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

  • Jing Li1 and
  • Robert L Ferris2
Journal for ImmunoTherapy of Cancer20142(Suppl 3):P221

https://doi.org/10.1186/2051-1426-2-S3-P221

Published: 6 November 2014

Keywords

Tumor MicroenvironmentTumor Infiltrate LymphocyteCytolytic ActivityCoated BeadCell Exhaustion
Rejection of tumor cells by a robust cellular immune response relies on production of type 1 cytokines (such as IFN-γ) and cytolytic activity of T cells. Programmed Death 1 (PD-1), a co-inhibitory receptor proposed to represent T cell dysfunction, is highly expressed on tumor infiltrating lymphocytes (TIL) [1], and may reflect T cell exhaustion marked by decreased proliferation, production of type 1 cytokines and poor cytolytic activity [2]. T-bet, a T-box transcription factor which can be activated by phosphorylated signal transducers and activators of transcription 1 (p-STAT1), plays an important role in Tc1/Th1 skewing. Although anti-PD-1 antibodies enhance IFN-γ secretion after TCR stimulation [3], the mechanistic link between PD-1 and Tc1/Th1 skewing remains unclear. In prospectively collected cancer tissues, TIL manifested dampened Tc1/Th1 skewing and activation compared to PBL (Figure 1 and 2). In addition, PD-1 triggering using PD-L1 coated beads further suppressed TCR-stimulated upregulation of p-STAT1, T-bet and p-S6 as well as Th1 cytokines, while PD-1 blockade reversed suppressive effects of PD-1: PD-L1 ligation (Figure 3). We also found that Src homology-2 domain-containing phosphatase (SHP-2) is higher in TIL than in PBL, tightly correlates with PD-1 expression (Figure 4), and negatively regulates STAT1 and T-bet activation (Figure 5). Thus, the PD-1/SHP-2/p-STAT1/T-bet axis provides an important mechanism for PD-1 suppression of type 1 immunity at tumor sites. PD-1 blocking Abs, which are clinically effective in several solid cancers, should improve T cell-based cancer immunotherapy by restoring robust type 1 immunity and T cell activation to reverse immunosuppression in the tumor microenvironment. SHP-2 inhibitory strategies may also be useful to improve type 1-biased TIL.
Figure 1
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 HNC 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
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 HNC 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.

Figure 3
Figure 3

PD-1 ligation with bead-coated PD-L1 suppressed TCR-stimulated up-regulation of p-STAT1, T-bet and production of Th1 cytokines, 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. Summary data of frequency of p-STAT1 (Y701)+ (A), T-bet+ (B) and p-S6 (S235/236)+ (C) in CD8+ and CD4+ TIL with indicated conditions is shown (n=7). Supernatants of TIL 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) were collected and stored at -80°. Th1 (IFN-γ and IL-2) and Th2 (IL-10) cytokines in the supernatants were determined by Luminex. Summary data of amount of IFN-γ (D), IL-2 (E) and IL-10 (F) in the supernatants of TIL cultured under indicated conditions is shown. The graphs present the mean ± SEM from 8 HNC patients. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p<0.05, **p<0.01.

Figure 4
Figure 4

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 100 ug/mL anti-PD-1 blockade (BMS-936558) for 48 h in the presence of 50 uM fusaruside or DMSO. Then p-STAT1 and T-bet were analyzed by flow cytometry. Supernatants were collected and stored at -80°C Th1 (IFN-γand 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 HNC patients. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p < 0.05, **p < 0.01.

Figure 5
Figure 5

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 100 ug/mL anti-PD-1 blockade (BMS-936558) for 48 h in the presence of 50 uM fusaruside or DMSO. Then p-STAT1 and T-bet were analyzed by flow cytometry. Supernatants were collected and stored at -80. 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 HNC patients. Statistical significance was determined by Wilcoxon (non-parametric paired) test. *p < 0.05, **p < 0.01.

Authors’ Affiliations

(1)
Department of Pharmacy, School of Medicine, Tsinghua University, Beijing, Pittsburgh, China, USA
(2)
University of Pittsburgh Cancer Institute, Pittsburgh, USA

References

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Copyright

© Li and Ferris; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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