Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice

Abstract

CD4+ T cells play a central role in the induction and persistence of CD8+ T cells in several models of autoimmune and infectious disease. To improve the efficacy of a synthetic peptide vaccine based on the self-Ag, gp100, we sought to provide Ag-specific T cell help. To identify a gp100 epitope restricted by the MHC class II allele with the highest prevalence in patients with malignant melanoma (HLA-DRB1*0401), we immunized mice transgenic for a chimeric human-mouse class II molecule (DR4-IE) with recombinant human gp100 protein. We then searched for the induction of CD4+ T cell reactivity using candidate epitopes predicted to bind to DRB1*0401 by a computer-assisted algorithm. Of the 21 peptides forecasted to bind most avidly, murine CD4+ T cells recognized the epitope (human gp10044-59, WNRQLYPEWTEAQRLD) that was predicted to bind best. Interestingly, the mouse helper T cells also recognized human melanoma cells expressing DRB1*0401. To evaluate whether human CD4+ T cells could be generated from the peripheral blood of patients with melanoma, we used the synthetic peptide h-gp10044-59 to sensitize lymphocytes ex vivo. Resultant human CD4+ T cells specifically recognized melanoma, as measured by tumor cytolysis and the specific release of cytokines and chemokines. HLA class II transgenic mice may be useful in the identification of helper epitopes derived from Ags of potentially great clinical utility.

FIGURE 1

Murine CD4⁺ T cells lines recognize intact human melanoma. Line A9895 was generated by vaccinating DR4-IE Tg mice with recombinant h-gp100 protein, followed by serial restimulation with peptide h-gp100_46–58 in vitro. Line C1056 was generated by vaccinating DR4-IE Tg mice with peptide h-gp100_46–58, followed by serial restimulation with h-gp100_46–58 in vitro. A, A9895 line recognizes peptides h-gp100_46–58 h-gp100_44–59 (100 μM peptides) pulsed onto DR4-IE splenocytes. There was no evidence of reactivity against control peptides HA_306–318 and murine-gp100_46–58. B, A9895 line recognizes recombinant h-gp100 protein (10 μg) and human melanoma lysate (1300 Mel; known to express h-gp100) pulsed onto DR4-IE splenocytes. There was no evidence of reactivity against the control breast carcinoma lysate MDA-231. T cell lines A9895 (C) and C1056 (D) react with intact DR4⁺ human melanomas 697 Mel, 1498 Mel, and 1102 Mel. In both cases, control tumors 888 Mel and 697 EBV-B, mismatched for the appropriate restriction element and Ag expression, respectively, were not recognized by either murine CD4⁺ T cell line. For all experiments, T cells and targets were cocultured for 24 h and supernatants were measured for IFN-γ secretion by ELISA. All experiments were repeated at least three times with similar results using different but comparable targets, thus demonstrating the generalizability of the experimental findings.

FIGURE 2

Characterization of CD4⁺ T cell line A9895. A, CD4⁺ T cell line A9895 exhibits a Th1-like cytokine profile. Secretion of cytokines IFN-γ, GM-CSF, IL-2, and TNF-α, but not IL-5, is seen against target melanoma line (1102 Mel). There was no reactivity seen against the control target (1102 EBV-B). The line also produces significant amounts of RANTES against melanoma target (1102 Mel), but not against control target (*, 888 Mel). Background reactivity for each cytokine and chemokine (baseline tumor production at 24 h) were subtracted from individual total values, respectively. B, Reactivity of line A9895 is MHC class II restricted. T cells were coincubated with targets in the presence of anti-HLA-A, -B, -C, anti-DR, and anti-murine CD4 mAbs. As a control for mAb blocking, the h-gp100_209–217-specific, HLA-A*0201-restricted, human CD8⁺ T cell clone (K4/H5) was cocultured with identical targets. Recognition of 697 Mel and 1102 Mel by line A9895 was blocked by mAbs anti-DR and anti-CD4, but unaffected by mAb anti-HLA-A, -B, -C. In the control group, mAb anti-HLA-A, -B, -C blocked recognition of 697 Mel and 1102 Mel (both express HLA-A*0201), but was unaffected by mAbs anti-DR and anti-CD4. C, Line A9895 specifically lyses DR4⁺ human melanoma (1102 Mel), but not control melanoma (888 Mel), in a 15-h ⁵¹Cr release assay. For A and B, T cells and targets were cocultured for 24 h and supernatants were measured for individual cytokine and chemokine secretion by ELISA. All experiments were repeated twice with similar results using different but comparable targets, thus demonstrating the generalizability of the experimental findings.

FIGURE 3

Characterization of the HLA-DRB1*0401 immunodominant epitope for h-gp100. A, The naturally processed 16-aa epitope h-gp100_44–59 provides stronger stimulation for the CD4⁺ T cell line A9895 than the nested variant h-gp100_46–58 used in the initial screening of candidate epitopes. Peptide-pulsed 1102 EBV-B cells were prepared at titrating peptide concentrations. B and C, Determinations of the P1 and P9 anchor positions for the DRB1*0401-restricted h-gp100 epitope. 1102 EBV-B cells were pulsed with indicated peptides at 10 μM (B) and 100 μM (C). T cells and targets were cocultured for 24 h and supernatants were measured for IFN-γ secretion by ELISA. All experiments were repeated three times with similar results using different but comparable targets, thus demonstrating the generalizability of the experimental findings.

FIGURE 4

Three different human CD4⁺ T cell lines from three separate patients recognize human melanoma. A and B, Lines Th and Wa recognize h-gp100_44–59 pulsed on DR4⁺ EBV-B cells and DR4⁺ intact human melanoma cells expressing h-gp100 endogenously. There was no reactivity detected against control targets 888 Mel (DR4⁻) and 1102 EBV-B pulsed with the irrelevant peptide (HA_306–318). C, Line Te-22 recognizes h-gp100_44–59, h-gp100 protein, h-gp100 EBV-B transfectants, and DR4⁺ intact human melanoma cells. There was no evidence of cytokine release against the irrelevant peptide (HA_306–318), irrelevant protein (GFP protein), control transfectant (GFP), and control tumor targets (888 Mel and 1102 EBV-B). Peptides (100 μM) and protein (10 μg/ml) were pulsed onto APCs overnight, and effectors were added at 1:1 ratio (10⁵/well). D, Experiment 1, Line Te-22 displays a Th1 cytokine profile with secretion of IFN-γ, GM-CSF, and IL-2, but not IL-4 against 1102 Mel. D, Experiment 2, Line Te-22 secretes chemokines MIP-1α, MIP-1β, MIP-3β, and RANTES, but not MIP-3α against 1102 Mel. In both experiments, there was minimal cytokine and chemokine release against control tumor targets 1102 EBV-B and 888 Mel, respectively. Background reactivity for each cytokine and chemokine (baseline tumor production at 24 h) were subtracted from individual total values, respectively. E, Line Te-22 specifically lyses 1102 Mel in a 12-h ⁵¹Cr release assay. There was no evidence of lysis against control tumor 1102 EBV-B. All experiments were repeated at least twice with similar results using different but comparable targets, thus demonstrating the generalizability of the experimental findings.