Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy

Abstract

Background: Homocitrullination is the post-translational modification of lysine that is recognized by T cells.

Methods: This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry.

Results: Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment.

Conclusion: We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

Keywords: CD4-positive T-lymphocytes; cellular; immunity; immunization; immunotherapy; vaccination.

Figure 1

Immunization with homocitrullinated peptides can induce modification-specific immune responses. IFNγ ELISpot responses were assessed for HHDII/DP4 mice immunized with individual peptides containing Hcit residues (A) or lysine residues (WT) (B). Aldo238-256wt was not tested. Symbols represent mean response for individual mice, line represents median value between mice. Significant p values are shown. Data are representative of independent studies where n=3. ELISpot, enzyme-linked immunosorbent spot assay; Hcit, homocitrulline; IFNγ, interferon gamma; WT, wild type.

Figure 2

Hcit-specific immune responses are CD4 mediated and mediate efficient tumor therapy in vivo. IFNγ ELISpot responses were assessed for HHDII/DP4 mice immunized with homocitrullinated peptides. Splenocytes from Aldo74-93^Hcit, Aldo140-157^Hcit Cyk371-388^Hcit, Bip328-346^Hcit, Bip562-579^Hcit or Eno156-176^Hcit immunized mice were restimulated with peptides alone or in combination with anti-CD4 or anti-CD8 blocking antibodies ex vivo (A) or after 7-day culture (B). Data are collated from at least two independent studies where n=3. In vivo tumor survival studies were carried out by implanting mice with HLA-matched B16F1 cells with constitutive MHC-II (B16 HHDII/DP4) or IFNγ-inducible MHC-II (B16 HHDII/iDP4). Mice were then given three immunizations of homocitrullinated or WT peptides. Survival was assessed for aldolase peptides in mice implanted with the B16 HHDII/DP4 (C) and B16 HHDII/iDP4 (D) melanoma lines. In the B16 HHDI/iDP4 model, survival was also determined for Bip328-346^Hcit, Bip562-579^Hcit and Eno156-176^Hcit (E) and Cyk371-388^Hcit (F) peptides. Statistical analysis compared survival in immunized mice with unimmunized mice. A representative data set is shown in which for each group n=10 and significant p values are shown. ELISpot, enzyme-linked immunosorbent spot assay; Hcit, homocitrulline; HLA, human leukocyte antigen; IFNγ, interferon γ; MHC-II, major histocompatibility complex class II; WT, wild type.

Figure 3

Tumor survival is dependent on MDSCs, CD4 cells and MHC-II presentation by tumor. Mice implanted with B16 HHDII/DP4 were treated with either anti-CD4 or anti-CD8 antibodies in combination with Aldo74-93^Hcit and Aldo140-157^Hcit immunizations. Survival was assessed in mice treated with antibodies (A). The statistical difference in survival between vaccinated mice and mice treated with each depletion antibody in combination with vaccination is shown. Mice implanted with B16 melanoma knocked out for mouse class II (B16 HHDII/MHC-II K/O) were also assessed for survival (B). For this study, statistical analysis compared control unimmunized mice with immunized mice, no statistical difference was observed. For all groups, n≥10. Hcit, homocitrulline; MDSCs, myeloid-derived suppressor cells; MHC-II, major histocompatibility complex class II.

Figure 4

Humans have a repertoire of CD4 T cells that can recognize homocitrullinated peptides. PBMCs were isolated from 15 healthy donors and 11 patients with cancer and assessed for proliferative responses to homocitrullinated peptides. Example plots shown for healthy donor BD0051 (A) with gating strategy (i, iii), proliferation (CFSE) versus CD4 or CD8 gated on total population (ii) and proliferation gated on either CD4+ or CD8+ cells (iv). Values displayed in (iv) represent the per cent of proliferating cells among CD4+ or CD8+ populations. Graphs show percentage CD4 proliferation in healthy donors (B) and patients (E) and paired analysis comparing media control to each peptide stimulation. Restimulation assays were performed on healthy donor PMBCs that had been cultured with modified peptides for 14 days. IFNγ ELISpot showed restimulation responses to modified and wild-type peptides (C). Example spots and summary data with significant p values are shown. TCR α and β repertoire diversity was assessed in the CD4+ CFSE^high/low cells in one healthy donor that showed responses to both peptides (D). Tree maps depict TCR α and β chain CDR3 clonotype usage in relation to repertoire size (i, ii). Each rectangle represents a unique CDR3 nucleotide sequence and rectangle size denotes the relative frequency of an individual sequence. Colors are randomly assigned and thus do not represent the same CDR3 sequence between plots. Diversity index for each population is shown in (iii). CFSE, carboxyfluorescein succinimidyl ester; ELISpot, enzyme-linked immunosorbent spot assay; IFNγ, interferon γ; PBMCs, peripheral blood mononuclear cells.

Figure 5

MPO in the TME in necessary for the antitumor effect of homocitrullinated peptides. Survival was determined in mice where immunization was delayed till day 14 (A). Mice were implanted with B16 HHDII/iDP4, then given a single immunization once tumor size reached 5×5–8×8 mm. Tumors were removed 7 days later for analysis. Tumor growth prior to cull is shown for each group (Bi) and waterfall plot shows the change in tumor growth from vaccination to day mice were culled for analysis (Bii). Data are collated from two independent studies in which n>5. Post-translational homocitrullination occurs as a result of increased cyanate levels, either due to high urea concentrations or more commonly in tissue due to the breakdown of thiocyanate by the MPO enzyme. Build-up of cyanate and its active form isocyanic acid drives homocitrullination which converts lysine residues in to Hcit (C). Flow cytometry was used for analysis of tumor-infiltrating MPO+ cells (D). Correlation of change in tumor volume between immunization and tumor analysis to MPO expression was assessed by linear regression analysis and significant p values are shown. Mice implanted with B16 HHDII/iDP4 were treated daily with the MPO inhibitor daily for 2 weeks and/or Aldo74-93^Hcit and Aldo140-157^Hcit immunizations on days 4, 11 and 18 (E). Statistical analysis compared the combination with individual treatments, n≥10. Hcit, homocitrulline; MPO, myeloperoxidase; TME, tumor microenvironment.

Figure 6

Tumor-infiltrating MDSCs are a source of MPO and essential for vaccine-mediated tumor therapy. Flow cytometry was used to assess MPO expression on in vitro grown B16F1 HHDII/DP4 (A) or CD45+ tumor-infiltrating (B) cells from in vivo grown B16F1 HHDII/DP4 cells. The proportion of MPO+ cells which express PMN-MDSC (Ly6G+Ly6C^low) or M-MDSC (Ly6G−Ly6C^high) markers was assessed in the spleen and tumor (C). Mice implanted with B16 HHDII/DP4 were treated with either anti-Ly6C or anti-Ly6G antibodies in combination with Aldo74-93^Hcit and Aldo140-157^Hcit immunizations. Staining shows the populations of splenic Ly6G+Ly6C^low (PMN-MDSCs) or CD11b+Ly6G−Ly6C^high (M-MDSC) after treatment with depletion antibodies (D). Survival was assessed in mice treated with antibodies (E). MDSCs, myeloid-derived suppressor cells; M-MDSC, monocytic MDSC; MPO, myeloperoxidase; PMN-MDSCs, polymorphonuclear MDSCs.

Figure 7

In vitro recombinant aldolase and B16F1 cell lines can be homocitrullinated FCS, recombinant aldolase protein, B16F1 cell lysate or whole B16F1 cells were incubated for 10 hours with media only or cyanate. Anti-Hcit ELISA was performed to determine the level of homocitrullination on control of cyanate treated samples (A). Mean, SD and p values are shown, n≥3. Homocitrullinated lysine residues were identified by mass spectrometry analysis (B). Three independent experiments were performed each with multiple injections. Homocitrulline residues (gray) are shown. Some lysine residues were not covered by the stochastic analysis (hashed). In vitro bone marrow-derived MDSCs (BM-MDSCs) were generated from transgenic mice. Cell phenotypes were defined as either CD11b+Ly6G+Ly6C^low (PMN-MDSCs) or CD11b+Ly6G−Ly6C^high (M-MDSC). Example plots are shown (C). B16F1 cells were incubated with untreated BM-MDSCs, H₂0₂ and thiocyanate overnight. The proportion of CD45− cells that stained positive for anti-Hcit antibody was then assessed by flow cytometry (D). Mean and individual experiments are shown after normalization to the untreated sample. Statistical analysis was performed. FCS, foetal calf serum; Hcit, homocitrulline; LPS, lipopolysaccharide; MDSCs, myeloid-derived suppressor cells.