Gamma delta T cells provide an early source of interferon gamma in tumor immunity

Abstract

Interferon (IFN)-gamma is necessary for tumor immunity, however, its initial cellular source is unknown. Because gammadelta T cells primarily produce this cytokine upon activation, we hypothesized that they would provide an important early source of IFN-gamma in tumor immunosurveillance. To address this hypothesis, we first demonstrated that gammadelta T cell-deficient mice had a significantly higher incidence of tumor development after challenge with a chemical carcinogen methylcholanthrene (MCA) or inoculation with the melanoma cell line B16. In wild-type mice, gammadelta T cells were recruited to the site of tumor as early as day 3 after inoculation, followed by alphabeta T cells at day 5. We then used bone marrow chimeras and fetal liver reconstitutions to create mice with an intact gammadelta T cell repertoire but one that was specifically deficient in the capacity to produce IFN-gamma. Such mice had a higher incidence of tumor development, induced either with MCA or by inoculation of B16 melanoma cells, compared with mice with IFN-gamma-competent gammadelta T cells. Moreover, genetic deficiency of gammadelta T cells resulted in impaired IFN-gamma production by tumor antigen-triggered alphabeta T cell upon immunization with tumor lysate. These results demonstrate that gammadelta T cells can play a necessary role in tumor immunity through provision of an early source of IFN-gamma that in turn may regulate the function of tumor-triggered alphabeta T cells.

Figure 1.

γδ T cells are necessary to prevent tumor development. (A) Protective role of γδ T cells in the MCA-induced tumor model. Sex- and age-matched B6 wild-type (WT, n = 28) and B6 TCRδ-deficient mice (TCRδ^−/−, n = 24) were injected with 75 μg MCA/mouse in the flank. Tumor development was recorded weekly after injection. Tumor size >4 × 4 mm was considered positive as previously described (reference 3). (B) Protective role of γδ T cells and IFN-γ in the B16 melanoma transfer model. Four groups of age- and sex-matched mice (B6 wild-type mice, n = 12; B6 TCRβ-deficient mice, n = 18; B6 TCRδ-deficient mice, n = 21; B6 IFN-γ–deficient mice, n = 15), all at 8 wk of age were injected subcutaneously with 5 × 10⁴ B16 F0 melanoma cells and tumor growth was recorded daily. The percentage of mice with tumors 21 d after injection is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01.

Figure 2.

γδ T cells are recruited earlier than αβ T cells into tumor sites. B6 wild-type mice were inoculated with B16 F0 melanoma cells (5 × 10⁴ cells/mouse) and at different time points after inoculation, the tumor injection site was frozen and stained with specific antibodies against TCR γδ, CD4, and CD8 as described in Materials and Methods. (A) One slide from each time point depicting the staining for each cell type is shown. Examples of positive cells are indicated by arrows. (B) Summary of mean ± SD of the number of positive cells per high power field is shown. (C) Cell suspensions were prepared from pools of 10 tissues collected from tumor injection sites and cells were stained with anti-CD3 and anti–γδ TCR. A representative example of a flow cytometry plot is shown. (D) Stained cells (from C) were fixed and permeabilized for intracellular IFN-γ (PE) staining. A representative plot is shown.

Figure 2.

γδ T cells are recruited earlier than αβ T cells into tumor sites. B6 wild-type mice were inoculated with B16 F0 melanoma cells (5 × 10⁴ cells/mouse) and at different time points after inoculation, the tumor injection site was frozen and stained with specific antibodies against TCR γδ, CD4, and CD8 as described in Materials and Methods. (A) One slide from each time point depicting the staining for each cell type is shown. Examples of positive cells are indicated by arrows. (B) Summary of mean ± SD of the number of positive cells per high power field is shown. (C) Cell suspensions were prepared from pools of 10 tissues collected from tumor injection sites and cells were stained with anti-CD3 and anti–γδ TCR. A representative example of a flow cytometry plot is shown. (D) Stained cells (from C) were fixed and permeabilized for intracellular IFN-γ (PE) staining. A representative plot is shown.

Figure 2.

γδ T cells are recruited earlier than αβ T cells into tumor sites. B6 wild-type mice were inoculated with B16 F0 melanoma cells (5 × 10⁴ cells/mouse) and at different time points after inoculation, the tumor injection site was frozen and stained with specific antibodies against TCR γδ, CD4, and CD8 as described in Materials and Methods. (A) One slide from each time point depicting the staining for each cell type is shown. Examples of positive cells are indicated by arrows. (B) Summary of mean ± SD of the number of positive cells per high power field is shown. (C) Cell suspensions were prepared from pools of 10 tissues collected from tumor injection sites and cells were stained with anti-CD3 and anti–γδ TCR. A representative example of a flow cytometry plot is shown. (D) Stained cells (from C) were fixed and permeabilized for intracellular IFN-γ (PE) staining. A representative plot is shown.

Figure 3.

Generation and analysis of bone marrow chimeras. (A) Composition of αβ and γδ T cells in wild-type mice versus an experimental group of bone marrow chimeras. Bone marrow chimeras were prepared as described in Materials and Methods using donors and recipients (refer to Table I). At the end of the experiments, splenocytes from the experimental group and wild-type mice were prepared and stained with FITC anti–γδ TCR (GL3), PE anti–αβ TCR (H57-597), and CyChrome anti-CD3 (145-2C11). After gating on CD3⁺ T cells, the composition of γδ and αβ T cells was analyzed. Two controls and six reconstituted mice are shown. (B) IFN-γ production by γδ and αβ T cells in bone marrow chimeras. Splenocytes from bone marrow chimeras (experimental group, γδ IFN-γ²) were cultured with anti-CD3 and anti-CD28 in the presence of IL-12 for 5 d. Cells were then restimulated with anti-CD3 and anti-CD28 in the presence of brefeldin A for 6 h. After staining with FITC anti–TCR γδ and CyChrome anti–TCR αβ, cells were fixed, permeabilized, and further stained with PE anti–IFN-γ. After gating on αβ and γδ T cells, the percentage of IFN-γ⁺ cells was detected (see representative histogram).

Figure 4.

IFN-γ produced by γδ T cells is critical for protective immune responses against tumors. (A) Development of MCA-induced tumors in bone marrow chimeras and in unmanipulated mice. Four groups of age- and sex-matched mice (B6 wild-type mice, n = 10; B6 TCRβ-deficient mice, n = 13; B6 TCRδ-deficient mice, n = 11; B6 IFN-γ–deficient mice, n = 19; all at age 6 wk), and two groups of age- and sex-matched bone marrow chimeras (experimental group, n = 9; control group, n = 8; 10 wk after reconstitution) were injected with MCA (75 μg/mouse in the flank). Animals were observed weekly and tumor size was measured with a calimeter. The percentage of mice that developed tumors is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01. (B) B16 melanoma model in bone marrow chimeras. Bone marrow chimeras reconstituted as in A were injected with 5 × 10⁴ B16 F0 cells/mouse and tumor growth was recorded. The percentage of mice that developed tumors 21 d after inoculation is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01. (C) B16 melanoma model in mice reconstituted with fetal liver cells. TCRδ^−/− mice were reconstituted with fetal liver cells either from B6 TCRβ^−/− mice (γδ⁺ IFN-γ⁺) or from TCRβ^−/− IFN-γ^−/− (γδ⁺ IFN-γ⁻). 8 wk after reconstitution, mice were inoculated with 5 × 10⁴ B16 F0 cells/mouse and tumor growth was recorded. The percentage of mice that developed tumors 21 d after inoculation is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01. (D) Time course of B16 tumor development in bone marrow chimeras (B) and fetal liver reconstituted mice (C).

Figure 4.

IFN-γ produced by γδ T cells is critical for protective immune responses against tumors. (A) Development of MCA-induced tumors in bone marrow chimeras and in unmanipulated mice. Four groups of age- and sex-matched mice (B6 wild-type mice, n = 10; B6 TCRβ-deficient mice, n = 13; B6 TCRδ-deficient mice, n = 11; B6 IFN-γ–deficient mice, n = 19; all at age 6 wk), and two groups of age- and sex-matched bone marrow chimeras (experimental group, n = 9; control group, n = 8; 10 wk after reconstitution) were injected with MCA (75 μg/mouse in the flank). Animals were observed weekly and tumor size was measured with a calimeter. The percentage of mice that developed tumors is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01. (B) B16 melanoma model in bone marrow chimeras. Bone marrow chimeras reconstituted as in A were injected with 5 × 10⁴ B16 F0 cells/mouse and tumor growth was recorded. The percentage of mice that developed tumors 21 d after inoculation is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01. (C) B16 melanoma model in mice reconstituted with fetal liver cells. TCRδ^−/− mice were reconstituted with fetal liver cells either from B6 TCRβ^−/− mice (γδ⁺ IFN-γ⁺) or from TCRβ^−/− IFN-γ^−/− (γδ⁺ IFN-γ⁻). 8 wk after reconstitution, mice were inoculated with 5 × 10⁴ B16 F0 cells/mouse and tumor growth was recorded. The percentage of mice that developed tumors 21 d after inoculation is shown. Data represent mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.01. (D) Time course of B16 tumor development in bone marrow chimeras (B) and fetal liver reconstituted mice (C).

Figure 5.

γδ T cells regulate IFN-γ production by tumor antigen–triggered αβ T cells. B6 and B6 TCRδ^−/− mice (n = 5 for both strains) were immunized with tumor lysates in CFA and rechallenged in vitro with identically prepared lysates for 24 h. Brefeldin A was added for the last 4 h of culture and cells were then used for intracellular cytokine staining. After gating on CD3⁺ and CD8⁺ T cells, the percentage of IFN-γ–producing cells is shown. (A) The percentage of IFN-γ–producing cells from both CD8⁺ and CD4⁺ cells is shown (CD8⁻, lower left quadrant of dot plot shown in B). Data is mean ± SD of five different mice. **, P < 0.01; ***, P < 0.05. (B) One example of intra-cellular cytokine staining from wild-type (WT) and TCRδ^−/− mice is shown.