow cytometry. Each symbol is the value of an individual animal, measure of center is the mean. a Quantification of CD8+ T cells in lymph nodes and tumors by frequency (left). **p = 0.00765, ****p = 0.000013. Absolute cell number in the lymph node or per mm2 in the tumor (right). *p = 0.043 (LN) and 0.0172 (tumor). b The frequency of p15E tetramer positive CD8+ T cells determined by FACS analysis in lymph nodes and tumors of WT and ALK5ΔCD8 mice. c The frequency of effector memory (CD44hi/CD62L−), central memory (CD44hi/CD62L+), naïve (CD44lo/CD62L+) or effector (CD44lo/CD62L−) CD8+ T cells. *p = 0.01, **p = 0.0092. d The frequency of Ly6C+CD62L− CD8+ T cells in MC38 tumors or tumor draining inguinal lymph nodes from C57BL/6 or ALK5ΔCD8 mice. ****p = 0.000046. e The median fluorescent intensity of granzyme B (GnzB) expression gated on CD44loCD62L−CD8+ T cells in MC38 tumors, presented as mean +/− SD. **p = 0.00316 (LN) and 0.0011 (tumor). f Specific cytotoxic killing of tumor cells was measured with the Incucyte live cell imaging system in co-cultures of MCA205-OVA or Panc02 tumor cells with CFSE-labeled C57BL/6 WT and ALK5ΔCD8 CD8+ T cells isolated from spleens of ΔactA-OVA vaccinated mice. Shown is the cumulative % dead tumor cells as determined by cytotox red reagent uptake of total tumor cells seeded following 24 h of co-culture presented as mean +/− SD. N = 3/condition. *p = 0.0344. g CD8+ T cells purified from spleens of naïve WT and ALK5ΔCD8 mice and activated with increasing amounts of plate bound anti-CD3 and 10μg/ml anti-CD28. Presented as mean +/− SD. N = 3/condition (i) Proliferation index measured by CFSE dye dilution, ****p = 0.000015 and 0.000043. (ii) IFN-γ secretion, **p = 0.00658 (10−1), 0.00924 (100), ***p = 0.00016, and (iii) TNF-α secretion **p = 0.00828 (100), 0.00182 (101), ***p = 0.00096 (10−1). Each graph is reflective of two independent experiments. P-values derived using unpaired, two-tailed t-test, except in g, which utilized 1-way ANOVA with multiple comparisons.
