Fig. 4: PTENα promotes cell resistance to T cell cytotoxicity.

a–e Wild-type and Ptenα^−/− mice were i.v. infected with 1 × 10⁶ PFU LCMV-Cl13. On day 7 post-infection, lungs were harvested from the mice. a The lungs were subjected to hematoxylin and eosin (H&E) staining. The image is representative of four mice with similar results. Scale bar: 200 μm. b TUNEL staining of lungs from wild-type and Ptenα^−/− mice on day 7 post-infection. The image is representative of four mice with similar results. The red arrow indicates TUNEL positive cells. c The tissue interstitial fluids were isolated from the lungs and subjected to MS analysis. The differential proteins were analyzed using GSEA with GO gene sets. ES is an abbreviation of enrichment score, and NES represents normalized enrichment score. KO refers to Ptenα^−/−. Each sample is a mixture of fluids from three mice. d and e The interstitial fluids were isolated from the lungs, and used for immunoblot analysis with indicated antibodies (d), and ATP content assay (e) (n = 4 mixed samples from three mice, mean ± SD, ***P = 0.0002). For immunoblot analysis, equal amounts of loading proteins were showed by the Ponceau S staining. The relative ratios of Hsp70, Hsp90, and HMGB1 to Ponceau S-stained total proteins were indicated. UT, untreatment. f–h In vitro cytotoxicity assay of B16 cells. Mock or PTENα expressing Pten^−/− B16 cells pulsed with GP_33-41 peptide were used as target cells. Effector cells and target cells were incubated at indicated ratios. Effector-uninfection refers to T cells from uninfected mice and acts as a negative control. f 20 h post-incubation, B16 cells were washed to remove lymphocytes and counted using CCK-8 (n = 3 cell cultures, mean ± SD, ***P (2) = 0.0006, ***P (25) = 0.0003, ****P < 0.0001). g Culture medium was collected 20 h post-incubation and centrifuged to remove cells and debris. The supernatant was subjected to ATP content assay (n = 3 cell cultures, mean ± SD, ns, not significant, **P = 0.0078, ****P < 0.0001). Untreatment control refers to pulsed B16 cells without effector incubation. h 12 h post-incubation, B16 cells were harvested and stained with DCFDA, using for flow cytometry analysis. Cells with higher intensity of FSC and SSC were gated and considered as B16 cells. Mean fluorescence intensity (MFI) of DCFDA was used for statistical analysis (n = 3 cell cultures, mean ± SD, *P = 0.0255). Untreatment control refers to pulsed B16 cells without effector incubation. Statistical significance was assessed by two-tailed unpaired Student’s t test (h) or one (e) or two (f and g)-way ANOVA followed by Tukey’s multiple comparisons test. Data are representative of two (a, b, d–h) independent experiments. Source data are provided as a Source Data file. See also Supplementary Fig. 7.