Fig 2: SHR-1701 inhibited TGF-ß signaling pathway, activated PI3K/Akt/Erk signaling pathway and rescued the anti-tumor function of peripheral CD8+ T cells isolated from lung cancer patients with impaired lymphocyte recovery. (A-B) Flow analysis of Ki-67 expression (A) and IFN-? secretion (B) of peripheral CD8+ T cells isolated from patients with satisfied/poor lymphocyte recovery after pre-treatment of SHR-1701 or anti-PD-1 Abs. (C-D) Western blotting analysis of the protein levels including TGF-ßR, pSmad2, and total Smad2 in peripheral CD8+(C) or CD4+ (D) T cells from patients with poor/satisfied lymphocyte recovery. (E) Western blotting analysis of the activation of pAkt and pErk after treatment of anti-PD-1 blocking Abs/SHR-1701/anti-PD-1 blocking Abs + TGF-ßR inhibitors/PBS in peripheral CD8+ T cells sorted from patients. (F) Western blotting analysis of the expression of Foxp3 after treatment of SHR-1701/TGF-ßR inhibitors/PBS in peripheral CD4+ T cells sorted from patients. (G) SHR-1701 reverses the tumor's response to PD-1 blockade immunotherapy, from resistant to sensitive, by regulating the balance between CD8+ T cells and Tregs. A-B: n = 6 for each group. C-D: n = 5 for each group. E, F: The examination was repeated in 3 patients with poor recovery and 3 patients with satisfied recovery. One-way ANOVA statistical tests were adopted for A and B. *, P < 0.050; **, P < 0.010. Abbreviations: n.s., not significant; PD-1, programmed cell death-protein 1; TGF-ßR, transforming growth factor-beta receptor; TGF-ß, transforming growth factor-beta; IFN-?, interferon-?

Fig 3: Treatment response of 6 different groups (control, DDP, anti-PD-1, DDP + anti-PD-1, DDP + anti-PD-1 + anti-TGF-ß, DDP + SHR-1701) in an orthotopic mouse model. (A) CMT167 cells were transplanted into the lungs of C57BL/6 mice. Follow-up treatment (injected with DDP, anti-PD-1, anti-TGF-ß, SHR-1701, or isotype control antibody) was then performed in six groups as shown. (B) Images and quantification of in vivo bioluminescence imaging of the six groups (control, DDP, anti-PD-1, DDP + anti-PD-1, DDP + anti-PD-1 + anti-TGF-ß, DDP + SHR-1701) at the indicated time points. The P-value represents a comparison of bioluminescence intensity on the 15th day. (C) Effect of different treatments (control, DDP, anti-PD-1, DDP + anti-PD-1, DDP + anti-PD-1 + anti-TGF-ß, DDP + SHR-1701) on survival of CMT167 orthotopic tumor-bearing mice. (D-E) Representative images of IHC staining for tumor-infiltrating Foxp3+ Treg cells (D) and CD8+ T cells (E). Scale bars: 100 µm, upper panel; 40 µm, lower panel. (F) Orthotopic tumors were analyzed by flow cytometry for GITR (left panel), IFN-? (middle panel), and CD69 (right panel) expression on CD8+ T cells after stimulation with CD3 and CD28 (both 1 mg/mL) for 48 h. (G) IFN-? (left panel) and CD69 (right panel) expression on CD8+ T cells in spleens were analyzed. Bar graphs represent the means ± SEM for each group. B-G: n = 5 for each group. One-way ANOVA statistical tests were adopted for more than two groups (B, D, E, F, and G). Log-rank tests were used in C. *, P < 0.050; **, P < 0.010; ***, P < 0.001. Abbreviations: n.s., not significant; PD-1, programmed cell death-protein 1; DDP, cisplatin; TGF-ß, transforming growth factor-beta; Treg, regulatory T cell; IFN-?, interferon-?; SEM, standard error of the mean; BL, bioluminescence

Fig 4: The PD-1 signal pathway participates in ECM development. C57BL/6 male mice and Pdcd1-/- C57BL/6 male mice (6–8 weeks old) were infected i. p. with 1 × 107 pRBCs. Survival was monitored daily (A). Pdcd1-/- mice showed exacerbated symptoms of ECM, and their survival time was shorter than that of WT mice (log-rank test, n = 10, P = 0.0046). Splenic CD8+ T cells were isolated from infected Pdcd1-/- and WT mice 5 dpi, co-incubation with sub-optimal concentrations of anti-CD3 and anti-CD28 mAbs (0.03 µg/ml) after CFSE staining. After 4 days of culture. Results of flow cytometry (B), divided index (C) and percentage of divided cell (C) analyzed by Flowjo software showed higher proliferation ability of CD8+ T cells in Pdcd1-/- mice than in WT mice. Proliferation assay used by CCK-8 showed the same results (E). (F) qPCR showed that the expression levels of PDL1 and ICAM-1 were highly increased in primary BMECs stimulated by IFN-? and pRBCs. (G) The ratio of splenic PD-1+ CD8+ T cells to total CD8+ T cells was monitored daily in ECM mice. Results are from three independent experiments. Data are from three independent experiments.* indicates that differences are significant (unpaired t-test, n = 10, 0.01 < P < 0.05).

Fig 5: The activation of peripheral, splenic and tumor-infiltrated CD8+ T cells accessed by GSEA and flow cytometry from DDP-treated and control mice. (A) Percentage of Ki-67+CD8+ T cells in the peripheral blood are shown for the control (Ctrl) and DDP-treated mice. (B-C) Changes in the percentage of Ki-67+CD8+ T cells after CD3 and CD28 (both 1 mg/mL) co-stimulation in tumors (B) and spleens (C) harvested from DDP-treated and control (Ctrl) mice. (D-E) Proportions of PD-1+CD8+ T cells and PD-1+CD4+Foxp3+ T cells in tumors harvested from DDP-treated and control (Ctrl) mice. (F) The ratio of PD-1+ CD8+ T cells to PD-1+ Treg cells in TME of DDP-treated and control (Ctrl) mice. (G) GSEA plot showing NESs for DDP-inhibited activation status, as well as the function of CD8+ T cells using RNA-seq data from tumor infiltrated CD8+ T cells. For all panels, bar graphs represent the means ± SEM for each group. Statistically significant differences are indicated as the results of unpaired Student's t-test (A, D, E, and F) or one-way ANOVA (B and C). A-F: n = 5; G: n = 2. **, P < 0.010; ***, P < 0.001. Abbreviations: n.s., not significant; PBMC, peripheral blood mononuclear cell; DDP, cisplatin; SEM, standard error of the mean; FSC, forward scatter; SSC, side scatter; GSEA, gene set enrichment analysis; ES, enrichment score; NES, normalized enrichment score; PD-1, programmed cell death-protein 1