Vaccination with post-translational modified, homocitrullinated peptides induces CD8 T-cell responses that mediate antitumor immunity

Abstract

Background: Post-translational modification of proteins has the potential to alter the ability of T cells to recognize major histocompatibility complex (MHC) class -I and class-II restricted antigens, thereby resulting in altered immune responses. One such modification is carbamylation (homocitrullination) that results in the formation of homocitrulline (Hcit) residues in a non-enzymatic reaction of cyanate with the lysine residues in the polypeptide chain. Homocitrullination occurs in the tumor microenvironment and CD4-mediated immune responses to Hcit epitopes can target stressed tumor cells and provide a potent antitumor response in mouse models.

Methods: Homocitrullinated peptides were identified and assessed in vitro for HLA-A2 binding and in vivo in human leukocyte antigen (HLA) transgenic mouse models for immunogenicity. CD8 responses were assessed in vitro for cytotoxicity and in vivo tumor therapy. Human tumor samples were analyzed by targeted mass spectrometry for presence of homocitrullinated peptides.

Results: Homocitrullinated peptides from aldolase and cytokeratin were identified, that stimulated CD8-mediated responses in vivo. Modified peptides showed enhanced binding to HLA-A2 compared with the native sequences and immunization of HLA-A2 transgenic mice generated high avidity modification specific CD8 responses that killed peptide expressing target cells. Importantly, in vivo the homocitrullinated aldolase specific response was associated with efficient CD8 dependent antitumor therapy of the aggressive murine B16 tumor model indicating that this epitope is naturally presented in the tumor. In addition, the homocitrullinated aldolase epitope was also detected in human tumor samples.

Conclusion: This is the first evidence that homocitrullinated peptides can be processed and presented via MHC-I and targeted for tumor therapy. Thus, Hcit-specific CD8 T-cell responses have potential in the development of future anticancer therapy.

Keywords: CD8-positive T-lymphocytes; antigens; immunogenicity, vaccine; immunotherapy.

Figure 1

Long homocitrullinated peptides stimulate CD8-mediated immune responses in HLA-A2 transgenic mice. (A) Immunization schedule. Immune responses in HLA-A2 transgenic (HHDII/DR1) mice immunized with individual peptides containing Hcit residues (B) were assessed by IFNγ ELISpot assay. Mice were immunized on days 1, 8 and 15 and splenocytes analyzed on day 21. Splenocytes from individual HHDII/DR1 mice immunized with Aldo 140-157Hcit, Aldo 204-219Hcit, Cyk8 112-132Hcit or Cy8 371-388Hcit, were subjected to peptide restimulation alone or in combination with CD4 or CD8 blocking ex vivo (C). Symbols represent mean IFNγ spots/million splenocytes for individual mouse (B and C). Aldo 204-219Hcit and Cy8 112-132Hcit responses (pooled splenocytes within each group where n=3) were also assessed with or without CD4 or CD8 antibody after 7-day in vitro culture (D). Peptide specific responses were assessed by IFNγ ELISpot assay. Data are collated from or representative of independent studies where n=3. Significant p values (p<0.05) are shown where statistical analysis was performed using paired (B and C) or unpaired (B and D) ANOVA. Aldo, aldolase A; ANOVA, analysis of variance; AV, average;Cyk8, cytokeratin 8; ELISpot, enzyme linked immunosorbent spot; Hcit, homocitrulline; HLA, human leukocyte antigen; IFNγ, interferon γ.

Figure 2

Homocitrullinated peptide immunization can induce modification specific CD8 responses. IFNγ ELISpot responses were examined in HLA-A2 transgenic (HHDII/DR1) mice on day 21 after three immunizations (days 1, 8 and 15) with Aldo 204-219Hcit or Cyk8 112-132Hcit peptide (A, C and D). Immune responses to vaccination with Aldo 204–219 wt or Cyk8 112–132 wt peptide were assessed in HHDII/DR1 mice (B). IFNγ responses were tested against the long Hcit and native (wt) sequences (A and B) and short Hcit 9aa CD8 peptides (B and C). LPS was added as positive control to confirm splenocyte viability (B). Symbols represent mean IFNγ spots/million splenocytes for individual mouse and the line represents median value between mice. Significant p values are shown from paired ANOVA analysis. Titrations of the short and long Aldo Hcit (Di) or Cyk8 Hcit (Dii) peptides were performed to determine the avidity of both the longer and shorter peptides (in HHDII/DR1 mice). For each concentration of peptide, data from three individual mice were collated and normalized avidity graphs are shown. Aldo, aldolase A; ANOVA, analysis of variance; AV, average; Cyk8, cytokeratin 8; Hcit, homocitrulline; IFNγ, interferon γ; wt, wild type.

Figure 3

Modified peptides show better binding to MHC class I. Binding affinity of the shorter Aldo and Cyk8 peptides selected based on IEDB predicted scores to HLA-A2 was determined in T2 binding assay (A and B) at 100 µg/mL of peptide concentration. Data from two independent experiments are presented. Paired t-test was performed to derive statistical significance. HLA-A2 binding affinity of Aldo 209-217Hcit or Cyk8 117-125Hcit was compared with the corresponding wt peptides at different peptide concentrations (C and D). Data from three independent experiments are presented and two-way ANOVA has been performed for statistical analysis. Aldo, aldolase A; ANOVA, analysis of variance; Cyk8, cytokeratin 8; Hcit, homocitrulline; HLA, human leukocyte antigen; IEDB, Immune Epitope Database; MFI, median fluorescence intensity; MHC, major histocompatibility complex; wt, wild type.

Figure 4

Aldo 209-217Hcit specific immune responses mediate efficient anti-tumor therapy in vivo. Expression of Hcit proteins in in-vivo grown B16 tumor was assessed by staining with anti-Hcit antibody. Paired analysis was performed comparing secondary (2’) antibody control to samples stained with anti-Hcit (anti-carbamyl) antibody for multiple tumors (Ai). An example of gating strategy is shown (Aii) where sequential gating was performed. FSC/SSC gated cells were doublet excluded using FSC-A/FCS-H gating. Events were then gated to include live cells negative for CD45 expression. Live/CD45− cells were then assessed for staining with anti-Hcit antibody (green histogram) or the secondary antibody alone (red histogram) (Aii). In vivo antitumor study was performed in HHDII mice implanted with B16HHDII tumor on day 1. Mice were then immunized with Aldo 209-217Hcit or Cyk8 117-125Hcit on day 4, 8 and 11 as shown in the schematic (B). Statistical analysis compares overall survival between unimmunized control mice and peptide immunized mice (Cii). Study is a representative data set for which each group contained n≥10 mice and significant p values are shown (Mantel-Cox test). Tumor growth for individual mice is shown for each group (Cii) and the number of tumor-free mice out of the total number of mice per group used in the experiment is given in brackets. Immune responses in the survivors (n=5) from Aldo 209-217Hcit immunized group was evaluated in IFNγ ELISpot assay (D). Symbols represent mean IFNγ spots/million splenocytes for individual mice and line represents median value between mice. Significant p values are shown (paired ANOVA). HHDII mice implanted with B16 HHDII cells were treated with either anti-CD4 or anti-CD8 antibody with or without Aldo 209-217Hcit immunization (E). The statistical difference in survival between peptide vaccinated mice and mice receiving anti-CD8 depletion antibody in combination with peptide is presented where for all the groups, n≥5 (Mantel-Cox test). Aldo, aldolase A; ANOVA, analysis of variance; Cyk8, cytokeratin 8; FSC, forward scatter light; FSC-A, FSC-Area; FSC-H, FSC-Height; ELISpot, enzyme linked immunosorbent spot; Hcit, homocitrulline; HLA, human leukocyte antigen; IFNγ, interferon γ, SSC, side scatter light.

Figure 5

Response avidity and cytotoxicity are not responsible for absence of tumor therapy from Cyk8 117-125Hcit vaccination. HLA-A2 transgenic mice (HHDII) were immunized with either Aldo 209-217Hcit or Cyk8 117-125Hcit peptide and peptide titrations of short and long Hcit peptides were performed in IFNγ ELISpot (A). Data from three independent experiments are shown for each concentration of peptide and normalized avidity graphs are presented. Splenocyte cultures expanded in vitro in the presence of Aldo 209-217Hcit or Cyk8 117-125Hcit were restimulated with T2 cells pulsed with Hcit or wt peptides. The ability to kill T2 cells pulsed with Hcit or wt peptide was tested in a non-radioactive cell killing assay (B). Data are shown where error bars indicate the variations between quadruplicate assay wells. Aldo, aldolase A; Cyk8, cytokeratin 8; ELISpot, enzyme linked immunosorbent spot; E:T, effector:target; Hcit, homocitrulline; HLA, human leukocyte antigen; IFNγ, interferon γ; wt, wild type.

Figure 6

Aldo 209-217Hcit sequence is detected in human tumor samples. Representative fragment ion traces for sequence Aldo 209-217Hcit from four samples. Upper panels show the endogenous tumor channel, lower panel the respective internal standard. Aldo, aldolase A; Hcit, homocitrulline.