Figure 7.
IL-4 is essential for anti-PD-1–mediated enhancement of tumor-specific CD8+T cell responses. (A) C57BL/6 mice were injected s.c. with MC38-OVA tumor cells. After 10 days, mice were treated or not with anti-PD-1 mAb (250 µg, i.v.). Heat map showing the cytokine landscape (as detected by multiplex protein assay) of the draining lymph nodes, 3 days after anti-PD-1 mAb treatment. Each value was normalized to the mean value of control samples (untreated). Compiled from three independent experiments with a total of 10 mice per group. (B) Tumor-bearing mice were treated or not with anti-PD-1 mAb (250 µg, i.v.) and received either two doses of the BCL6 inhibitor FX1 or a vehicle as a control. IL-4 concentration was measured in the draining lymph node 3 days after treatment. Compiled from two independent experiments with a total of at least five mice per group. (C and D) Tumor-bearing mice were transferred with CTV-labeled OT-I CD8+ T cells and treated with anti-PD-1 Ab or left untreated and received either two doses of anti-IL-4 Ab or an isotype control. (C) Representative histograms showing CTV dilution in OT-I CD8+ T cells in the indicated group. (D) Quantification of OT-I CD8+ T cell replication index. Compiled from three independent experiments with a total of 9–10 mice per group. (E–G) Tumor-bearing mice were transferred with CTV-labeled OT-I CD8+ T cells and mice were treated (or not) with anti-PD-1 mAb (250 µg, i.v.) or received one dose of IL-4 complexes. (E) Representative histograms showing CTV dilution in OT-I CD8+ T cells. (F) Quantification of OT-I CD8+ T cell replication index. Compiled from four independent experiments with a total of 10–12 mice per group. (G) Pie charts and quantification showing TNF-α and IFN-γ production in OT-I CD8+ T cells. Compiled from two independent experiments with a total of five to six mice per group. (H and I) C57BL/6 mice were injected s.c. with MC38-OVA tumor cells. After 10 days mice were treated with anti-PD-1 mAb with or without anti-IL4 mAb. Control mice received the appropriate isotype controls (250 µg, i.v.). Detection of p-STAT6 in OT-I CD8+ T cells 2 days after treatment. RFI, ratio of fluorescence intensity. (H) Representative dot plots showing p-STAT6 staining in each cell generation identified by CTV dilution. (I) MFI of p-STAT6 staining on OT-I CD8+ T cells normalized to that measured with an isotype control. Representative of two independent experiments with four to five mice per group in each experiment. (J–L) OT-I CD8+ T cells were labeled by CTV and in vitro activated with OVA peptide. IL-4 was added on day 1 and 2 and CTV dilution was assessed on day 3. (J) Quantification of OT-I CD8+ T cell replication index in the presence or absence of IL-4. (K) Absolute number of OT-I CD8+ T cells in the presence or absence of IL-4. (L) The average size of divided OT-I CD8+ T cells was estimated by flow cytometry. Results from four independent experiments are shown. Each dot represents the mean of at least three replicates measured in each experiment. Statistical analyses were performed using two-way ANOVA (B, D, F, and I) and paired t test (J–L). *, P < 0.05; **, P < 0.01.

IL-4 is essential for anti-PD-1–mediated enhancement of tumor-specific CD8 + T cell responses. (A) C57BL/6 mice were injected s.c. with MC38-OVA tumor cells. After 10 days, mice were treated or not with anti-PD-1 mAb (250 µg, i.v.). Heat map showing the cytokine landscape (as detected by multiplex protein assay) of the draining lymph nodes, 3 days after anti-PD-1 mAb treatment. Each value was normalized to the mean value of control samples (untreated). Compiled from three independent experiments with a total of 10 mice per group. (B) Tumor-bearing mice were treated or not with anti-PD-1 mAb (250 µg, i.v.) and received either two doses of the BCL6 inhibitor FX1 or a vehicle as a control. IL-4 concentration was measured in the draining lymph node 3 days after treatment. Compiled from two independent experiments with a total of at least five mice per group. (C and D) Tumor-bearing mice were transferred with CTV-labeled OT-I CD8+ T cells and treated with anti-PD-1 Ab or left untreated and received either two doses of anti-IL-4 Ab or an isotype control. (C) Representative histograms showing CTV dilution in OT-I CD8+ T cells in the indicated group. (D) Quantification of OT-I CD8+ T cell replication index. Compiled from three independent experiments with a total of 9–10 mice per group. (E–G) Tumor-bearing mice were transferred with CTV-labeled OT-I CD8+ T cells and mice were treated (or not) with anti-PD-1 mAb (250 µg, i.v.) or received one dose of IL-4 complexes. (E) Representative histograms showing CTV dilution in OT-I CD8+ T cells. (F) Quantification of OT-I CD8+ T cell replication index. Compiled from four independent experiments with a total of 10–12 mice per group. (G) Pie charts and quantification showing TNF-α and IFN-γ production in OT-I CD8+ T cells. Compiled from two independent experiments with a total of five to six mice per group. (H and I) C57BL/6 mice were injected s.c. with MC38-OVA tumor cells. After 10 days mice were treated with anti-PD-1 mAb with or without anti-IL4 mAb. Control mice received the appropriate isotype controls (250 µg, i.v.). Detection of p-STAT6 in OT-I CD8+ T cells 2 days after treatment. RFI, ratio of fluorescence intensity. (H) Representative dot plots showing p-STAT6 staining in each cell generation identified by CTV dilution. (I) MFI of p-STAT6 staining on OT-I CD8+ T cells normalized to that measured with an isotype control. Representative of two independent experiments with four to five mice per group in each experiment. (J–L) OT-I CD8+ T cells were labeled by CTV and in vitro activated with OVA peptide. IL-4 was added on day 1 and 2 and CTV dilution was assessed on day 3. (J) Quantification of OT-I CD8+ T cell replication index in the presence or absence of IL-4. (K) Absolute number of OT-I CD8+ T cells in the presence or absence of IL-4. (L) The average size of divided OT-I CD8+ T cells was estimated by flow cytometry. Results from four independent experiments are shown. Each dot represents the mean of at least three replicates measured in each experiment. Statistical analyses were performed using two-way ANOVA (B, D, F, and I) and paired t test (J–L). *, P < 0.05; **, P < 0.01.

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