PD-1/SHP-2 inhibits Tc1/Th1 phenotypic responses and the activation of T cells in the tumor microenvironment

Abstract

Immune rejection of tumors is mediated by IFNγ production and T-cell cytolytic activity. These processes are impeded by PD-1, a coinhibitory molecule expressed on T cells that is elevated in tumor-infiltrating lymphocytes (TIL). PD-1 elevation may reflect T-cell exhaustion marked by decreased proliferation, production of type I cytokines, and poor cytolytic activity. Although anti-PD-1 antibodies enhance IFNγ secretion after stimulation of the T-cell receptor (TCR), the mechanistic link between PD-1 and its effects on T-cell help (Tc1/Th1 skewing) remains unclear. In prospectively collected cancer tissues, we found that TIL exhibited dampened Tc1/Th1 skewing and activation compared with peripheral blood lymphocytes (PBL). When PD-1 bound its ligand PD-L1, we observed a marked suppression of critical TCR target genes and Th1 cytokines. Conversely, PD-1 blockade reversed these suppressive effects of PD-1:PD-L1 ligation. We also found that the TCR-regulated phosphatase SHP-2 was expressed higher in TIL than in PBL, tightly correlating with PD-1 expression and negative regulation of TCR target genes. Overall, these results defined a PD-1/SHP-2/STAT1/T-bet signaling axis mediating the suppressive effects of PD-1 on Th1 immunity at tumor sites. Our findings argue that PD-1 or SHP-2 blockade will be sufficient to restore robust Th1 immunity and T-cell activation and thereby reverse immunosuppression in the tumor microenvironment.

Figure 1. CD8⁺ TIL have dampened Tc1 phenotypic responses and activation compared to PBL

p-STAT1, T-bet, p-STAT4 and p-S6 levels in CD8⁺ PBL and TIL from HNSCC patients were analyzed by intracellular flow cytometry. Representative figures (A) and summary data (B) show percentage of p-STAT1 (Y701)+ (n=15), T-bet+ (n=16), p-STAT4 (Y693)+ (n=7) and p-S6 (S235/236)+ (n=13) cells in CD8⁺ TIL compared with paired PBL at baseline. Total PBL and TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell= 10:1) for 48hrs and then p-STAT1, T-bet and p-S6 were tested by flow cytometry. Representative figures (C) and summary data (D) of percentage of p-STAT1 (Y701)+, T-bet+ and p-S6 (S235/236)+ (n=6) cells in CD8⁺ TIL compared with paired PBL post-stimulation are shown. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. CD4⁺ TIL show abortive Th1 differentiation and low activation compared with PBL

p-STAT1, T-bet, p-STAT4 and p-S6 levels in CD4⁺ PBL and TIL from HNSCC patients were analyzed by intracellular flow cytometry. Representative figures (A) and summary data (B) show percentage of p-STAT1 (Y701)+ (n=15), T-bet+ (n=16), p-STAT4 (Y693)+ (n=7) and p-S6 (S235/236)+ (n=13) cells in CD4⁺ TIL compared with paired PBL at baseline. Total PBL and TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell= 10:1) for 48hrs and then p-STAT1, T-bet and p-S6 were tested by flow cytometry. Representative figures (C) and summary data (D) of percentage of p-STAT1 (Y701)+, T-bet+ and p-S6 (S235/236)+ (n=6) cells in CD4⁺ TIL compared with paired PBL post-stimulation are shown. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01. p>0.05 was considered to be not significant (n.s.).

Figure 3. PD-1⁺ TIL co-localize with PD-L1⁺ HNSCC cells in the tumor microenvironment

H&E (left panels) and PD-1/PD-L1 double immunoperosidase staining (right panels) were performed, and representative matching areas in five (n=5) tumors are shown. PD-1-positive lymphocytes are labeled with a red chromogen, and PD-L1 positive HNSCC cells are labeled with a brown chromogen. Images were taken at 200×.

Figure 4. PD-1 ligation with bead-coated PD-L1 suppresses p-STAT1, T-bet, p-S6 and production of Th1 cytokines upon TCR stimulation, while anti-PD-1 blockade could reverse the suppressive effects of PD-1

Total TIL were stimulated with anti-CD3/-CD28/hIgG1 or anti-CD3/-CD28/PD-L1 coated beads (bead: cell=10:1) for 48h in the presence of 100ug/mL hIgG4 or anti-PD-1 (BMS-936558), then p-STAT1, T-bet and p-S6 were analyzed by flow cytometry. Supernatants from each condition were collected and stored at −80°C. Th1 (IFN-γ and IL-2) and Th2 (IL-10) cytokines in the supernatants were determined by Luminex. A) Representative data showing p-STAT1 (Y701), T-bet and p-S6 (S235/236) levels in CD8⁺ TIL under the described conditions. Summary data of frequency of p-STAT1 (Y701)+ (B), T-bet+ (C) and p-S6 (S235/236)+ (D) in CD8⁺ and CD4⁺ TIL with indicated conditions is shown (n=7). E) Summary data of amount of IFN-γ, IL-2 and IL-10 in the supernatants of TIL cultured under indicated conditions is shown. The graphs present the mean ± SEM from 8 HNSCC patients. F) Immunohistochemistry analysis of PD-1 and IFN-γ in serial sections of representative HNSCC tumors. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01. p>0.05 was considered to be not significant (n.s.).

Figure 5. SHP-2 is overexpressed in TIL and correlates with PD-1⁺ expression

Expression level of SHP-2 in TIL and paired PBL from HNSCC patients (n=10) was assessed by flow cytometry. A) Representative figure (upper panel) and summary data (lower panel) showing MFI of SHP-2 in CD8⁺ and CD4⁺ TIL compared with PBL. B) Representative figure (upper panel) and summary data (lower panel) showing MFI of SHP-2 in PD-1⁻ and PD-1⁺ CD8⁺ and CD4⁺ TIL. Statistical significance was determined by Wilcoxon (non-parametric paired) test. **p<0.01. C) Expression levels of PD-1 and SHP-2 (shown by MFI) in tumor infiltrating T cells from 5 HNSCC patients.

Figure 6. SHP-2 activation by fusaruside suppresses p-STAT1/T-bet and production of Th1 cytokines upon TCR stimulation

Total TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell=10:1) or anti-CD3/-CD28/PD-L1 beads plus 100ug/mL anti-PD-1 blockade (BMS-936558) for 48h in the presence of 50uM fusaruside or DMSO. Then p-STAT1 and T-bet were analyzed by flow cytometry. Supernatants were collected and stored at −80°C. Th1 (IFN-γ and IL-2) and Th2 (IL-10) cytokines in the supernatants were determined by Luminex. A) Summary data of frequency of p-STAT1+ and T-bet+ cells in CD8⁺ and CD4⁺ TIL at different conditions is shown (n=6). B) Summary data of amount of IFN-γ (n=8), IL-2 (n=4) and IL-10 (n=8) in the supernatants of TIL cultured under indicated conditions. The graphs present the mean ± SEM from different HNSCC patients. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01. p>0.05 was considered to be not significant (n.s.).