Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: Treg cell depletion unleashes IFNγ-producing γδ T cell responses. (A) Schematic representation of the experimental approach. 1 × 10 6 of E0771 breast cancer cells were inoculated subcutaneously in the mammary fat pad of Foxp3-DTR mice. DTx in PBS was administered intraperitoneally on days 7, 9, 11 (50 μg/kg), and 14 (25 μg/kg) after tumor inoculation. (B) Percentages of Treg cells in tumors at day 15 after tumor inoculation ( n = 12 mice per group), represented as means ± SEM and analyzed by Mann–Whitney U test. (C) Tumor growth of PBS- or DTx-treated mice ( n = 14 mice per group). Means ± SEM, repeated measures two-way ANOVA with Sidak’s multiple comparisons test. (D) Fold change of numbers of γδ T cells per milligram of tumor ( n = 22 control and 23 DTx mice), normalized over controls, represented as means ± SEM and analyzed by the Mann–Whitney U test. Data in B–D represent a pool of three independent experiments. (E) Unsupervised hierarchical clustering of tumor-infiltrating γδ T cells from PBS (pool of n = 3 mice) or DTx-treated mice (pool of n = 3 mice), based on spectral flow cytometry data. (F) Distribution of clusters across the individual mice ( n = 6). (G) Protein expression levels of different markers across clusters. Data in E–G represent one representative experiment out of >3 independent experiments. (H) Representative density plots (gated on γδ T cells) and quantification of IFNγ + γδ T cells in tumors ( n = 22 control and 23 DTx mice). Fold change of percentage, numbers per milligram, and mean fluorescence intensity (MFI) over controls are represented. (I) Fold change of percentage of IL-17A + γδ T cells and numbers per milligram in tumors ( n = 22 Control and 23 DTx mice). (J) γδ T cell polarization measured by the ratio of percentages of IFNγ + versus IL-17A + γδ T cells (γδIFN/γδ17) ( n = 18 control and 19 DTx mice). Data in H–J are a pool of three different experiments. (K) Representative density plots (gated on IFNγ + γδ T cells) and quantification of proliferation of tumor IFNγ + γδ T cells, measured by Ki-67 ( n = 5 control 7 DTx mice) and BrdU ( n = 7 mice per group) staining. One representative out of two independent experiments. (L) Fold change of percentages of IFNγ + γδ T cells in the tumor dLNs spleen of control and DTx-treated mice ( n = 22 control and 23 DTx mice). (M) Fold change of percentages of Vγ subsets (within γδ T cells) in the tumors ( n = 22 control and 23 DTx mice). Data in L and M are a pool of three different experiments. (N) Fold change of percentages of granzyme B + cells within γδ T cells in tumors ( n = 10 control and 12 DTx mice), one representative out of three independent experiments. Data in H-N represented as means ± SEM and analyzed by unpaired t test for normal distributions or Mann–Whitney U test for non-normal distributions. (O) Quantification of tumor cell death of E0771 cells over a 24-h killing assay in the presence of γδ T cells, Treg cells, or both, measured by percentage of annexin V + cells ( n = 3–4 replicates). Data are represented as means ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. One representative out of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: MANN-WHITNEY, Control, Flow Cytometry, Expressing, Fluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: IFNγ + γδ T cell expansion upon Treg cell depletion in MC38 colon cancer model and in tumor-free mice. (A) Schematic representation of the experimental approach. 2 × 10 6 of MC38 colon cancer cells were inoculated subcutaneously in the flank of Foxp3-DTR mice. DTx in PBS was administered intraperitoneally on days 6, 8, 10 (50 μg/kg), and 12 (25 μg/kg) after tumor inoculation. (B) Tumor growth of PBS- or DTx-treated mice ( n = 6 mice per group). Means ± SEM, repeated measures two-way ANOVA with Sidak’s multiple comparisons test. (C) Quantification of the frequency of IFNγ + γδ T cells and of proliferation of IFNγ + γδ T cells, measured by Ki-67 ( n = 6) staining, in tumors. (D) Schematic representation of the experimental approach. Foxp3-DTR mice were injected DTx in PBS on days 0, 2, 4 (1.5 μg), and 7 (0.75 μg). ( E ) Quantification of percentages of Treg cells and γδ T cells within CD45 + cells and quantification of IFNγ and IL-17A expression by γδ T cells in the spleen, inguinal LN, mammary fat, lung, and colonic lamina propria of DTx-treated or control mice. Data are represented as means ± SEM and analyzed by unpaired t test (for normal distributions) or Mann–Whitney U test (for non-normal distributions). Data are representative of two independent experiments. Data are represented as means ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: Staining, Injection, Expressing, Control, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: Treg cell depletion increases systemic IFNγ-producing αβ T cells and reveals the anti-tumor contribution of CD8 T cells. (A–C) Quantification of IFNγ + CD8 and CD4 (Th1) T cells as a fold change of percentage and numbers per milligram in (A) tumors or as a fold change of percentages in (B) dLNs and (C) spleen of DTx-treated over control mice. Data are represented as means ± SEM of a pool of three independent experiments and analyzed by unpaired t test for normal distributions or Mann–Whitney U test for non-normal distributions. (D) Schematic representation of the experimental approach. 1 × 10 6 of E0771 breast cancer cells were inoculated in the mammary fat pad of Foxp3-DTR mice. DTx in PBS was administered intraperitoneally on days 7, 9, 11 (1.5 μg), and 14 (0.75 μg) after tumor inoculation. On the same days, 100 μg of αCD8β mAb or isotype control was administered intraperitoneally. (E) Percentages of CD8 T cells (within CD45 + T cells) in tumors ( n = 6 isotype, 5 DTx + isotype, 6 αCD8β, and 5 DTx + αCD8β). Data are represented as means ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (F) Tumor growth of mice treated with isotype ( n = 7), αCD8β ( n = 5), DTx + isotype ( n = 6), or DTx + αCD8β ( n = 5), analyzed by repeated measures two-way ANOVA with Sidak’s multiple comparisons test. (G) Percentages of γδ T cells (within CD45 + T cells) and IFNγ+ within γδ T cells ( n = 6 isotype, 5 DTx + isotype, 6 αCD8β, and 5 or DTx + αCD8β) in tumors. Data are representative of two independent experiments, are represented as means ± SEM, and analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: Control, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: IFNγ-biased Vγ1 + γδ T cells are anti-tumor effectors of Treg depletion therapy. ( A ) Schematic representation of the experimental approach. 1 × 10 6 of E0771 breast cancer cells were inoculated in the mammary fat pad of Foxp3-DTR mice. DTx in PBS was administered intraperitoneally on days 7, 9, 11 (1.5 μg), and 14 (0.75 μg) after tumor inoculation. On the same days, 100 μg of αVγ1 mAb or isotype control was administered intraperitoneally. (B) Tumor growth of mice treated with PBS + isotype ( n = 10), DTx + isotype ( n = 9), PBS + αVγ1 ( n = 9), or DTx + αVγ1 ( n = 7). Pool of two independent experiments, repeated measures two-way ANOVA with Sidak’s multiple comparisons test. (C) Percentages of Treg cells in tumors at day 15 after tumor inoculation. (D) Percentages of γδ T cells (within CD45 + T cells) and Vγ1 + and Vγ4 + cells within γδ T cells ( n = 10 PBS + isotype, 9 DTx + isotype, 9 PBS + αVγ1, and 7 DTx + αVγ1). Data in C and D, represented as means ± SEM, are a pool of two independent experiments and analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (E) Percentage of IFNγ + cells within γδ T cells and representative density plots. (F) Percentage of IFNγ-producing CD4 and CD8 αβ T cells. Data in E and F, represented as means ± SEM ( n = 5 PBS + isotype, 4 DTx + isotype, 6 PBS + αVγ1, and 3 DTx + αVγ1), are representative of two independent experiments and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: Control

Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: Inhibition of IL-10, IL-35, and adenosine pathways does not impact Treg-mediated suppression of IFNγ + γδ T cells. (A) Quantification of proliferation index of IFNγ + γδ T cells in the presence or absence of Treg cells and in the presence of αIL-10– or αIL-35–neutralizing antibody or the CD39 inhibitor ARL67156 (fold change over γδ T cells only is represented). Data are representative of two independent experiments. (B) Schematic representation of the experimental approach. 1 × 106 of E0771 breast cancer cells were inoculated in the mammary fat pad of Foxp3-DTR mice (without any DTx administration). In addition, 200 μg of αIL-10–neutralizing antibody was administered intraperitoneally on days −1, 0, 1, and 3 relative to tumor inoculation, followed by intratumoral injections on days 7, 10, and 13. (C) Tumor growth of αIL-10–treated or control mice ( n = 7 mice per group). Data are analyzed by repeated measures two-way ANOVA with Sidak’s multiple comparisons test without reaching significant differences between the two groups. (D) Percentages of γδ T cells within CD3 + T cells ( n = 7 mice per group) and IFNγ + cells within γδ T cells ( n = 6 mice per group). Data are represented as means ± SEM and analyzed by unpaired t test. **P < 0.01 and ****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: Inhibition, Control

Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: IL-2 neutralization in the absence of Treg cells limits anti-tumor γδ T cell responses and impairs tumor control. (A) Schematic representation of the experimental approach. 1 × 10 6 of E0771 breast cancer cells were inoculated in the mammary fat pad of Foxp3-DTR mice. DTx in PBS was administered intraperitoneally on days 7, 9, 11 (1.5 μg), and 14 (0.75 μg) after tumor inoculation. Between days 7 and 14 a combo of two αIL-2 antibodies (100 μg of S46B6-1 and 100 μg of JES6-1A12) or isotype controls was administered intraperitoneally daily to neutralize IL-2. (B) Tumor growth of PBS + isotype- (control), DTx + isotype-, PBS + αIL-2–, or DTx + αIL-2–treated mice ( n = 5 mice per group). Data are representative of two independent experiments with similar trends and were analyzed by repeated measures two-way ANOVA with Sidak’s multiple comparisons test. (C) Percentages of Treg cells in tumors at day 15 after tumor inoculation. (D and E) (D) Percentages of γδ T cells within CD45 + T cells and (E) CD25 expression within γδ T cells. (F) Percentage of IFNγ + cells and granzyme B + cells within γδ T cells and representative density plots. Data in C–F correspond to a pool of two independent experiments with similar trends ( n = 10 PBS + isotype, 10 DTx + isotype, 5 PBS + αIL-2, or 9 DTx + αIL-2, represented as means ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons test). (G) Quantification of proliferation of IFNγ + γδ T cells by Ki-67 expression ( n = 5 mice per group). One representative out of two independent experiments, analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: Neutralization, Control, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Regulatory T cells sabotage anti-tumor γδ T cells by creating IL-2–deficient environments

doi: 10.1084/jem.20252133

Figure Lengend Snippet: IL-2Rβ γ c agonism promotes anti-tumor murine γδ T cell responses and tumor control. (A) Schematic representation of the experimental approach. 1 × 10 6 of E0771 breast cancer cells were inoculated in the mammary fat pad of Foxp3-DTR mice (without any DTx administration), and 10 μg of Neo2/15 was administered intraperitoneally daily between days 7 and 14 in the indicated group. (B) Tumor growth of Neo2/15-treated or control mice ( n = 9 mice per group). Data were analyzed by repeated measures two-way ANOVA with Sidak’s multiple comparisons test. (C) Percentages of Treg cells in tumors at day 15 after tumor inoculation ( n = 9 mice per group). (D and E) (D) Percentages of γδ T cells within CD45 + T cells and (E) CD25 expression within γδ T cells ( n = 9 mice per group). Data in B–E are represented as means ± SEM of a pool of two independent experiments and analyzed by unpaired t test. (F) Percentage of IFNγ + cells and granzyme B + cells within γδ T cells and representative density plots ( n = 5 control mice and 4 Neo2/15-treated mice). Data are represented as means ± SEM of one representative out of two independent experiments and analyzed by unpaired t test. (G) Schematic representation of the experimental approach to deplete Vγ1 + γδ T cells together with Neo2/15 administration. Briefly, 1 × 106 of E0771 breast cancer cells were inoculated in the mammary fat pad of Foxp3-DTR mice. 10 μg of Neo2/15 in PBS was daily administered intraperitoneally. In addition, 100 μg αVγ1 mAb or isotype control was intraperitoneally administered on days 7, 9, 11, and 14 after tumor inoculation. (H) Tumor growth of mice treated with isotype ( n = 5), Neo2/15 ( n = 5) or Neo2/15 + αVγ1 ( n = 5), analyzed by repeated measures two-way ANOVA with Sidak’s multiple comparisons test and were representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001.

Article Snippet: C57BL/6J Foxp3-DTR (B6.129 [Cg]-Foxp3tm3 [DTR/GFP] Ayr/J) (Foxp3-DTR [C57BL/6 background] [ ]) were obtained from The Jackson Laboratory and are backcrossed for at least eight generations to C57BL/6NTac mice, and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG-Tg[Hu-IL15] [NOD/ShiLtJ background]) were obtained from The Jackson Laboratory, and C57BL/6J FOXP3-hCD2/IL-17A-GFP (CD57BL/6 background) mice were bred in house from C57BL/6J FOXP3-hCD2 mice, kindly provided by Prof. Shohei Hori (University of Tokyo, Japan), and C57BL/6J IL-17A-GFP mice were obtained from Biocytogen.

Techniques: Control, Expressing