STAT1 inhibits T-cell exhaustion and myeloid derived suppressor cell accumulation to promote antitumor immune responses in head and neck squamous cell carcinoma

Abstract

Cancers of the oral cavity remain the sixth most diagnosed cancer worldwide, with high rates of recurrence and mortality. We determined the role of STAT1 during oral carcinogenesis using two orthotopic models in mice genetically deficient for Stat1. Metastatic (LY2) and nonmetastatic (B4B8) head and neck squamous cell carcinoma (HNSCC) cell lines were injected into the oral cavity of Stat1 deficient (Stat1^-/- ) and Stat1 competent (Stat1^+/+ ) mice. Stat1^-/- mice displayed increased tumor growth and metastasis compared to Stat1^+/+ mice. Mechanistically, Stat1^-/- mice displayed impaired CD4+ and CD8+ T-cell expansion compared to Stat1^+/+ mice. This was associated with enhanced T-cell exhaustion, and severely attenuated T-cell antitumor effector responses including reduced expression of IFN-γ and perforin at the tumor site. Interestingly, tumor necrosis factor (TNF)-α production by T cells in tumor-bearing mice was suppressed by Stat1 deficiency. This deficiency in T-cell expansion and functional responses in mice was linked to PD-1 and CD69 overexpression in T cells of Stat1^-/- mice. In contrast, we observed increased accumulation of CD11b+ Ly6G+ myeloid derived suppressor cells in tumors, draining lymph nodes, spleens and bone marrow of tumor-bearing Stat1^-/- mice, resulting in a protumorigenic microenvironment. Our data demonstrates that STAT1 is an essential mediator of the antitumor response through inhibition of myeloid derived suppressor cell accumulation and promotion of T-cell mediated immune responses in murine head and neck squamous cell carcinoma. Selective induction of STAT1 phosphorylation in HNSCC patients could potentially improve oral tumor outcomes and response to therapy.

Keywords: cancer; cytokine; oral; signal; transducer.

Figure 1:. STAT1 deficiency promotes HNSCC tumor growth and metastasis.

(A) Tumor volumes in LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice (B) Mouse weights in LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice (C) Tumor weights at terminal sacrifice in LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice (D) Representative images of LY2 or B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. Images were taken at 20 days (LY2) and 41 days (B4B8) post injection. (E) Representative images of excised tumors measured at the time of harvest in LY2 or B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. (F) Representative histological images of lymph node sections from tumor bearing Stat1+/+ and Stat1−/− mice stained with hematoxylin and eosin. Metastatic regions in Stat1−/− lymph nodes are shown. (G) Lungs of LY2 tumor bearing Stat1^−/− and Stat1^+/+ mice. Metastatic nodules are shown in white circles. (H) Bar graph showing incidence of lymph node and lung metastases in LY2 tumor bearing Stat1^−/− and Stat1^+/+ mice. (I) Mortality of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. (J) Gene expression analysis of Mmp1 and Mmp9 mRNA transcripts in tumor tissues of LY2 tumor bearing Stat1^−/− and Stat1^+/+ mice, determined by RT-qPCR. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.005 for comparisons between Stat1^−/− and Stat1^+/+ mouse groups using Student’s T test. Stat1^−/− LY2 (n = 4), Stat1^+/+ LY2 (n = 7); Stat1^−/− B4B8 (n = 8), Stat1^+/+ B4B8 (n = 7).

Figure 2:. Deficiency of STAT1 limits CD4+ and CD8+ T lymphocyte expansion during metastatic HNSCC.

(A–B) Flow cytometric analysis of CD4+ T cells and CD8+ T cells in lymph nodes of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. (C – D) Flow cytometric analysis of CD4+ T cells and CD8+ T cells in spleens of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice as determined by flow cytometry. Representative contour plots are shown as well as graphs showing percentages of respective T cell populations. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.005 for comparisons between Stat1^−/− and Stat1^+/+ mouse groups using Student’s T test.

Figure 3:. STAT1 deficiency promotes T cell exhaustion during HNSCC.

(A-C) PD-1 expression in CD4+ T cells and CD8+ T cells in the (B) lymph nodes and (C) spleens of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. (D-F) CD69 expression in CD4+ T cells and CD8+ T cells in the (E) lymph nodes and (F) spleens of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. (G and H) TIM3 expression in PD-1+ CD4+ T cells in the draining lymph nodes of LY2 tumor bearing and non-tumor bearing Stat1^−/− and Stat1^+/+ mice. Representative histogram plots (A and D) and zebra plots (G) of lymph node populations are shown as well as graphs showing percentages of positive cell populations. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.005 for comparisons between Stat1^−/− and Stat1^+/+ mouse groups using Student’s T test.

Figure 4:. T cells of HNSCC tumor bearing Stat1−/− mice have impaired effector functions.

(A – B) TNFα production by CD4+ and CD8+ T cells in lymph nodes and spleens of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. (C – D) IFNγ production by CD4+ and CD8+ T cells in lymph nodes and spleens of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice. Cells were stimulated with PMA and ionomycin with brefeldin for 4 hours, and cytokine detection was determined flow cytometric analysis of intracellularly stained cells. Cells are gated on CD3 and CD4 for CD4+ T cells and CD3 and CD8 for CD8+ T cells. Representative contour plots are shown as well as graphs showing frequencies of cytokine producing T cells. (E) Cellular damage on CD3 stimulated and non-stimulated lymph node and spleen cells of LY2 tumor bearing Stat1^−/− and Stat1^+/+ mice as detected by LDH assay. (F) Gene expression of T-bet, Ifng, Perforin, Klrc1, Tnfa and Bcl2 in tumors of LY2 bearing Stat1^−/− and Stat1^+/+ mice as determined by RT-qPCR. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.005 for comparisons between Stat1^−/− and Stat1^+/+ mouse groups using Student’s T test.

Figure 5:. STAT1 deficiency increases the accumulation of myeloid cell populations during HNSCC.

(A-D) Analysis of CD11b+ Ly6Ghi Ly6Cint populations in draining lymph nodes, spleen, bone marrow and primary tumors of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice, as determined by flow cytometry. Representative contour plots are shown as well as graphs showing frequencies of respective myeloid populations. (E and F) Gene expression analysis of (E) Cxcl1 and (F) Il-1 in tumors of LY2 bearing Stat1^−/− and Stat1^+/+ mice as determined by RT-qPCR. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.005 for comparisons between Stat1^−/− and Stat1^+/+ mouse groups using Student’s T test.

Figure 6:. Myeloid cells in tumor microenvironment promote tumor progression in Stat1^−/− mice.

(A-B) Expression of PDL1 in tumors and spleens of LY2 tumor bearing Stat1^−/− and Stat1^+/+ mice, as determined by flow cytometry. Representative contour plots are shown as well as graphs showing frequencies of respective PDL1+ myeloid cell populations. (C) Sorted CD11b+ Ly6G+ cells from Stat1^−/− and Stat1^+/+ mice were co-cultured with CFSE labelled T cells activated with CD3 and CD28. Proliferation of CFSE labelled cells were determined by flow cytometry and proliferation indices are shown on bar graph. Negative controls are CFSE labelled T cells activated with CD3 and CD28. (D) Production of IL-17 by CD3 activated T cells from lymph node and spleens of tumor bearing Stat1^−/− and Stat1^+/+ mice. (E–F) Analysis of F4/80+ CD206+ macrophages in draining lymph nodes and spleen of LY2 and B4B8 tumor bearing Stat1^−/− and Stat1^+/+ mice, as determined by flow cytometry. Representative contour plots are shown as well as graphs showing frequencies of myeloid populations. Cells are gated on CD11b+ cells. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.005 for comparisons between Stat1^−/− and Stat1^+/+ mouse groups using Student’s T test.