Defective removal of invariant chain peptides from MHC class II suppresses tumor antigen presentation and promotes tumor growth

Abstract

Tumor-draining lymph node dendritic cells (DCs) are poor stimulators of tumor antigen-specific CD4 T cells; however, the mechanism behind this defect is unclear. We now show that, in tumor-draining lymph node DCs, a large proportion of major histocompatibility complex class II (MHC-II) molecules retains the class II-associated invariant chain peptide (CLIP) fragment of the invariant chain bound to the MHC-II peptide binding groove due to reduced expression of the peptide editor H2-M and enhanced activity of the CLIP-generating proteinase cathepsin S. The net effect of this is that MHC-II molecules are unable to efficiently bind antigenic peptides. DCs in mice expressing a mutation in the invariant chain sequence that results in enhanced MHC-II-CLIP accumulation are poor stimulators of CD4 T cells and have diminished anti-tumor responses. Our data reveal a previously unknown mechanism of immune evasion in which enhanced expression of MHC-II-CLIP complexes on tumor-draining lymph node DCs limits MHC-II availability for tumor peptides.

Keywords: CLIP; CP: Cancer; CP: Immunology; MHC class II; antigen presentation; dendritic cells; invariant chain; tumor immunology; tumor-draining lymph node.

Figure 1.. Antigen presentation to CD4 T cells by DCs in tdLNs is defective

(A) Proliferation of OVA-specific T cells transferred to tumor-bearing mice in response to immunization with OVA/alum was determined by flow cytometry. Control mice were injected with PBS/alum. Three recipient mice per group were injected in each experiment, and the data shown are representative of 3 independent experiments. (B) The ability of DCs to form MHC-II-Eα_(52–68) complexes was determined by fluorescence-activated cell sorting (FACS). Four mice were injected with OVA-Eα fusion protein in each experiment, and the data shown are representative of 2 independent experiments. (C) DCs from tdLNs and ndLNs were cultured with T cells in the presence of OVA. The amounts of IFNγ and IL-2 released by T cells were measured by ELISA in triplicate. The data shown are representative of 3 independent experiments. (D) DCs from tdLNs and ndLNs were incubated with OVA-Eα fusion protein together with OVA-AF594 (D) or with Eα_(52–68) peptide alone (E), and their ability to form MHC-II-Eα _(52–68) complexes was analyzed by flow cytometry. The experiments were performed in triplicate, and data shown are representative of 3 independent experiments. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Figure 2.. Tumors enhance MHC-II-CLIP expression on cDC2s in tdLNs

(A) Gating strategy for phenotypic analysis of DC subsets in skin-draining LNs by flow cytometry (B) MHC-I, CD40, and MHC-II expression in migratory and resident DCs in tdLNs and ndLNs was determined by flow cytometry. Four mice were analyzed in each experiment, and the results shown are representative of 3 independent experiments. Isotype control staining from both ndLN and tdLN samples was identical. (C) MHC-II-CLIP expression in tdLN and ndLN DCs was determined by flow cytometry. The ratio of MHC-II-CLIP relative to the total MHC-II in each sample was calculated and normalized to ndLNs. Four mice were analyzed in each experiment, and the results shown are representative of 3 independent experiments. See also Figure S1. (D) Fluorescently labeled DCs were injected into tumor-bearing mice both i.t. and s.c. on the contralateral flank. MHC-II and MHC-II-CLIP expression on transferred DCs that migrated to the LNs was analyzed by flow cytometry. The ratio of MHC-II-CLIP amount relative to the total amount of MHC-II present in each sample was calculated and normalized to ndLN DCs. Four mice were analyzed in each group, and the data shown are representative of 2 independent experiments. Created with BioRender. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. tdLN cDC2s have reduced H2-M expression and enhanced cathepsin S activity

(A) The expression of H2-M in cDC subsets in tdLNs and ndLNs was determined by flow cytometry. Four mice were analyzed in each experiment, and the results shown are representative of 3 independent experiments. (B) Surface expression of MHC-II-CLIP in DC subsets in H2-M^+/+ or H2-M^+/− mice was analyzed by flow cytometry. Three mice from each strain were analyzed in each experiment, and the results shown are representative of 3 independent experiments. (C) Gating strategy used for the purification of DC subsets from tdLNs and ndLNs by flow cytometry. (D) The enzymatic activity of cathepsin S in cell lysates of FACS-sorted DCs from tdLNs and ndLNs was measured in a fluorometric assay. The results shown are from 4 independent experiments combined. (E) The expression of cystatin C in FACS-sorted DCs from tdLNs and ndLNs was determined by immunoblot analysis. The results shown are from 3 independent experiments combined. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01. See also Figures S4 and S6.

Figure 4.. Increased CLIP retention on MHC-II impairs antigen presentation to CD4 T cells

(A) The CLIP peptide sequence in WT Ii and M98A Ii mice. (B) Surface expression of MHC-II and MHC-II-CLIP complexes on WT Ii or M98A Ii DC subsets was determined by flow cytometry. Four mice were analyzed in each experiment, and the results shown are representative of 3 independent experiments. (C) The ability of WT Ii and M98A Ii DCs to form MHC-II-Eα_(52–68) and MHC-I-OVA_(257–264) complexes was measured by flow cytometry. Four mice were analyzed in each experiment, and the results shown are representative of 3 independent experiments. (D) The proliferation of OVA-specific T cells adoptively transferred to WT Ii or M98A Ii mice following immunization with OVA/alum was determined by flow cytometry. Three mice of each strain were injected in each experiment, and the data shown are representative of 3 independent experiments. (E) WT Ii or M98A Ii DCs were cultured with OT-II T cells either in the presence of OVA protein or OVA_(323–339) peptide in triplicate. The amount of IL-2 released by proliferating T cells was measured by ELISA. The data shown are representative of 3 independent experiments. (F) WT Ii or M98A Ii DCs were cultured with OT-II T cells in the presence of OVA protein under Th2 skewing conditions in triplicate. T cell proliferation and IL-4 production by T cells were analyzed by FACS. The data shown are representative of 3 independent experiments. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5.. Accumulation of MHC-II-CLIP in DCs diminishes anti-tumor responses

(A) S.c. B16F10-OVA tumor growth in WT Ii and M98A Ii mice was measured twice a week. Six mice of each strain were injected in each experiment, and the data shown are representative of three independent experiments. The statistical significance of differences in tumor growth between WT Ii and M98A Ii mice at each time point is indicated. (B) Proliferation of OT-II T cells cultured with DCs purified from tdLNs of B16F10-OVA tumor-bearing WT Ii or M98A Ii mice was measured by flow cytometry. In parallel experiments, IL-2 released into the culture medium by proliferating T cells was measured by ELISA in triplicate. The data shown are representative of 3 independent experiments. (C) Lung B16F10 melanoma metastasis was induced in WT Ii and M98A Ii mice, and metastatic lung foci were counted on the lung sections using the multipoint tool in the ImageJ software. Four mice of each strain were used in each experiment. The data shown are representative of 3 independent experiments. Representative images of H&E-stained lung sections for each mouse strain are shown. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01.