TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer - PubMed (original) (raw)

. 2016 Dec;6(12):1366-1381.

doi: 10.1158/2159-8290.CD-15-1347. Epub 2016 Sep 28.

Zachary T Freeman 1, Ali Ghasemzadeh 2, Michael A Chattergoon 1, Alleluiah Rutebemberwa 1, Jordana Steigner 1, Matthew E Winter 1, Thanh V Huynh 3, Suzanne M Sebald 3, Se-Jin Lee 3, Fan Pan 2, Drew M Pardoll 2, Andrea L Cox 4 2

Affiliations

TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer

Benjamin V Park et al. Cancer Discov. 2016 Dec.

Abstract

Programmed death-1 (PD-1) is a coinhibitory receptor that downregulates the activity of tumor-infiltrating lymphocytes (TIL) in cancer and of virus-specific T cells in chronic infection. The molecular mechanisms driving high PD-1 expression on TILs have not been fully investigated. We demonstrate that TGFβ1 enhances antigen-induced PD-1 expression through SMAD3-dependent, SMAD2-independent transcriptional activation in T cells in vitro and in TILs in vivo The PD-1hi subset seen in CD8+ TILs is absent in Smad3-deficient tumor-specific CD8+ TILs, resulting in enhanced cytokine production by TILs and in draining lymph nodes and antitumor activity. In addition to TGFβ1's previously known effects on T-cell function, our findings suggest that TGFβ1 mediates T-cell suppression via PD-1 upregulation in the tumor microenvironment (TME). They highlight bidirectional cross-talk between effector TILs and TGFβ-producing cells that upregulates multiple components of the PD-1 signaling pathway to inhibit antitumor immunity.

Significance: Engagement of the coinhibitory receptor PD-1 or its ligand, PD-L1, dramatically inhibits the antitumor function of TILs within the TME. Our findings represent a novel immunosuppressive function of TGFβ and demonstrate that TGFβ1 allows tumors to evade host immune responses in part through enhanced SMAD3-mediated PD-1 expression on TILs. Cancer Discov; 6(12); 1366-81. ©2016 AACRThis article is highlighted in the In This Issue feature, p. 1293.

©2016 American Association for Cancer Research.

PubMed Disclaimer

Figures

Figure 1

Figure 1

TGF-β1 enhances PD-1 expression on human T cells in a dose-dependent manner. Human CD3+ T cells were isolated from healthy donor PBMCs and were activated with αCD3/αCD28-conjugated beads with or without TGF-β1 (50 ng/ml). (a) Representative plots PD-1 (Y-axis) vs. CFSE (X-axis) are shown for different conditions. (b) PD-1 MFI is shown as filled circle lines (αCD3/αCD28) and open-circle lines (αCD3/αCD28 + TGF-β1). The percentage of cells in each CFSE generation (G0, G1, G2, G3 and G4) is shown as black bar graphs (αCD3/αCD28) and white bar graphs (αCD3/αCD28 + TGF-β1). The data represent combined results of two independent trials. (c) Naïve and memory subset of T cells (CD4+ and CD8+ T cells) were isolated based on CCR7 and CD45RA expression and treated with αCD3/αCD28 activation in the presence or absence of TGF-β1. The representative histogram of PD-1 is shown: shaded histogram (Isotype); thin histogram (αCD3/αCD28); and bold histogram (αCD3/αCD28+TGF-β1). (d) PD-1 MFI was assessed on each subset (X-axis) of CD8+ (left) and CD4+ (right) T cells; αCD3/αCD28 alone (black bars); αCD3/αCD28 with TGF-β1 (grey bars). The data represent combined results of two independent trials. (e) Isolated human CD3+ T cells were activated with αCD3/αCD28-conjugated beads in the presence of varying concentrations of TGF-β1 (5 to 5 × 104 pg/ml). Mean fluorescent intensity (MFI) of PD-1 (left) and HLA-DR (right) expressions were assessed in CD4+ (grey bars) and CD8+ (black bars) T cells. The shown result is the representative of at least three independent trials. (f) Pdcd-1 transcript levels of human CD3+ T cells under different treatments were normalized to that of resting CD3+ T cells. The result is shown as mean +/− SEM of technical replicates and representative of at least three independent trials. The data were analyzed using Student’s t-test and considered significant if *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Figure 2

Anti-TGF-β1 neutralizing antibody and a TGF-βRI kinase inhibitor negate TGF-β1 mediated PD-1 enhancement (a, b) Enriched human CD3+ T cells were activated with αCD3/αCD28-conjugated beads and TGF-β1 under varying concentrations of TGF-β1 nAb (a) or SB431542 (b) and PD-1 MFI was assessed: medium alone (closed circles) αCD3/αCD28 only (open circles); αCD3/αCD28 + TGF-β1 (closed triangles). The result shown is representative of at least three independent trials. (c) Western-blot analysis of phosphorylated-Smad2 (p_Smad2_) in human CD3+ T cells treated with varying concentrations of TGF-βRI kinase inhibitor (SB431542). (d) Enriched human CD3+ T cells were activated with αCD3/αCD28-conjugated beads and TGF-β1 under increasing concentrations SB431542. Pdcd-1 transcript levels in each condition were normalized to that of resting human CD3+ T cells. The result is shown as mean +/− SEM of technical replicates and representative of at least three independent trials.

Figure 3

Figure 3

Smad3 directly binds to the Smad-binding elements (SBEs) and regulates PD-1 promoter activity (a) Schematic illustration of the proximal region of human Pdcd-1 promoter. Two Smad-binding-elements (SBEs) are located at 1.2 kb (SBE-D) and 1.0 kb (SBE-P) upstream of the Pdcd-1 transcription start site. NFATc1 consensus sequence is located in immediate proximity to SBE-P. Wild-type (WT) and mutated (Mut) sequences of both SBE-D and SBE-P are shown. (b) Jurkat T cells were transfected with luciferase reporter vectors containing wild-type (WT), mutant SBE-D, mutant SBE-P, and mutant SBE-D/P sequences of PD-1 promoter (1.9 kb). After co-transfection with TGF-βRI and RII expression plasmids, the cells were activated with plate-bound αCD3 and soluble αCD28 in the absence (grey bars) or presence (white bars) of TGF-β1 (50 ng/ml). Luciferase activity was measured as described in the method. (c) Isolated human CD3+ T cells were activated under different conditions: medium alone (white bars); αCD3/αCD28 alone (black bars); αCD3/αCD28 with TGF-β1 (hatched bars); αCD3/αCD28 + TGF-β1 with SB431542 (grey bars). Immunoprecipitated DNA was subjected for qPCR and fold enrichment of binding relative to IgG is shown as mean +/− SEM of triplicate results. (d) Jurkat T cells were co-transfected with 1.5 μM of siRNA against Smad2 or Smad3 and PD-1 promoter-driven luciferase activity was measured in relative luciferase units (ratio of firefly to renilla luciferase activity). (e) Transfected Jurkat T cells were treated with 10μM of specific inhibitor of Smad3 (SIS3) and PD-1 promoter-driven luciferase activity was measured after activation. The results are shown as mean +/− SEM of technical replicates and is representative of at least three independent trials. The data were analyzed using two-way analysis of variance (ANOVA) and considered significant if *p<0.05.

Figure 4

Figure 4

TGF-β1-dependent Smad3 enhances PD-1 expression on human and murine T cells (a) Human CD3+ T cells from healthy donors were isolated and pretreated with SIS3 at varying concentrations. Subsequently, the cells were activated with αCD3/αCD28-conjugated beads with or without TGF-β1. Mean fluorescence intensity (MFI) of PD-1 expression in different conditions was assessed: αCD3/αCD28 (closed circles); αCD3/αCD28 + TGF-β1 (open circles). The result shown is representative of at least three independent trials. (b) CD4+ T cells were isolated from wild-type (WT), Smad2 f/f; _Cd4_-cre (Smad2 cKO), Smad3 f/f; _Cd4_-cre (Smad3 cKO) mice and activated with plated-coated αCD3 and soluble αCD28 with or without TGF-β1 (50 ng/ml). PD-1 expression is shown as overlaid histograms with shaded histogram (isotype control), thin histogram (αCD3/αCD28), bold histogram (αCD3/αCD28+TGF-β1). PD-1 MFI is also shown as mean +/− SEM and represents combined results of two independent trials (bar graphs). (c) Isolated WT, Smad2 cKO, and Smad3 cKO OT-1 cells were activated with type-1 ovalbumin in the presence of irradiated splenocytes. PD-1 MFI is shown as mean +/− SEM and represents combined results of two independent trials (bar graphs). (d) Growth kinetics of B16 melanoma in WT (n=7), Smad2 cKO (n=10), and Smad3 cKO (n=11) mice are shown as the mean volume +/− SEM on different days. The data represent the combined results of two independent experiments. (e) Average CD8+ PD-1+ percentages in Smad2 cKO and Smad3 cKO TIL are shown as normalized values to WT CD8+ PD-1+ percentages. The data were analyzed using Student’s t-test and considered significant if *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Figure 5

Adoptive transfer of Smad3 cKO CD8+ T cells results in reduced tumor burden and PD1hi subset relative to transfer of WT CD8+ T cells (a) Growth kinetics of B16-Ova in CD45.1 congenic mice that received no T cells (closed circles), WT OT-1 (open circles) or Smad3 cKO OT-1 (triangles). C57/BL6 expressing CD45.1 congenic markers were challenged with 1 × 105 B16-Ova melanoma cell line on Day 0. On Day 10, 107 CD45.2 CD8+ OT-1 T cells from WT (n = 7) or Smad3 cKO (n = 5) mice were adoptively transferred into the mice with comparable tumor sizes. Tumor volume (mm3) is shown as mean +/− SEM on different days, and the data represent combined results of two independent experiments. The data were analyzed using one-way ANOVA and considered significant if *p<0.05, **p<0.01. (b, c) CFSE-labelled tumor infiltrating WT OT-1 (top) or cKO OT-1 (bottom) were isolated from B16-Ova 5 days after adoptive transfer, and tumor infiltrating lymphocytes (TIL) proliferation was assessed: Smad3 cKO (b) and Smad2 cKO OT-1 (c). CD45.2+ donor population was gated from a plot of CD8 (Y-axis) and CD45.2 (X-axis) (left), and a representative histogram of CFSE (right) is shown from pooled TIL from n = 6 mice per group. (d, e) Contour plots of PD-1 (top) and LAG3 (bottom) among the proliferated cells (i.e CFSE negative populations) are shown as isotype (left), WT (middle) and cKO (right): Smad3 cKO (d) and Smad2 cKO (e). The data are representative of two independent experiments.

Figure 6

Figure 6

Loss of Smad3 in CD8+ T cells leads to enhanced cytokine production. (a) Growth kinetics of MC-38 in WT (n=7), Smad2 cKO (n=6), or Smad3 cKO cells (n=6) are shown as the mean volume +/− SEM on different days. Data is representative example from 2 independent experiments. (b) Average CD8+ CD44+ PD-1 MFI in Smad2 cKO and Smad3 cKO TIL are shown as normalized values to WT CD8+ PD-1+ percentages. The data were analyzed using Student’s t-test and considered significant if *p<0.05. (c) Percentage of CD8 T cells producing IFNγ, TNFα, or IL-2 in the spleen, draining lymph node, and tumor. The data were analyzed using Student’s t-test and considered significant if *p<0.05.

Figure 7

Figure 7

Anti-PD-1 blocking antibody enhances anti-tumor immune responses in WT mice, but has minimal effect in _Smad3_-deficient mice. WT and Smad3 cKO were challenged with B16-melanoma cell line on Day 0. WT and Smad3 cKO mice were treated with either isotype-matched control IgG (circles) or anti-PD-1 antibody (squares) from the day of tumor implantation until Day 17. Tumor volume (mm3) on Day 17 is shown as mean +/− SEM on and the data represent combined results of two independent experiments. The data were analyzed using two-way analysis of variance (ANOVA) and considered significant if *p<0.05.

Similar articles

Cited by

References

    1. Agata Y, Kawasaki A, Nishimura H, Ishida Y, Tsubat T, Yagita H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. International Immunology. 1996;8(5):765–72. - PubMed
    1. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439(7077):682–87. - PubMed
    1. Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, et al. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009;114(8):1537–44. - PMC - PubMed
    1. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8(8):793–800. - PubMed
    1. Lipson EJ, Sharfman WH, Drake CG, Wollner I, Taube JM, Anders RA, et al. Durable cancer regression off-treatment and effective reinduction therapy with an anti-PD-1 antibody. Clin Cancer Res. 2013;19:462–8. United States: 2012 Aacr. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources