Murine CD27(-) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages

Abstract

Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17-deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27((-)) subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17-secreting CD27((-)) Vγ6((+)) γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17-dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.

Keywords: gamma-delta T cells; tumor immunology.

Fig. 1.

γδ T cells infiltrate ID8 tumors and enhance ovarian cancer cell growth in vivo. (A) ID8 tumor growth in C57BL/6 WT (n = 6) and TCRδ^−/− female mice (n = 8), measured by luciferase bioluminescence at the indicated weeks posttransplantation. (B) Representative FACS plots for γδ T cells in peritoneal exudates of ID8-bearing mice or PBS controls (at week 6 postinoculation). (C and D) Absolute numbers of total and BrdU⁺ γδ T cells in ID8-bearing mice or PBS controls. BrdU was provided during a period of 2 wk before analysis. Each dot represents one animal. (E) Representative immunofluorescence imaging of γδ T cells in ID8 tumor foci. Data are representative of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

γδ T cells are major providers of IL-17A, which promotes ovarian cancer cell growth. (A) Representative FACS plots of intracellular IL-17A and IFN-γ stainings in γδ T cells isolated from the peritoneal cavity at weeks 2 and 6 post-ID8 tumor cell inoculation. (B) Absolute numbers of IL-17⁽⁺⁾ or IFN-γ⁽⁺⁾ γδ T cells at weeks 2 and 6 after inoculation of PBS or ID8 tumor cells. Each dot represents one animal. (C) Total numbers of BrdU⁽⁺⁾ cells within IL-17A⁽⁺⁾ or IFN-γ⁽⁺⁾ γδ T subsets. BrdU was provided for a period of 2 wk before analysis. (D) Representative plot, absolute numbers, and mean fluorescence intensity (MFI) for IL-17 in total IL-17⁽⁺⁾ cells and respective contributions of CD4 and γδ T cells in the peritoneal cavity of tumor-bearing mice (n = 5) at weeks 2 and 6 postinoculation. (E) MFI for IL-17 in total IL-17⁽⁺⁾ cells in peritoneal exudates of WT or TCRδ^−/− tumor-bearing mice (n = 5) at weeks 2 and 6 postinoculation. (F) ID8 tumor growth in C57BL/6 WT and IL-17A^−/− mice, measured by luciferase bioluminescence. Statistical analysis was performed using the Mann–Whitney test. Data are representative of two to four independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

IL-17 production by γδ T cells is essentially restricted to a distinctive CD27⁽⁻⁾ Vγ6⁽⁺⁾ cell subset. Peritoneal exudates were analyzed at week 2 and week 6 postinoculation of ID8 cells or PBS. (A) Representative FACS plots of Vγ1 and Vγ4 stainings within IL-17A⁽⁺⁾ or IFN-γ⁽⁺⁾ γδ T cells in tumor-bearing mice (at week 6). (B) FACS staining with GL3 and 17D1 monoclonal antibodies to detect Vγ6⁽⁺⁾ γδ T cells. (C) Histogram overlay of CD27 staining in Vγ1, Vγ4, and Vγ6 subsets of γδ T cells in tumor-bearing mice (at week 6). (D) Absolute numbers of Vγ1, Vγ4, and Vγ6 subsets of IL-17A⁽⁺⁾ or IFN-γ⁽⁺⁾ γδ T cells in ID8 tumor-bearing mice or in PBS-injected controls (at week 6). Each dot represents one animal. (E) Representative intracellular IL-17A and IFN-γ stainings (Top), BrdU incorporation (Middle), and absolute numbers (Bottom) of Vγ1, Vγ4, and Vγ6 subsets of γδ T cells in ID8 tumor-bearing mice or in PBS-injected controls (at weeks 2 and 6). Data are representative of three independent experiments; *P < 0.05.

Fig. 4.

γδ T cells and IL-17 selectively mobilize a population of small peritoneal macrophages. (A) Quantitative PCR expression of vegfa and ang2 in peritoneal exudates of C57BL/6 wild-type (WT), IL-17A^−/−, and TCRδ^−/− mice, at week 6 of ID8 tumor development. (B) Percentage of F4/80^hi (large peritoneal macrophages, LPMs) and F4/80^lo (small peritoneal macrophages, SPMs) cells in WT, TCRδ^−/−, and IL-17A^−/− mice, at week 6 post-ID8 tumor cell inoculation. (C) Representative FACS staining of F4/80^hi (LPM) and F4/80^lo (SPM) cells in ID8- or PBS-injected mice (Left) and summary graphs for the percentage of SPMs at week 6 (Center) and from weeks 5 to 8 of tumor development (Right). (D and E) Forward scatter (FSC) and MHC class II expression in LPM and SPM cells, and respective MFI (n = 5), at week 6 of tumor development. Data are representative of three independent experiments; *P < 0.05, **P < 0.01.

Fig. 5.

Small peritoneal macrophages express a proangiogenic gene profile and directly promote tumor cell proliferation. (A) F4/80^hi (LPM) and F4/80^lo (SPM) macrophages from the peritoneal exudates of C57BL/6 wild type were FACS sorted after 6 wk of ID8 tumor development and analyzed by RT-qPCR for tnfa, tgfb, vegfa, cxcl1, cxcl8, il10, il1b, il6, il17ra, mif, tie2, and cd163 expression, normalized to the housekeeping gene hprt. Bars represent SD; *P < 0.05, **P < 0.01, ***P < 0.005. (B and C) ID8 tumor cell numbers (at 24, 48, and 96 h) and BrdU incorporation (at 48 h) upon noncontact coculture (at 1:1 ratio) with LPMs or SPMs, sorted from ID8 tumor-bearing mice (at week 6). Data are representative of two to three independent experiments; **P < 0.01.

Fig. 6.

CD27⁽⁻⁾ Vγ6⁽⁺⁾ γδ T cells and IL-17 up-regulate proangiogenic and proinflammatory mediators in SPMs. FACS-sorted SPMs from peritoneal exudates of week 6 ID8-bearing C57BL/6 wild-type mice were (A) cocultured for 24 h with Vγ6⁽⁺⁾ γδ T cells (in a Transwell system) at 1 γδ:25 SPM ratio or (B) incubated for 24 h with recombinant IL-17. Gene expression analysis for tgfb, vegfa, cxcl1, mif, il1b, gata6, il17ra, and cd163 was performed by RT-qPCR and normalized to the housekeeping gene hprt. Bars represent SD; *P < 0.05, **P < 0.01, ***P < 0.005. Additional data from an independent experiment are shown in Fig. S7.