MAGE-A3161–175 contains an HLA-DRβ4 restricted natural epitope poorly formed through indirect presentation by dendritic cells

Abstract

We report here that HLA-DRbeta4*01 restricted MAGE-A3(161-175 )specific CD4(+) T cells from a healthy donor recognize a naturally processed epitope formed through the exogenous but not the endogenous pathway. However, the intensity of recognition of the native epitope by MAGE-A3(161-175 )specific CD4(+) T cells strongly depends on the antigen presenting cells and the amount of protein available for processing. EBV-transformed lymphoblastoid cells (LCLs) and melanoma cells engineered to express MAGE-A3 in the endosomal/lysosomal compartment were strongly recognized while autologous dendritic cells loaded with lysate from MAGE-A3 expressing cells were, although significantly, poorly recognized. To prove that the amount of antigen available for processing was a key factor determining the different response LCLs were sorted by MAGE-A3 expression. The response intensity correlated with the amount of MAGE-A3 expressed by the cells. Collectively, these results suggest that different antigen presenting cells with different amount of antigen available for processing as well as protease activity are important factors in determining the epitope repertoire produced in vivo, and therefore reliable tools should be used when testing recognition of native epitopes by peptide specific CD4(+) T cells.

Endosomal Proteases Influence the Repertoire of MAGE-A3 Epitopes RecognizedIn vivoby CD4+ T Cells

“…HT144 (HT144IiM3) and DAS (LCL-IiM3) cells expressing MAGE-A3 in the endosomal/lysosomal compartment were described in ref. 13. The HLAclass II type of cells used was identified by molecular typing and is reported in Table 1 for melanoma patients.…”

“…Peptide stimulated CD4 + T cells from in vitro restimulation assays or CD4 + T-cell clones were cultured in triplicate in 96 U-bottomed plates in the presence of APCs used at different T cells to APCs ratios as described in ref. 13 To study the role of endosomal/lysosomal enzymes in the presentation of MAGE-A3 epitopes APCs (dendritic cells, LCL-IiM3, and HT144IiM3) were treated with specific inhibitors. The inhibitors used were: leupeptin (Sigma) and pepstatin A (Calbiochem).…”

“…MAGE-A3 161-175 -specific CD4 + T cells were generated from a healthy donor and have been described in detail in ref. 13. MAGE-A3 111-125 -specific CD4 + T cells were generated from melanoma patient 011 (Table 1).…”

“…MAGE-A3 161-175 -specific CD4 + T cells have been described in ref. 13. MAGE-A3 111-125 -specific CD4 + T-cell clones were obtained from patient 011 by limiting dilution after 3 weeks of stimulation with the specific peptide as described in (28).…”

“…Several MAGE-A3 CD4 + T-cell epitopes have been identified (6)(7)(8)(9)(10)(11)(12)(13). Little is known about the repertoire of CD4 + T-cell epitopes recognized in vivo by neoplastic patients and even less on how antigen processing during priming in dendritic cells influences their formation.…”