Figure 6. Downregulation of IFNAR1 on cytotoxic T lymphocytes undermines their survival within tumor microenvironment.

(A) Immunoblot analysis of Bcl-xl, cleaved caspase 3 levels and β-actin (loading control) in splenocytes from WT and SA OT-1 mice activated with SIINFEKL peptide (0.5µg/mL for 48 hr) and then cultured for indicated times.

(B) Viability of activated CD3⁺CD8⁺ cells in the presence of media supplemented or not with either IL2 (100 U/ml) or anti-CD25 antibody (100 µg/ml) as indicated was determined by flow cytometry analysis after indicated times. Mean ±SD (triplicates per each mouse spleen – average from 3 mice) are shown. Asterisks denote statistical significance (p<0.05) between WT and SA, between WT and WT treated with IL2 and between SA and SA treated with anti-CD25 antibody.

(C) Flow cytometry analysis of the fraction of viable OT-1 WT (CD45.1) or SA (CD45.2) CTL in the MC38OVA tumors 72 hr after injection (1:1 ratio) intravenously (i.v.) or directly into the tumors (i.t.) of Rag1^−/− mice bearing MC38OVA tumors.

(D) Quantitation of the experiments shown in 6C (Mean % of viable cells from tumors from 3–5 mice). Similar results were obtained at least in 2 independent experiments.

(E) Quantitation of flow cytometry analysis of the fraction of viable FAP-CAR EGFP⁺ WT (CD45.1) or EGFP⁺ SA (CD45.2) CTL in the MC38 tumors 72 hr after injection (1:1 ratio) intravenously (i.v.) or directly into the tumors (i.t.) of WT MC38 tumor bearing mice. Data are shown as Mean % of viable cells (n=5).

See also Figures S6 and S7.