A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

Abstract

CD4(+) T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8(+) T cell epitope, MELOE-1(36-44), in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8(+) T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4(+) T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1(26-46) revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4(+) T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4(+) T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1(22-46), containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4(+) and CD8(+) T cell responses in vitro, making it a potential candidate for melanoma vaccination.

Fig. 1

Expansion and TCR characterization of MELOE-1_32–46 and MELOE-1_26–40 specific CD4⁺ clones. a PBMC from healthy donor DA3 were cultured for 14 days with 10 μM MELOE-1_32–46 and the presence of specific CD4⁺ T cells was assessed by CD4/IFN-γ double staining after restimulation with peptide (left panel). Specific T cells were cloned by limiting dilution and specificity of the clones was also assessed by CD4/IFN-γ double staining (right panel clone 1A3). b TCR sequences of the MELOE-1_32–46 specific CD4⁺ T clones. c PBMC from donor DC1 were stimulated as in a with MELOE-1_26–46 (left panel) and cloned. Specificity of the clones was assessed after restimulation with MELOE-1_26–40 (right panel clone 1C9). d TCR sequences of MELOE-1_26–40 specific CD4⁺ T clones

Fig. 2

Recognition of DC loaded with MELOE-1 whole protein by the CD4⁺ T cell clones. a Negative control. DC were matured with TNF-α and PolyI:C for 4 h, fixed and then incubated in serum-free medium with MELOE-1 for 4 h, to verify the absence of exogenous loading on class II HLA molecules. b DC were loaded with MELOE-1 whole protein or with the control peptide Melan-A_16–40 A27L, for 4 h in serum-free medium with TNF-α and PolyI:C prior to fixation. Co-cultures with clones 1C9 or 1A3 were performed at a 1:1 clone:DC ratio and responses of each clone was assessed by TNF-α and IFN-γ intracellular staining and flow cytometry analysis. A representative experiment is shown out of six performed. ND not done

Fig. 3

HLA class II restrictions and optimal epitopes recognized by the two clones. a Clones 1A3 and 1C9 were stimulated by MELOE-1_32–46 and MELOE-1_26–40, respectively, in the presence or not of decreasing concentrations of anti-HLA-DP, -DQ or -DR mAbs. b The HLA class II-restricting alleles were determined by assessing 1A3 and 1C9 TNF- α responses against unpulsed (grey histogram) or peptide-pulsed (dotted line) EBV-B cells expressing HLA-DQ or -DR alleles from the donors DA3 and DC1. c Clone 1C9 was incubated at a 1:1 ratio with the HLA-DRβ1*1101+ C3 EBV-B cell-line loaded with various concentrations of the indicated peptides and TNF production was measured in the WEHI biological assay. d As in c with clone 1A3 and the HLA-DQβ1*0603+ BL30/95 EBV-B cell-line

Fig. 4

Functional characterization of the CD4⁺ T cell clones. a Cytotoxicity of CD4⁺ T cell clones 1A3 and 1C9 against peptide-pulsed EBV-B cells was assessed by a standard chromium release assay. b Representative examples of HLA-DQ or HLA-DR expression by melanoma cell-lines after incubation with (bold line) or without (thick line) 500 IU/ml IFN-γ for 48 h. c IFN-γ production by CD4⁺ T cell clones 1A3 and 1C9 in response to HLA class II-matched melanoma cell-lines, treated or not with IFN-γ and loaded or not with MELOE-1 peptides. Shown are representative responses of 1A3 and 1C9 T cell clones toward one out of three HLA-DQβ1*0603+ and one out of five HLA-DRβ1*1101+ MELOE-1-expressing melanoma cell-lines tested, respectively. d IFN-γ production by clone 1A3 in response to HLA-matched DC loaded with M117 or SW480 cell lysate or the MELOE-1_32–46 peptide as positive control (n = 2)

Fig. 5

Stimulation of PBL from a HLA-A*0201+ HLA-DRβ1*1101+ donor with MELOE-1_22–46-loaded autologous DC. a Immature DC were loaded with MELOE-1_22–46 for 4 h, washed and matured with TNF-α and PolyI:C for 12 h. Efficiency of MELOE-1_22–46 processing and presentation or cross- presentation was assessed by coculturing loaded DC with the CD4⁺ T cell clone 1C9 or with the MELOE-1_36–44-specific CD8⁺ T cell clone M170.48 at a 1:1 ratio for 5 h. b PBL of a HLA-A*0201+ HLA-DRβ1*1101+ healthy donor (DS7) were stimulated by MELOE-1_22–46–loaded DC at a 10:1 PBL:DC ratio and cultured for 14 days in the presence of IL-2. c PBL from donor DS7 were stimulated by MELOE-1_22–46–loaded DC at a 10:1 PBL:DC ratio in the presence of IL-6 and IL-12 for 7 days, restimulated with loaded DC and cultured for 7 more days in the presence of IL-2. In both cases, the presence of MELOE-1_24–37 specific CD4⁺ T cells and MELOE-1_36–44 specific CD8⁺ T cells was assessed by challenging T cells with MELOE-1_24–37 or MELOE-1_36–44 peptides, respectively, followed by CD4/IFN-γ or CD8/IFN-γ double staining and flow cytometry analysis. Examples of wells with MELOE-1_24–37 (left panels) and/or MELOE-1_36–44 (right panels) specific T cells expansions are shown. Percentage of specific T cells among CD8⁺ or among CD4⁺ T cells are indicated