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. Author manuscript; available in PMC: 2021 Mar 15.
Published in final edited form as: Int J Cancer. 2019 Nov 29;146(6):1717–1729. doi: 10.1002/ijc.32781

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

Nathan Ryan 1, Kelvin Anderson 1, Greta Volpedo 1,2, Omar Hamza 1, Sanjay Varikuti 1, Abhay R Satoskar 1,2, Steve Oghumu 1
PMCID: PMC7366342  NIHMSID: NIHMS1605260  PMID: 31709529

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 non-metastatic (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 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 pro-tumorigenic micro-environment. Our data demonstrates that STAT1 is an essential mediator of the anti-tumor 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: Oral, Cancer, Signal, Transducer, Cytokine

INTRODUCTION:

Cancers of the oral cavity and pharynx remain among the most prevalent cancers1, with over 300,000 reported cases and an annual mortality of approximately 145,000 deaths worldwide2. Head and neck squamous cell carcinoma (HNSCC) is complicated by tumor recurrence and lymph node metastasis, which is commonly associated with poor prognosis and a 5 year survival rate of 55–65%2, 3. There is therefore an urgent need to clarify the molecular and immunological mechanisms underlying head and neck carcinogenesis and identify novel targets for oral cancer prevention and therapy4–6.

Signal transducer and activator of transcription 1 (STAT1) is a transcription factor involved in a wide variety of immunological responses. It transduces signals from type I, II and III interferons (IFN), interleukin (IL)-21, IL-27 and IL-35. In response to IFN, STAT1 mediates CD4 and CD8 T cell activation and differentiation towards a T helper 1 (Th1) immune response7–9 STAT1 is also involved in macrophage activation10–12. Interestingly, head and neck squamous cancer cells also express STAT1, and STAT1 activation in cancer cells has been shown to be associated with immune evasion and metastasis

There are a few reports which describe the contribution of STAT1 to HNSCC. Recent studies using immunohistochemical staining suggested that activated STAT1 expression was predictive of improved survival and response to adjuvant therapy in oral cavity squamous cell carcinoma patients13. In contrast, other studies suggest that upregulation of Stat1 gene expression is associated with HNSCC development14. Indeed, STAT1 induction has been reported to enhance the production of PDL1 and IDO, which are recognized to contribute to an immunosuppressive tumor microenvironment and promote HNSCC15–17. Therefore, despite the evidence for STAT1 as a mediator of tumor suppression in the context of HNSCC18, STAT1 activity has paradoxically been shown to also function as an oncogene, mediating immune escape, cancer cell proliferation and invasion in HNSCC16, 17, 19. These conflicting findings are indicative of the need for further study into the role STAT1 plays during HNSCC.

While previous studies might suggest that the contrasting roles of STAT1 on HNSCC are dependent on whether STAT1 is expressed on tumor cells or within the tumor microenvironment, there are currently no studies that address the contribution of STAT1 expression on cells of the tumor microenvironment during head and neck carcinogenesis. In this study, we investigate the role of host STAT1 expression during experimental HNSCC using two orthotopic murine BALB/c models with metastatic LY2 and non-metastatic B4B8 cancer cells. LY2 cells were derived from PAM 212 squamous cell carcinoma cells which develop rapid tumors in the oral cavity with lymph node metastases, while B4B8 cells were derived from BALB/c oral keratinocytes treated with the oral carcinogen 4NQO20–22. These models provide ideal syngeneic in vivo systems to examine the role of immunological mediators during HNSCC in immunocompetent mice. We also examine the underlying cellular and molecular mechanisms behind host STAT1 expression on HNSCC tumor growth. Our results demonstrate that STAT1 inhibits myeloid derived suppressor cell accumulation and promotes T-cell mediated anti-tumor immune responses.

MATERIALS AND METHODS

Mice

Male and female BALB/c wild type (Stat1+/+) mice, 7–8 weeks old, were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Male and female BALB/c Stat1−/− mice were generated as described previously23. All animals were housed in an Ohio State University animal facility in accordance with all guidelines set forth by University Laboratory Animal Resources (ULAR). Animal experiments were approved by the Institutional Animal Care and Use Committee (Protocol #2018A00000054) and Institutional Biosafety Committee of the Ohio State University.

Cell Lines

Murine metastatic Pam LY-2 (RRID:CVCL_Z594) and non-metastatic B4B8 (RRID:CVCL_0B35) oral squamous cell carcinoma cells, were a generous gift from Dr. Vigneswaran20, 21, and were cultured as monolayers in advanced DMEM/F12 media (Life Technologies, Waltham, MA, USA) supplemented with 2% fetal bovine serum (Corning, Corning, NY, USA), 100 μg/mL penicillin G, 100 μg/mL streptomycin, and 2 mM L-glutamine (Life Technologies) at 37°C and 5% CO2. All experiments were performed with mycoplasma-free cells.

Orthotopic Cell injections

LY2 cells were grown to 75% confluence and harvested by trypsinization. Cells were resuspended in serum-free advanced DMEM/F12 media. Prior to injection, cell suspensions were mixed 1:1 with Matrigel (Corning). A total of 5.0*10^5 cells were injected in a volume of 40 μL into the right buccal mucosa of Stat1−/− and Stat1+/+ mice that were anesthetized by isoflurane. Throughout the duration of the experiment, tumor-bearing mice were allowed access to food and water ad libitum. Weights of the mice were taken twice weekly. Tumor sizes were also recorded twice weekly, with the tumor volumes being estimated using the formula V=(A*B2)2 mm3, where A = the longer diameter of the tumor and B = the shorter diameter. At terminal sacrifice, primary tumors, draining lymph nodes, spleens, lungs, and bone marrow were harvested. Harvested lungs were placed in Bouin’s solution (MilliporeSigma, Burlington, MA, USA) for later examination of any metastatic nodules.

Flow Cytometry

Single cell suspensions were generated from tumors, spleens, draining lymph nodes, and bone marrow, by passing through a 70 μL nylon mesh. Cells were incubated with fluorochrome conjugated antibodies for CD11b, Ly6G, Ly6C, CD11c, F4–80, PD-L1, CD206, CD3, CD4, CD8 and PD-1. In some experiments, cells were stimulated with a cell activation cocktail containing PMA and ionomycin (Biolegend, San Jose, CA), and intracellularly stained for IFN-γ and TNF-α cytokine production. Samples were run using BD FACS Calibur or BD FACS Aria (BD Biosciences, San Jose, CA) and subsequent analysis performed using FlowJo software (Tree Star, Inc., Ashland, OR).

Lymph Node Histopathology

Lymph nodes harvested from LY2 tumor-bearing mice were fixed in 10% neutral buffered formalin, followed by paraffin embedding. The paraffinized tissues were sectioned into 5 μm thick cuts, followed by standard processing and staining with hematoxylin and eosin. Lymph node metastasis was validated by microscopic analysis of the sectioned tissue as described previously21.

Real Time PCR

Total RNA was extracted from tumors using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA). RNA procured from primary tumor tissues was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Primer sequences were generated using the IDT RealTime qPCR Tool website (https://www.idtdna.com/scitools/Applications/RealTimePCR/, Integrated DNA Technologies, Coralville, IA). PCR amplification was performed in duplicate using the PowerUp SYBR Green Master Mix (BioRad, Hercules, CA), using beta actin (Actb) as reference gene. Genes investigated included Mmp1, Mmp9, Prf1, Tbet, Ifng, Tnf, Il1, Cxcl1 and Bcl2.

T cell stimulation, LDH and ELISA

Single cell suspensions were prepared from spleens and lymph nodes. Cells were incubated with plate bound αCD3 (1μg/ml) and αCD28 (1μg/ml) antibodies (Biolegend SanDiego, CA) for 72 hours. Cell supernatants were analyzed for IL-17, TNFα and IFNα production. Capture and detection antibodies were purchased from Biolegend (San Diego, CA). Cellular damage of plated spleen and lymph node cells was determined by quantification of lactose dehydrogenase (LDH) in cell supernatants using the Pierce LDH cytotoxicity assay kit (Thermo Scientific, Rockford, IL).

MDSC suppression assay

T cells from spleens and lymph nodes of tumor bearing mice were isolated using nylon wool24. Isolated T cells were stained with CFSE and then seeded in 96 well plates, containing plate bound αCD3 and αCD28 antibodies. CD11b+ Ly6G+ Ly6Cint were sorted from LY2 tumor bearing spleens of Stat1−/− and Stat1+/+ mice, and co-cultured with CFSE labelled T cells. After 72 hour incubation, T cells were analyzed by flow cytometry and proliferating cells were determined by proliferation indices using Flowjo software (Tree Star, Inc., Ashland, OR).

Statistics

Statistically analyses were performed using GraphPad Prism v8.0.2 (GraphPad Software, San Diego, CA, USA). Student’s T test was used to determine statistically significant differences between groups with a p value cut off of 0.05.

Data availability

Data will be made available upon reasonable request.

RESULTS

STAT1 deficiency promotes HNSCC tumor growth

We determined the involvement of STAT1 in oral tumor growth using metastatic and non-metastatic HNSCC cell lines LY2 and B4B8 orthotopically injected into the buccal mucosa of Stat1−/− and Stat1+/+ BALB/c mice. Primary tumor growth occurred very rapidly in LY2 injected Stat1−/− mice compared to Stat1+/+ mice (Figure 1A and D). These mice were sacrificed at 3 weeks post LY2 injection, having met early removal criteria. At this time, tumor volumes of LY2 bearing Stat1−/− mice were five times larger than in Stat1+/+ counterparts. This was accompanied by a significant reduction in weights of LY2 cancer bearing Stat1−/− mice but not Stat1+/+ mice (Figure 1B). Tumor weights were similarly larger in Stat1−/− than in Stat1+/+ mice (Figure 1C and E). Tumor growth in the non-metastatic B4B8 cancer cell injected mice progressed less rapidly than in the metastatic LY2 injected mice for both Stat1−/− and Stat1+/+ mice. However, as in the LY2 injected mice, tumor volumes were significantly higher in Stat1−/− mice compared to Stat1+/+ mice (Figure 1A and D). Similarly, after 5 weeks post cancer cell injections, mouse weights in tumor bearing Stat1−/− mice were significantly lower than in Stat1+/+ mice (Figure 1B), while tumor weights were significantly higher in Stat1−/− mice compared to Stat1+/+ mice (Figure 1C and E). Taken together, our results demonstrate that Stat1 deficiency in the host promotes the growth of HNSCC in orthotopic models of both metastatic and non-metastatic HNSCC cells.

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

Figure 1:

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(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).

Lymph node metastasis is enhanced in the absence of STAT1

We employed two orthotopic models of murine HNSCC in order to determine the role of STAT1 both in primary tumor growth as well as subsequent lymph node and lung metastasis. The LY2 HNSCC cell line metastasizes to draining cervical lymph nodes and lungs of BALB/c mice20. Histological analysis of Stat1−/− and Stat1+/+ mice revealed draining lymph node metastasis in 86% of tumor bearing Stat1−/− mice at 3 weeks post LY2 injection, while Stat1+/+ mice displayed 0% lymph node metastasis (Figure 1F and H). Lungs from Stat1−/− tumor-bearing mice showed metastatic nodules in 14%, while 0% of tumor bearing Stat1+/+ mice showed lung metastasis (Figure 1G and H). Further evidence for increased metastatic potential observed in Stat1−/− mice during HNSCC was displayed by a significant increase in matrix metalloproteinases Mmp1 and Mmp9 transcripts in primary tumors, biomarkers associated with cancer cell infiltration and metastasis (Figure 1J). Mortality occurred only in 14% of LY2 tumor bearing Stat1−/− mice (Figure 1I). Our data demonstrates that STAT1 inhibits lymph node metastasis in an orthotopic LY2 HNSCC model.

Deficiency of STAT1 limits CD4+ and CD8+ T lymphocyte expansion during metastatic HNSCC

We determined potential mechanisms underlying STAT1 mediated susceptibility to HNSCC. STAT1 plays an important role in mounting and maintaining effective T cell mediated anti-tumor immune responses25. Therefore, we examined CD4+ and CD8+ T lymphocyte populations in secondary lymphoid organs of metastatic (LY2) and non-metastatic (B4B8) tumor bearing Stat1−/− and Stat1+/+ mice. Draining lymph nodes of LY2 tumor-bearing Stat1−/− mice displayed significantly lower proportions of CD4+ and CD8+ T lymphocytes compared to Stat1+/+ mice (Figure 2A and B). Similar results were obtained in the spleens of LY2 tumor-bearing Stat1−/− and Stat1+/+ mice, which reflects a diminished systemic T cell response in the absence of STAT1 (Figure 2C and D). Interestingly, proportions of CD4+ and CD8+ T cell populations in the lymph nodes and spleens of non-metastatic B4B8 tumor bearing Stat1−/− and Stat1+/+ mice were mostly similar, with a slight increase in splenic CD8+ T cells of Stat1−/− mice (Figure 2A–D). These results indicate a role for STAT1 in effective CD4+ and CD8+ T cell recruitment during metastatic HNSCC.

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

Figure 2:

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(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.

STAT1 deficiency promotes T cell exhaustion during HNSCC

Next, we determined potential mechanisms behind the reduced T cell infiltration observed in draining lymph nodes and spleens of LY2 tumor bearing Stat1−/− mice. PD-1 expression on activated cells has been shown to promote T cell apoptosis and suppression of the adaptive immune response26. Therefore, we analyzed PD-1 expression by T cells in the tumor draining lymph nodes and spleens of LY2 tumor bearing Stat1−/− and Stat1+/+ mice. In the draining lymph node, PD-1 expressing CD4+ and CD8+ T cells were significantly higher in LY2 tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 3A and B). In the spleen, PD-1 expressing CD4+ T cells were identical in LY2 tumor bearing Stat1−/− and Stat1+/+ mice, while PD-1 expressing CD8+ T cells were slightly increased in LY2 tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 3C). In non-metastatic B4B8 tumor bearing mice, we observed increased PD-1 expression in splenic but not lymph node CD4+ and CD8+ T cells of Stat1−/− mice compared to Stat1+/+ mice (Figure 3B and C).

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

Figure 3:

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(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.

We also determined the expression of CD69, a T cell activation marker known to promote T cell exhaustion in breast cancer. Our data revealed increased CD69 expression in lymph node CD4+ and CD8+ T cells as well as splenic CD8+ T cells of LY2 tumor bearing Stat1−/− mice compared to Stat1+/+ controls (Figure 3D–F). Similarly, in B4B8 tumor bearing Stat1−/− mice, CD69 expression was increased on lymph node CD4+ T cells and splenic CD4+ and CD8+ T cells, compared to B4B8 tumor bearing Stat1+/+ mice (Figure 3D–F). Finally, we analyzed expression of TIM3 in CD4+ T cells in the draining lymph nodes of tumor bearing and non-tumor bearing Stat1+/+ and Stat1−/− mice. Our results show enhanced TIM3 expression in PD-1+ CD4+ T cells of tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 3G and H). Taken together, our data demonstrates that enhanced PD-1, TIM3 and CD69 expression in tumor bearing Stat1−/− mice is associated with the diminished CD4+ and CD8+ T cell expansion and responses at the draining lymphoid organs. The enhanced T cell exhaustion mediated by PD-1 and TIM3 potentially contributes to enhanced susceptibility to metastatic HNSCC in the absence of STAT1.

T cells of HNSCC tumor bearing Stat1−/− mice have impaired effector functions

The observed deficiency in T cell numbers associated with enhanced PD-1 expression in T cells of tumor bearing Stat1−/− mice led us to investigate effector functions of these T cells during experimental HNSCC in our orthotopic murine model. We examined anti-tumor T cell expressing cytokines including TNF-α, IFNγ and IL-17, as well as the cytotoxic molecule Perforin using flow cytometry and RT-qPCR. TNF-α signaling activates the mitochondrial apoptosis pathway and has been shown to be directly cytotoxic to tumor cells27. Our analysis demonstrated a significant attenuation of TNF-α production by CD4+ and CD8+ T cells in the draining lymph nodes and spleens of tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 4A and B). This was true for both metastatic LY2 and non-metastatic B4B8 orthotopic HNSCC tumor models (Figure 4A and B). IFN-γ production by T cells in the draining lymph nodes were similar between Stat1−/− and Stat1+/+ mice in both metastatic and non-metastatic tumor models. However, IFN-γ production by splenic CD4+ T cells was lower in LY2 tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 4C and D).

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

Figure 4:

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(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.

To confirm the effects of STAT1 deficiency on T cell functionality during experimental HNSCC, we analyzed the release of lactose dehydrogenase (LDH) in CD3 stimulated T cells harvested from lymph nodes and spleens of tumor bearing Stat1−/− and Stat1+/+ mice. Interestingly, CD3 stimulated cells from the lymph node and spleens of tumor bearing Stat1−/− mice showed greater LDH release compared to Stat1+/+ mice, indicating increased T cell damage (Figure 4E). Further, in draining lymph nodes of tumor bearing Stat1−/− mice, cellular damage (determined by LDH assay) was evident in non CD3 stimulated cells, but not in Stat1+/+ lymph node cells (Figure 4E). These data further suggest impaired T cell functionality in the absence of STAT1 during HNSCC.

We next analyzed T cell associated markers in the tumors of LY2 bearing Stat1−/− and Stat1+/+ mice by RT-qPCR. Transcripts for both the Th1 transcription factor, T-bet as well as Ifng were found to be significantly downregulated in Stat1−/− mice compared to Stat1+/+ mice (Figure 4F). Perforin, a CD8+ T cell cytotoxicity molecule and Klrc1, also expressed on activated CD8 T cells, was significantly downregulated in Stat1−/− mice compared to Stat1+/+ mice (Figure 4F). Interestingly we observed similar Tnf-α mRNA transcripts in the tumors of both Stat1+/+ and Stat1−/− tumor-bearing mice (Figure 4F). Finally, the anti-apoptotic marker Bcl2 was downregulated in Stat1−/− mice compared to their Stat1+/+ counterparts (Figure 4F). Taken together, our results demonstrate that STAT1 is essential for the development of T cell-mediated cytotoxicity and Th1 anti-tumor effector responses during HNSCC.

STAT1 deficiency increases the accumulation of myeloid cell populations during HNSCC

The defective T lymphocyte effector responses in tumor bearing Stat1−/− mice led us to examine potential immunosuppressive myeloid cell populations in the tumors, draining lymph nodes, spleens and bone marrows of tumor bearing mice. CD11b+Ly6G+Ly6Cint and CD11b+Ly6G−Ly6Chi myeloid cell populations, which are canonical markers of granulocytic and monocytic myeloid derived suppressor cells (MDSCs) respectively are pro-tumorigenic via immunosuppression of anti-tumor T cell responses28. Our analysis revealed a significantly increased accumulation of CD11b+Ly6G+Ly6Cint populations in primary tumors, draining lymph nodes, spleens and bone marrow of LY2 tumor-bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 5A–D). Similar trends were found in the spleens, bone marrow and tumors of B4B8 tumor bearing Stat1−/− mice, although to a lesser extent (Figure 5B and C). CD11b+Ly6G−Ly6Chi cell populations were increased in lymph nodes, spleens and bone marrow of LY2 but not B4B8 tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Supplemental Figure 1).

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

Figure 5:

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(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.

To corroborate these findings, we analyzed gene expression of inflammatory cytokines and chemokines that promote the accumulation of granulocytic myeloid derived suppressor cells in the tumor microenvironment of LY2 tumor bearing mice. We observed significantly increased levels of Cxcl1 and Il-1 transcripts in tumor-bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 5E and F). The increased presence of these immunosuppressive myeloid cell populations in the tumor microenvironment and in primary and secondary lymphoid organs of tumor bearing Stat1−/− mice suggests a role for STAT1 in promoting anti-tumor T cell responses and consequent reduction in tumor burdens.

Myeloid cells in tumor microenvironment promote tumor progression in Stat1−/− mice

Our observation of increased accumulation of CD11b+Ly6G+Ly6Cint populations in tumors and lymphoid organs Stat1−/− mice led us to determine whether the absence of STAT1 interferes with the immunosuppressive properties to this population of cells. Of note, PDL1 expression among CD11b+ cells in the tumors were similar for both Stat1−/− and Stat1+/+ mice, but was increased in the lymph nodes and spleens of LY2 tumor bearing Stat1−/− mice (Figure 6A and B). To determine the immunosuppressive properties of granulocytic MDSCs in mice with metastatic HNSCC, we sorted CD11b+Ly6G+ cells from LY2 tumor bearing Stat1−/− and Stat1+/+ mice and co-cultured them with CFSE labelled T cells activated with CD3 and CD28 antibodies. Our data demonstrated that CD11b+Ly6G+ cells from tumor bearing Stat1+/+ and Stat1−/− mice were equally able to suppress T cell proliferation (Figure 6C). This suggests that the ability to suppress anti-tumor T cell responses by granulocytic MDSCs in HNSCC is not inhibited by the absence of STAT1.

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

Figure 6:

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(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.

Tumor associated macrophages (TAMs), constituting a major immune cell population in the oral tumor microenvironment, are known to suppress anti-tumor immunity, and have been shown to be correlated with a poor prognosis in HNSCC29. Therefore, we analyzed TAMs within the tumor, and lymphoid organs of HNSCC tumor bearing mice, using flow cytometry. We observed significantly increased expression of CD11b+F4/80+CD206+ cells (marker for M2-polarized macrophages) in the spleen and draining lymph nodes of Stat1−/− tumor bearing mice compared to Stat1+/+ mice (Figure 6E and F). Our results are consistent with our data that IL-17 production by re-stimulated T cells was significantly higher in tumor bearing Stat1−/− mice compared to Stat1+/+ mice (Figure 6D), and IL-17 is known to be linked with the induction of an M2-like phenotype in macrophages30. Taken together, our results demonstrate that immunosuppressive myeloid cell populations in the tumor microenvironment and draining lymphoid organs promote tumor progression in Stat1−/− mice.

DISCUSSION

The results of our study clarify the role of STAT1 expression by cells of the tumor microenvironment in host immune responses during HNSCC. Using metastatic (LY2) and non-metastatic (B4B8) orthotopic models of HNSCC in immunocompetent BALB/c mice, we show that deletion of Stat1 results in significantly increased tumor growth, morbidity, metastatic potential and immune suppression, mediated by diminished anti-tumor T lymphocyte responses and accumulation of tumor-promoting, immunosuppressive MDSCs and TAMs. Although it is possible that global defects in hematopoietic cell development in Stat1−/− mice could partly contribute to the observed differences between Stat1−/− and Stat1+/+ tumor bearing mice, our analysis of non-tumor bearing mice suggests that more than global defects in immune cell development are involved. Non tumor bearing Stat1−/− and Stat1+/+ mice have similar proportions of CD4+ and CD8+ T cells in their draining lymph nodes and spleens. Similarly, levels of myeloid immune cell populations in the lymph node, spleens and bone marrow were similar between non tumor bearing Stat1−/− and Stat1+/+ mice. These data agree with previous reports demonstrating normal lymphoid and monocyte development in Stat1−/− mice31, 32. Our data therefore suggest that the observed differences in tumor growth are due to a lack of STAT1. This previously uncharacterized role for STAT1 expression in T cell responses during HNSCC opens up additional targeted approaches for oral cancer immunotherapy.

Our orthotopic models of metastatic and non-metastatic HNSCC have been characterized previously20–22. LY2 HNSCC cell lines possess a more aggressive phenotype and metastasize to the lymph nodes and lungs within 6 – 7 weeks post injection. As such, we performed a more extensive characterization of cellular and molecular markers of invasion and metastasis in this model. Although we observed one instance of lung metastasis in LY2 tumor bearing Stat1−/− mice and no lung metastatic nodules in the Stat1+/+ mice at 3 weeks post injection, lymph node metastasis occurred in 87% of LY2 tumor bearing Stat1−/− mice at this time point. This alludes to the highly aggressive nature of this HNSCC tumor model, which is characteristic of a majority of clinical HNSCC cases. It was also striking that we observed lung metastasis in Stat1−/− mice within this short time frame (3 weeks), at which time primary tumors had advanced to a point requiring early termination of the experiment. The susceptibility of Stat1−/− mice to metastatic HNSCC demonstrates the important role of host STAT1 expression against HNSCC growth and metastasis.

Despite the evidence for STAT1 as an important mediator of anti-tumor immunity as demonstrated by our study and other tumors31, 33, 34, a number of studies suggest that STAT1 mediated signaling in cancer and immune cells promotes immunosuppression through PD-1/PDL1 interactions during HNSCC35. While STAT1 expression by HNSCC cells has been confirmed to correlate with tumor invasion, metastasis and immunosuppression through PDL1 expression17, 19, 35, 36 our orthotopic model using Stat1−/− mice suggests a reverse effect of STAT1 on PDL1 expression by myeloid cells and PD-1 expression by CD4+ and CD8+ T cells. This contrasts with previous work suggesting that STAT1 signaling promotes PD-1 expression in CD4+ and CD8+ T cells of HNSCC patients. It must be noted that in the previous study, immune cells were treated with IFNα, which induced PD-1 expression in CD4+, CD8+ T and NK cells of HNSCC patients, presumably through STAT1 signaling. In our study, PD-1 expression was enhanced in Stat1−/− CD4+ and CD8+ T cells of tumor bearing mice. It is therefore possible that IFNα mediated induction of PD-1 expression in lymphocytes of HNSCC patients may occur through STAT1 independent mechanisms. Other clinical studies support our results on the positive effects of STAT1 expression on epithelial and other solid tumors. For example, STAT1 expression in the tumor microenvironment has previously been linked to favorable prognoses in patients with esophageal squamous cell carcinoma or high-grade serous ovarian cancer, potentially stemming from its ability to downregulate NF-κB and STAT3 mediated activity37,38. Interestingly, STAT1 activation has been shown to be instrumental in positive patient responses to cetuximab, and anti-EGFR monoclonal antibody used in HNSCC treatment, where it plays a role in upregulating MHC class I antigen presentation and thus anti-tumor CTL activity39.

The increased expression of T cell exhaustion markers PD-1 and CD69 observed in Stat1−/− tumor bearing mice highlight a potential mechanism for STAT1-mediated anti-tumor activity during HNSCC and implicate STAT1 as essential to maintaining the anti-tumor immune response generated by T lymphocytes. Reduced accumulation of CD4+ and CD8+ T cells in our model of HNSCC are likely in part a result of upregulated PD-1, causing the loss of tumor infiltrating T cells. Interactions between PD-1 and its ligand PDL1 are a recognized mechanism for immune evasion by tumor cells. Upregulation of PD-1 promotes T lymphocyte apoptosis, resulting in the suppression of the adaptive immune response40. In a model of non-small cell lung cancer, Stat1 deficient mice upregulated the expression of both PD-1 and PDL1, resulting in a diminished anti-tumor immune response41. It was therefore not surprising that PD-1 expression on T cells was enhanced in Stat1−/− tumor bearing mice and correlated with impaired T cell expansion during HNSCC. CD69, a marker traditionally associated with activated T cells42, was recently shown to be associated with T cell exhaustion and enhanced tumor progression43. We found CD69 to be highly elevated in T lymphocytes isolated from tumor bearing Stat1−/− mice. Interestingly, our results further showed that the high levels of CD69 expression in Stat1−/− mice seem to directly correlate with high PD-1 expression and enhanced tumor progression in HNSCC. Similar to PD-1/PDL1 blockade therapy, blocking CD69 using anti-CD69 antibodies attenuated tumor progression in breast carcinogenesis43. Taken together, our results demonstrate an essential role for STAT1 in preventing T cell exhaustion and enhancing anti-tumor T cell immunity during HNSCC.

Although we did not observe any intrinsic deficiency of tumor bearing Stat1−/− T cells in producing IFNγ, we did observe a significant downregulation of Ifng mRNA in tumors of Stat1−/− mice compared to Stat1+/+ mice. This is likely a consequence of the attenuated T cell expansion observed in tumor bearing Stat1−/− mice. In contrast, we observed a striking abrogation of TNFα production by T cells deficient of STAT1. This deficiency in TNFα production was observed in draining lymph nodes of both metastatic (LY2) and non-metastatic (B4B8) tumor models. TNFα produced by activated T cells are an essential component of the anti-tumor response during HNSCC44, and about 30% of HNSCCs overexpress Fas Associated Death Domain (FADD), an important component of TNF receptor signaling. As a multifunctional cytokine, TNFα can exert pro-tumoral activities by promoting inflammation, cancer cell proliferation, invasion, metastasis and angiogenesis. On the other hand TNFα can induce cancer cell death via TNFR1 mediated apoptotic and necrotic pathways27. Interestingly, during metastatic HNSCC, TNFα gene expression at the tumor site was similar between Stat1−/− and Stat1+/+ mice, suggesting that STAT1 mediated TNFα production by T cells is regulated to maintain its tumor cytotoxic activity, but prevent its pro-inflammatory tumor promoting functions.

Further evidence for the vital role of STAT1 in promoting T cell expansion and functionality during HNSCC was demonstrated by the increased cellular damage in CD3 stimulated cells of tumor bearing Stat1−/− mice. A recently described mechanism of STAT1 mediated regulation of T cell responses involve protection from cellular cytotoxicity via NK cells45. It is likely that this process of STAT1 mediated T cell protection is involved in maintaining anti-tumor T cell responses against HNSCC. Additionally, the inability of Stat1−/− mice to effectively produce perforin at the tumor site indicates a deficiency of anti-tumor cytotoxic T cell responses. Taken together, our results highlight the essential role of STAT1 in maintaining T cell viability and anti-tumoral T cell effector function.

IFN-γ dependent signaling mediated by STAT1 is known to promote the development of myeloid type 1 dendritic cells46, 47, and reverse the immunosuppressive properties of pro-tumoral tumor associated macrophages48. In turn, this promotes anti-tumoral cytotoxic T lymphocyte activity. Indeed, recent reports demonstrate that STAT1 deficiency reprograms IFN-γ signaling in macrophages toward a suppressive phenotype mediated by STAT3, which impairs their ability to trigger an effective adaptive immune response11.

The accumulation of granulocytic MDSC and M2 polarized macrophage populations in tumors, draining lymph nodes, spleens and bone marrows of tumor bearing Stat1 deficient mice indicates a direct or indirect role for STAT1 in the generation of these tumor promoting myeloid cells during HNSCC. MDSCs and tumor associated M2 polarized macrophages have been shown to suppress the anti-tumor response, partially mediated by upregulated PDL1/PD-1 interactions49. Not surprisingly, we observed increased PDL1 expression in the spleens of LY2 tumor bearing Stat1−/− mice compared to Stat1+/+ mice. However, PDL1 expression among myeloid cells in the tumors, and the ability of CD11b+Ly6G+ cells to suppress T cell proliferation during HNSCC appeared independent of STAT1. These results suggest that the effect of these immunosuppressive myeloid populations on antitumor immune responses are due in part to their enhanced accumulation in the absence of STAT1. Interleukin-17 has been shown to promote MDSC accumulation and M2 macrophage polarization, and IL-17 production is associated with poor prognosis in HNSCC patients50. We observed increased production of IL-17 in Stat1−/− deficient T cells of LY2 tumor bearing mice. Similar findings were shown in our previous work in breast cancer tumors of Stat1 deficient mice31. Our data shows that in the absence of STAT1, IL-17 may be responsible for the increased accumulation of CD11b+ Ly6G+ cells and F4/80+ CD206+ tumor associated macrophages in HNSCC tumor-bearing mice. It would therefore be worth investigating the efficacy of IL-17 blockade therapy on HNSCC development and metastasis outcomes.

In conclusion, we demonstrate that host STAT1 expression is essential for an effective anti-tumoral immune responses against HNSCC. This response is mediated by promotion of T lymphocyte expansion and cytotoxic activity, prevention of T cell exhaustion, and inhibition of MDSC and M2 macrophage accumulation. While STAT1 expression by HNSCC cells can promote immune evasion and tumor promotion, STAT1 expression by immune cells within the tumor microenvironment is essential for optimal anti-tumor immune responses during HNSCC. Our results provide a rationale for exploring targeted approaches that promote STAT1 activation in immune cells, as a potentially viable strategy to HNSCC therapy.

Supplementary Material

Supplementary Figure 1

Novelty and Impact:

We report a novel role for STAT1 in mitigating T cell exhaustion during head and neck squamous cell carcinoma. Using metastatic and non-metastatic oral cancer cells in immunocompetent BALB/c mice we show that STAT1 suppresses T cell exhaustion, promotes TNF-α production in T cells, and inhibits accumulation of myeloid derived suppressor cells. Induction of STAT1 signaling in immune cells within the oral tumor microenvironment could potentially improve head and neck tumor outcomes.

Acknowledgements:

This research was supported by Award Number K01CA207599 from the National Cancer Institute (NIH/NCI), UL1TR001070 from the National Center for Advancing Translational Sciences (NIH/NCATS) and RSG-19-079-01-TBG from the American Cancer Society (ACS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or American Cancer Society.

Abbreviations:

STAT

Signal transducer and activator of transcription

HNSCC

Head and neck squamous cell carcinoma

IFN

Interferon gamma

TNF

Tumor necrosis factor

Footnotes

Conflict of Interest: The authors declare no potential conflicts of interest

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Supplementary Materials

Supplementary Figure 1
NIHMS1605260-supplement-Supplementary_Figure_1.pptx (420KB, pptx)

Data Availability Statement

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