Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11

Abstract

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4(+) T cells from the blood of a healthy donor. CD4(+) T cells strongly recognized MAGE-3281-295 and, to a lesser extent, MAGE-3141-155 and MAGE-3146-160. Moreover, CD4(+) T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4(+) T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3-positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4(+) T cells recognized MAGE-3281-295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4(+) T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4(+) T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4(+) T cell epitopes.

Figure 1

Proliferative activity of CD4⁺ T cells stimulated with MAGE-3 pool measured in 2-d microproliferation assays. The data are representative of n = x experiments, and are means of triplicate determinations ± SD. (A) Responses to MAGE-3 pool (0.01, 0.5, 0.1, 0.5, 1, and 5 μg/ml; n = 6). (B) Responses to rMAGE-3 protein (5, 10, and 20 μg/ml; n = 3). (C) Responses to the individual synthetic peptides forming the MAGE-3 pool (10 μg/ml; n = 7) at different weeks of propagation. The blank (i.e., the basal level of proliferation of CD4⁺ T cells in the presence of APCs only) was subtracted and was as follows: 2 wk, 30,866 ± 1,115; 4 wk, 7,106 ± 2,201; and 6 wk, 21,838 ± 2,767. Responses significantly higher than the blanks are indicated as *P < 0.001 and **P < 0.025 (determined by unpaired, one-tailed Student's t test). (D) Response to MAGE-3 pool (5 μg/ml; n = 5) (a) and to peptide corresponding to sequence 281–295 (b), in the presence of different doses of L243 mAb (0.25 and 0.5 μg/ml). The blank was 1,251 ± 444; the proliferation of CD4⁺ T cells in the presence of MAGE-3 pool was 28,191 ± 373; and the proliferation in the presence of sequence 281–295 was 22,504 ± 141.

Figure 2

Cytolytic activity of MAGE-3–specific CD4⁺ T cells. The data are representative of n = x experiments, and are means of triplicate determinations ± SD. (A) Lytic activity against different HLA-DR–matched and unmatched melanoma cells (n = 6). HLA-DR types of CD4⁺ T cells and melanomas are indicated at the bottom along with their symbols. (B) Cytofluorimetric analysis for HLA-DR (surface) and MAGE-3 (intracytoplasmic) expression in melanoma cells used as targets (n = 4). Filled histograms, stained sample; open histograms, background staining obtained with FITC-conjugated second-step reagent only.

Figure 2

Cytolytic activity of MAGE-3–specific CD4⁺ T cells. The data are representative of n = x experiments, and are means of triplicate determinations ± SD. (A) Lytic activity against different HLA-DR–matched and unmatched melanoma cells (n = 6). HLA-DR types of CD4⁺ T cells and melanomas are indicated at the bottom along with their symbols. (B) Cytofluorimetric analysis for HLA-DR (surface) and MAGE-3 (intracytoplasmic) expression in melanoma cells used as targets (n = 4). Filled histograms, stained sample; open histograms, background staining obtained with FITC-conjugated second-step reagent only.

Figure 3

CD4⁺ T cells recognize MAGE-3_281–295 in association with HLA-DR11 on OI TC cells. The data are representative of n = x experiments, and are means of triplicate determinations ± SD. (A) Lytic activity of CD4⁺ CTLs against LCL alone or LCL pulsed with MAGE-3_141–155, MAGE-3_146–160, and MAGE-3_281–295 (n = 3). (B) Cold target inhibition experiments (n = 3). Cold targets (OI TC [○] and LCL pulsed with MAGE-3_281–295 [□]) were used to inhibit the lytic activity of MAGE-3–specific CD4⁺ CTLs against hot OI TC (E/T ratio of 40:1). Percentage of specific lysis against OI TC cells in the absence of cold targets was 26 ± 1.2%.