Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

Figures

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  Figure 1. Homocitrullinated peptide responses in HLA-HHDII/DR1, HLA-DR4 and Balb/c mice. Mice were immunized with three doses of homocitrullinated peptides with the adjuvants CpG and MPLA. Responses to homocitrullinated vimentin (Vim) peptides were determined in HLA-HHDII/DR1 (A) and HLA-DR4 (B) mice. Responses to cytokeratin 8(Cyk8) peptides were determined in HLA-HHDII/DR1 mice (C). Aldolase (Aldo) responses were screened in HLA-HHDII/DR1 mice (D). Responding peptides were then also tested in HLA-HHDII/DP4 (E), C57Bl/6 (F) and Balb/c (G) mice. For each study n≥3. Mean and standard deviations and significant p values are shown; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

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  Figure 2. Characterization of the homocitrulline-mediated immune responses. Mice were immunized with homocitrullinated peptides then assessed for responses using IFNγ ELISpot assays. ELISpots were then performed with homocitrullinated (Hcit) and wild type (wt) peptides or homocitrullinated peptides in the presence of anti-CD4 or anti-CD8 blocking antibodies. Vim116-135Hcit responses were assessed in HLA-HHDII/DR1 (A), HLA-DR4 (B) and Balb/c (C) mice. Cyk8 371-388Hcit responses were assessed in HLA-HHDII/DR1 (D) and Balb/c (E) mice. Aldo 74-93Hcit responses were only assessed in Balb/c mice (F). Mice were also immunized with wild type Vim116-135 peptide and responses were assessed in HLA-HHDII/DR1 and HLA-DR4 mice (G). For each study n≥3, Homocitrulline immunized mice were assessed in ≥3 independent studies. Mean and standard deviations and significant p values are shown; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

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  Figure 3. Heathy Human Donors have a repertoire that can recognize the Vim116-135Hcit peptide. PBMCs were isolated from 17 healthy donors and assessed for proliferative responses to homocitrullinated peptides using a CFSE dilution assay. Example plots from healthy donor BD0051 (A) show proliferation (CFSE) vs CD4 or CD8 in media control (i) PHA stimulated (ii) or Vim116-135Hcit stimulated (iii) cells after gating on the total lymphocyte population. CFSE proliferation in the gated CD4 or CD8 populations was assessed (B). Given that the majority of proliferation in response to Vim116-135Hcit was found in the CD4+ population, the percentage of the CD4+ proliferation was assessed in individual healthy donors (Ci) and paired analysis comparing media control to peptide stimulation (Cii). Restimulation assays were performed on PMBCs from 5 healthy donor that had been cultured with Vim116-135Hcit peptide for 14 days. IFNγ ELISpot showed restimulation responses to homocitrullinated (hcit) and wild type (wt) peptides (D). Restimulation to Vim116-135Hcit was also performed for two donors in the presence of anti-MHC-I, MHC-II, DP, DR or isotype blocking antibodies (E). Summary data with significant p…

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  Figure 4. Human response to Vim116-135Hcit is oligoclonal. TCR α and β repertoire diversity was assessed in the CD4+ CFSEhigh/low cells in one healthy donor (BD016). Responses to vehicle (A), positive control CD3 and HepB (B) or Vim116-135Hcit peptide stimulation (C) were assessed. Tree maps depict TCR α and β chain CDR3 clonotype usage in relation to repertoire size. 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 (D).

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  Figure 5. Vim116-135Hcit responses are associated with an anti-tumor response. HLA-HHDII/DR1 (A) and HLA-DR4 (B) mice were implanted with HLA-matched B16 F1 cells expressing MHC-II under a constitutive (A, B, D, E) or inducible (C) promoter on day 1. On day 4, 11 and 18 mice were immunized with homocitrullinated (Hcit) or wild type (wt) Vim116-135 peptides or adjuvant only controls. Anti-tumor survival was compared to unimmunized control mice. Anti-tumor studies were also performed with Cyk8 371Hcit (D) and Aldo140-157Hcit (E) in HLA-HHDII/DR1 mice implanted with HLA-matched B16 cells. For each group and study n≥10, apart from CpG/MPLA control in panel B where n=5. significant p values are shown.

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  Figure 6. Anti-tumor response requires direct presentation on MHC-II by tumor. Anti-tumor studies were performed using tumor which expressed MHC-I but were knocked out for MHC-II. HLA-HHDII/DR1 (A) and HLA-DR4 (B) mice were challenged with B16 K/O MHC-II cell lines on day 1. On day 4, 11 and 18 mice were immunized with Vim116-135Hcit peptide or adjuvant only controls. Anti-tumor survival was compared to unimmunized control mice. For each group n=10, significant p values are shown.

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  Figure 7. Cancer Patients also have a repertoire that can recognize the Vim116-135Hcit peptide. PBMCs were isolated from 11 cancer patients (7 Lung cancer patients, 2 Ovarian patients and 1 Breast cancer patient) and assessed for proliferative responses by CFSE dilution assay to homocitrullinated peptide. Graph shows CD4 proliferation in individual patients (A) and paired analysis comparing media control to Vim16-135Hcit stimulation (B).