Extended Data Fig. 6: BFAR does not affect TCR and JAK1 downstream signaling activation.
From: Age-related decline in CD8+ tissue resident memory T cells compromises antitumor immunity
a, Immunoblot analysis of phosphorylated and total ZAP70, LAT, AKT, FOXO1, ERK and p65 in whole-cell lysates of WT and BFAR-deficient splenic naive CD8+ T cells that were stimulated with anti-CD3 plus anti-CD28 (α-CD3/CD28; 1 μg/ml) for the indicated time points. b, Flow cytometric analysis of the phosphorylated STAT1 in mouse splenic CD27+CD8+ T cells or CD27−CD8+ T cells that were cocultured with MB49 tumor cell supernatant in the presence of plate-bound anti-CD3 plus anti-CD28 (α-CD3/28; 1 μg/ml) for 3 days. c-f, Immunoblot analysis of phosphorylated and total STAT3, STAT5 and STAT1 in whole-cell lysates of WT and BFAR-deficient mouse splenic naive CD8+ T cells that were stimulated with IL-6 (100 ng/ml) or IL-2 (50 ng/ml) or IFN-α (100 ng/ml) or IFN-β (100 ng/ml) for the indicated time points. g, STAT1 luciferase activity in HEK293T cells transfected with luciferase reporter, together with the indicated plasmids. h,i, Flow cytometric analysis and the corresponding statistical analysis of the frequencies of IFNγ-producing and Granzyme B-producing CD8+ T cells in WT and BFAR-deficient mouse splenic CD8+ T cells that were treated with DMSO or NF-κB inhibitor (Pyrrolidinedithiocarbamate ammonium) or JAK2 inhibitor (XL019) and were stimulated by plate-bound anti-CD3 plus anti-CD28 (α-CD3/28; 1 μg/ml) for 3 days. j, Immunoblot analysis of phosphorylated and total Smad2/3 in whole cell lysates of WT and BFAR-deficient mouse splenic CD8+ T cells that were stimulated with α-CD3/28 (3 μg ml−1) overnight and then stimulated with TGFβ1 (5 ng ml−1) for the indicated time points. k,l, Tumor growth (k) and Flow cytometric statistical analysis (l) of the frequencies and absolute number of CD8+CD69+CD103+ TRM cells and IFNγ-producing CD8+ T cells of Rag1−/− mice that were adoptively transferred with Tgfbr1+/+ and Tgfbr1K268R mouse splenic CD8+ T cell and then s.c. inoculated with MB49 tumor cells at the same day (n = 5 mice/group). Bar graphs are presented as mean ± s.e.m. A two-tailed Student’s t-test was performed for comparisons. The data are representative of two (k,l) or three (a-j) independent experiments.
