Fig 1.
Tumor-associated neutrophils suppress γδ 17 T-cell responses.
Frequency of (A) total and IL-17+ γδT cells and (B) CD8+ and CD4+ T cells in the PEC of tumor-free and B16-F0 tumor-bearing mice. Data were pooled from four different experiments. (C) Representative FACS plots and summary of neutrophil, monocyte, and Treg cell frequency in the PEC of tumor-free and B16 tumor-bearing mice. Data were pooled from four independent experiments. (D) Frequency of IL-17+ γδ T cells in B16 tumor–bearing mice injected with vehicle (PBS) or mAb αGr-1, αLy6G, αCD115 + clodronate liposomes, and αCD25. (E) Representative FACS plots and frequency of neutrophils in tumor-free liver and within Hepa 1–6 intrahepatic tumor developed in C57BL/6 mice and (F) frequency of IL-17+ γδ T cells within Hepa 1–6 tumors developed in mice deficient/depleted for neutrophils (Neu −) or respective controls (Neu +). Red and blue circles represent αGr-1 mAb-treated or PBS-treated C57BL/6 mice, respectively, whereas red and blue triangles represent Genista homozygous or littermate controls, respectively. (G) Left: intrahepatic Hepa 1–6 tumor growth in mice with (heterozygous littermate control, n = 10) and without (Genista homozygous, n = 4) mature neutrophils. Data were pooled from two independent experiments. Right: intrahepatic Hepa 1–6 tumor growth in C57Bl/6J WT (n = 5) and Il17−/− (n = 5) mice treated with αGr-1. Data presented as mean ± SEM. Statistical analysis was performed using Student t test or Mann-Whitney test. Data are provided in S1 Data. Lipo, clodronate liposome; mAb, monoclonal antibody; PEC, peritoneal exudate cell; RLU, relative luminescence units; Treg, regulatory T; γδ17, IL-17–producing γδ T cell.
Fig 2.
Neutrophils selectively inhibit the proliferation of Vγ6+ γδ T cells.
Representative FACS plots and/or frequency (gated on CD45+ lymphocytes) of (A) γδ T cells, (B) CD4+ and CD8+ T cells, and (C) Vγ1−Vγ4− γδ T cells (gated on γδ T cells) in intraperitoneal B16 or intrahepatic Hepa 1–6 tumors, developed in mice deficient/depleted for neutrophils (Neu −) or respective controls (Neu +). Red and blue circles represent αGr-1 mAb-treated or PBS-treated C57BL/6 mice, respectively, whereas red and blue triangles represent Genista homozygous or littermate controls, respectively. Data were pooled from two (Hepa 2–6) and three to five (B16) independent experiments. (D) Representative FACS plots of γδ T-cell phenotype in PBS− (Neu +) or αGr-1 (Neu −) mAb-treated B16 tumor–bearing mice. (E) Frequency of BrdU+ Vγ6+ T cells in B16 tumor–bearing mice and of Ki67+ Vγ6+ T cells in Hepa 1–6 tumor–bearing mice at days 9 and 21 post–tumor inoculation, respectively. Statistical analysis was performed using Student t test or Mann-Whitney test. Data are provided in S1 Data. BrdU, bromodeoxyuridine; mAb, monoclonal antibody; TCR, T-cell receptor.
Fig 3.
Tumor-associated neutrophils inhibit CD27− Vγ6+ γδ T-cell proliferation by inducing oxidative stress.
(A) Representative histograms and summary of in vitro CD27− γδ T-cell proliferation cultured alone (n = 13), in the presence of neutrophils from BM of B16 tumor–free (n = 3) or tumor–bearing mice (n = 5), or with neutrophils from the PEC of B16 tumor–bearing mice (n = 7). Data were pooled from four independent experiments. (B) Total superoxide-positive cells in B16 tumor–bearing mice depleted (αGr-1 mAb, Neu −, n = 13) or not (Neu +, n = 8) for neutrophils. Data were pooled from three independent experiments. Total hydrogen peroxide levels in peritoneal supernatants of B16 tumor–bearing mice depleted (αGr-1 mAb, Neu −, n = 8) or not (Neu +, n = 7) for neutrophils. Data are representative of two independent experiments. (C) Protein oxidation assessed by flow cytometry in total γδ T cells from neutrophil-sufficient and neutrophil-depleted B16 tumor–bearing PEC. (D) Gene expression of oxidative stress–related genes in Vγ6+ T cells, CD4+, and CD8+ T cells sorted from B16 tumor–bearing PEC (Neu +), relative to the same populations sorted from neutrophil-depleted B16 tumor–bearing PEC (Neu −), normalized to Hprt. (E) Representative histograms and summary of in vitro CD27− γδ T-cell proliferation, cultured alone or in the presence of neutrophils from the PEC of C57Bl/6J or Cybb−/− (Nox2−/−) B16 tumor–bearing mice (n = 4, each). (F) Frequency of Vγ6+ and IL-17+ γδ T cells in PEC of C57Bl/6J and Cybb−/− (Nox2−/−) B16 tumor–bearing mice, 13 days post–tumor inoculation. Data were pooled from two independent experiments. (G) Frequency of Vγ6+ T cells and IL17+ γδ T cells in PEC of C57Bl/6J B16 tumor–bearing mice, treated with PBS or NAC. Data were pooled from two independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey HSD post hoc test, Student t test, or Mann-Whitney test. Data are provided in S1 Data. BM, bone marrow; CTV, cell trace violet; Cybb, cytochrome B(−245), β subunit; DNP, dinitrophenyl; Gclm, glutamate-cysteine ligase modifier subunit; Gcl, glutamate-cysteine ligase; Gpx, glutathione peroxidase; Gsr, glutathione reductase; Hprt, hypoxanthine-guanine phosphoribosyltransferase; mAb, monoclonal antibody; MFI, mean fluorescence intensity; NAC, N-acetylcysteine; Nfe2l2, nuclear factor, erythroid 2 like 2; Nox2, NADPH oxidase 2; PEC, peritoneal exudate cells; Prdx, peroxiredoxin; Sod1, superoxide dismutase 1; Srxn1, sulfiredoxin-1; Txn, Thioredoxin; Txnrd, Thioredoxin reductase.
Fig 4.
Murine CD27− γδ T cells and human Vδ1+ γδ T cells express low levels of glutathione and are highly susceptible to ROS.
(A) FACS-sorted CD27− and CD27+ γδ T cells were stimulated and proliferation was assessed by CTV dilution, with increasing concentrations of H2O2 (left, n = 2–3) or with different concentrations of the superoxide-generating system X/XO, right. (B) Total glutathione levels in CD27− γδ, CD27+ γδ, CD8+, and CD4+ T cells sorted from spleen and lymph nodes of tumor-free mice. Dotted lines link subsets from the same mouse. (C) Schematic representation of enzymes involved in redox metabolism. (D) Expression of redox-related genes in IL-17+ γδ T cells relative to IFN-γ+ γδ T cells at steady state, normalized to Hprt or β2microglobulin. (E) FACS-sorted Vδ1+, Vδ2+, CD8+, and CD4+ T cells (from buffy coats of healthy donors) were stimulated for 6 days in the presence of H2O2 (n = 4) and proliferation was assessed by CTV dilution, left. Total glutathione levels in Vδ1+, Vδ2+, CD4+, and CD8+ T cells (n = 5) sorted from buffy coats of healthy donors, right. Statistical analysis was performed Wilcoxon-matched-pairs signed rank test, Mann-Whitney test, and two-way ANOVA, followed by Tukey HSD post hoc test. Data are provided in S1 Data. CTV, cell trace violet; Gcl, glutamate-cysteine ligase; Gclc, glutamate-cysteine ligase catalytic subunit; Gclm, glutamate-cysteine ligase modifier subunit; Gpx, glutathione peroxidase; GSH, glutathione; Gsr, glutathione reductase; Gss, glutathione synthetase; Hprt, hypoxanthine-guanine phosphoribosyltransferase; Mdh2, malate dehydrogenase 2; Pgd, Phosphogluconate dehydrogenase; Prdx, Peroxiredoxin; ROS, reactive oxygen species; Txn, Thioredoxin; Txnrd, Thioredoxin reductase; X/XO, xanthine/xanthine oxidase.