Fig 2: Stress increases CD3+ T cell exhaustion. Flow cytometric analysis of the proportions of a tumor TNF-α+ CD3+, b IFN-γ+ CD3+, c Ki67+ CD3+, d CD107a+ CD3+, e PD-1+ CD3+, f LAG3+ CD3+ and g TIM-3+ CD3+ at the tumor endpoint between the control (Con) and acute restraint (AR) mice (n = 4). Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01 by an unpaired Student’s t-test

Fig 3: GRAP2 involves CD8+ T cell exhaustion in NSCLC. a UMAP plot in NSCLC_GSE149655 scRNA-seq dataset from TISCH2 database. The CD8Tex mainly consisted of cells from cluster 4 and cluster 13. GRAP2 expresses highly in both clusters CD8Tex. b UMAP plot in NSCLC_EMTAB6149 scRNA-seq dataset from TISCH2 database. The CD8Tex mainly consisted of cells from cluster 0, cluster 8 and cluster 12. GRAP2 expresses highly in all three cluster CD8Tex. (c-f) Correlation analysis of gene expression levels between GRAP2 and PDCD1 c, LAG3 d, HAVCR2 e and TIGIT f of NSCLC patients through TCGA database. g Hazard ratio (HR) of impact of GRAP2 expression levels in CD8Tex on the survival of different cancer patients. The high expression of GRAP2 in CD8Tex significantly increases the risk of shortened survival in lung adenocarcinoma patients (p < 0.05). h Summary of the mechanism of stress promoting CD8+ T cell exhaustion in NSCLC

Fig 4: Stress induces CD8+ T cell exhaustion. Flow cytometric analysis of the proportions of a tumor Ki67+ CD8+, b PD-1+ CD8+ and c TIM-3+ CD8+ at the tumor endpoint between the control (Con) and acute restraint (AR) mice (n = 4). d–e Immunofluorescence analysis of the expression levels of PD-1, TIM-3 and CD8a in tumor tissues (30x). Data are represented as the mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by an unpaired Student’s t-test

Fig 5: Corticosterone (COR) promotes CD8+ T cell exhaustion and NSCLC growth. a The LLC tumor growth curves and the endpoint tumor weights were displayed of control (Con) and corticosterone treatment (COR) mice (n = 5). (b-d) Flow cytometric analysis of the proportions of PD-1+ and LAG3+, granzyme B+ and IFN-γ+ of CD8+ T cells of tumor tissues from Con and COR mice (n = 4). (e-f) The LLC tumor growth curves, the endpoint tumor weights and tumor sizes were showed of acute restraint (AR+PBS) and acute restraint+RU486 treatment (AR+RU486) mice (n = 6). g CCK-8 detection of CD8+ T cell viability after 24 hours of treatment with different concentrations of COR. Flow cytometric analysis of the proportions of h IFN-γ+, i granzyme B+, j LAG3+, k PD-1+ and l TIM-3+ of CD8+ T cells among DMSO, COR and COR+RU486 treatment groups (n = 3). Data are represented as the mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by an unpaired Student’s T-test or one-way ANOVA