Group 2 innate lymphoid cells boost CD8+ T-cell activation in anti-tumor immune responses

Abstract

Group 2 innate lymphoid cells (ILC2s) are essential for orchestrating type 2 immune responses during allergic airway inflammation and infection. ILC2s have been reported to play a regulatory role in tumors; however, this conclusion is controversial. In this study, we showed that IL-33-activated ILC2s could boost CD8⁺ T-cell function through direct antigen cross-presentation. After activation by IL-33, ILC2s showed an enhanced potential to process antigens and prime CD8⁺ T cell activation. Activated ILC2s could phagocytose exogenous antigens in vivo and in vitro, promoting antigen-specific CD8⁺ T cell function to enhance antitumor immune responses. Administration of OVA-loaded ILC2s induces robust antitumor effects on the OVA-expressing tumor model. These findings suggested that the administration of tumor antigen-loaded ILC2s might serve as a potential strategy for cancer treatment.

Keywords: CD8 T cell; ILC2; anti-tumor response; antigen presentation.

Figure 1.

IL-33-activated ILC2s upregulated MHC-I molecules and molecules related to antigen processing and presentation. (a) GSEA enrichment analysis identifying the MHC-I protein complex in activated ILC2s. NES, normalized enrichment score; FDR, false discovery rate. (b) Heatmap of the log₂-transformed expression of selected MHC class I-associated molecules and costimulatory molecules from RNA-sequencing data across populations. (c) GSEA enrichment analysis showing enrichment of the antigen processing and presentation of peptide antigen via MHC class I pathway. (d) Heatmap of the log₂-transformed expression of selected cathepsin proteins and antigen processing-related genes. (e) RT-PCR analysis of the expression of the heavy and light chains of MHC-I: B2M, H2-D1, and H2-K1. (f) RT-PCR analysis of the expression of the heavy and light chains of MHC-1 in ILC2s stimulated with IL-33 for 3 days in vitro. (g) the levels of the heavy and light chains of MHC-I in IL-33-stimulated ILC2s were analyzed on different days by RT-PCR. (h) the level of CD80 in ILC2s was analyzed by RT-PCR. (i) Representative flow cytometric histogram overlays and quantitative analysis of CD80 expression on ILC2s from IL-33-treated mice. (j) the level of CD86 in ILC2s was analyzed by RT-PCR. (k) Representative flow cytometric histogram overlays and quantitative analysis of CD86 expression on ILC2s from IL-33-treated mice. For RNA seq and cytometric data, n = 2 mice per group; for RT-PCR, n = 2 replicates in each experiment. Data are shown as the means ± SEMs. The p value was determined by one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 2.

Ilc2s phagocytosed antigens in vitro and in vivo. (a) Flow cytometric histogram overlays and quantitative analysis depicting in vitro uptake of FITC-dextran by ILC2s (n = 3 independent wells). (b) Analysis of tumor-derived mCherry fluorescence in ILC2s cocultured with B16F10-SOVA tumor cells (n = 3 independent wells). (c) Schematic showing the strategy for the lung metastasis model. (d) Representative flow cytometry plot and gating of tumor-infiltrating immune cells. (e) Frequency of mCherry⁺ immune cells across tumors (n = 5 mice per group). (f) Representative flow cytometric histograms of tumor-derived mCherry fluorescence within tumor-infiltrating ILC2s from day 7 through day 28. (g) Frequency of mCherry⁺ ILC2s in the tumor over the course of tumor progression (n = 5 mice per group). (h) Flow cytometry plot and frequency of CD69⁺ CD8 were analyzed by flow cytometry (n = 3 independent wells). Data are shown as the means ± SEMs. The p value was determined by one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 3.

ILC2s formed cell clusters with CD8⁺ T cells in the presence of antigen. (a) the cells showing red fluorescence are ILC2s, whereas those with no fluorescence are CD8⁺ T cells. ILC2s (2 × 10 were cocultured with CD8⁺ T (6 × 10 cells for 20 hours. Live cell imaging of ILC2–CD8⁺ T-cell interactions at different time points revealed that the cells formed constant cell clusters during the coculture. Scale bar = 50 μm or 5 μm. (b) Statistical analysis of cell cluster number per field (n = 4 views per well). (c) Statistical analysis of cell cluster area percentage per field (n = 4 views per well). Data are shown as the means ± SEMs. The p value was determined by two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 4.

ILC2s promoted OT-I CD8⁺ T-cell proliferation and cytokine production with OVA-stimulation. (a) Analysis of CD8⁺ T-cell proliferation using CTV by flow cytometry as described in the Methods. CTV-labeled OT-I CD8⁺ T cells were cultured with ILC2s in the absence or presence of 10 μM OVA_257–264. CD8⁺ T-cell proliferation was measured by flow cytometry, as shown in the histograms. Column chart showing the percentages of proliferating OT-I CD8⁺ T cells (n = 3 independent wells). (b) CTV-labeled OT-I CD8⁺ T cells were cultured with ILC2s in the absence or presence of soluble OVA protein. CD8⁺ T-cell proliferation was measured by flow cytometry (n = 3 independent wells). (c) After pretreatment with or without the OVA protein, isolated ILC2s and supernatants were separately cultured with CD8⁺ T cells. CD8⁺ T-cell proliferation was measured by flow cytometry (n = 3 independent wells). (d) CFSE-labeled OT-I CD8⁺ T cells were cultured with ILC2s in the absence or presence of soluble OVA protein or MOG protein. CD8⁺ T-cell proliferation was measured by flow cytometry(n = 3 independent wells). (e–f) the IFN-γ and TNF-α protein levels in the supernatants were detected by ELISA (n = 3 independent wells). (g) the percentage of GZMB⁺ in CD8⁺ T cells were detected by flow cytometry(n = 3 independent wells). Data are shown as the means ± SEMs. The p value was determined by one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 5.

OVA-loaded ILC2s increased CD8⁺ T cells cytotoxicity and delayed antigen-specific tumor growth. (a) Schematic diagram of the experimental design. Activated ILC2s were loaded with OVA protein or MOG protein. The ratio of OT-I CD8⁺T cells to tumor cells was 10:1. (b) Representative data for LDH release from B16F10-OVA cells after treatment with OT-I CD8⁺ T cells induced by ILC2s (n = 3 independent wells). (c) Representative images of B16F10-OVA cells stained with crystal violet after co-culture with OT-I CD8⁺ T cells. Statistical analysis of the areas of living cells at the bottom of the plate was performed (n = 3 views per well). Data are shown as the mean ± SEMs. The p value was determined using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. (d) B16F10-OVA(2 × 10 mixed with OVA loaded or not ILC2s (2 × 10 were inoculated subcutaneously until the maximum tumor reached ~1000 mm³. (e–f) Tumor growth curve and volume in different treatment groups (n = 4–6 mice per group). Data are shown as the mean ± SEMs. The p value was determined using a two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. (g–i) the percentages of CD8⁺ T cells and Treg cells and cell ratio in the tumors of ILC2- and OVA-loaded ILC2-treated mice and control mice (n = 4–6 mice per group). Data are shown as the mean ± SEMs. The p value was determined by one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 6.

Adoptive transfer of OVA-loaded ILC2s inhibited B16F10-OVA lung metastasis. (a) Schematic diagram showing strategy for OVA-loaded ILC2s adoptive transfer treatment in lung metastasis model. (b) Representative pictures of lung tumor clones. (c) Representative photomicrographs of haematoxylin/eosin(HE)-stained lung sections. (d) the metastatic tumor nodules in lung in different treatment group (n = 6 mice per group). (e) the lung weight in different treatment group (n = 3 mice per group). (f–h) the percentages of CD8⁺ T cells and Treg cells and cell ratio in the tumors of ILC2- and OVA-loaded ILC2-treated mice and control mice (n = 3 mice per group). Data are shown as the means ± SEMs. The p value was determined by one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.