Detection of low‐frequency human antigen‐specific CD4⁺ T cells using MHC class II multimer bead sorting and immunoscope analysis

Abstract

MHC class II tetramers are attractive tools to study antigen-specific CD4 + T cell responses in various clinical situations in humans. HLA-DRA1*0101/DRB1*0401 MHC class II heterodimers were produced as empty molecules using the Drosophila melanogaster expression system. Peptide binding experiments revealed that these molecules could be loaded efficiently with appropriate MHC class II tumor epitopes. Interestingly, MHC class II tetramer staining was influenced by modifications in membrane lipid rafts, and could in itself induce activation changes of stained CD4 + T cells at 37 C. In order to increase the threshold of detection of poorly represented peripheral antigen-specific CD4 + T cells, we combined cell sorting using MHC class II multimer beads together with TCR analysis using the immunoscope technology. This strategy greatly increased the sensitivity of detection of specific CD4 + T cells to frequencies as low as 4Â10 -6 among peripheral blood mononuclear cells. Such a combined approach may have promising applications in the immunomonitoring of patients under vaccination protocols to tightly follow induced antigen-specific CD4 + T cells expressing previously identified TCR.

Tumor Antigen–Specific FOXP3+ CD4 T Cells Identified in Human Metastatic Melanoma: Peptide Vaccination Results in Selective Expansion of Th1-like Counterparts

“…As previously reported by us using a single Melan-A 25-36 -specific CD4 T-cell clone isolated from a melanoma patient, we further show here, using multiple independent DQ6/Melan-A 25-36 CD4 T-cell clones from additional patients, that they efficiently recognize the dodecapeptide Melan-A [25][26][27][28][29][30][31][32][33][34][35][36] , as well as the two amino acid shorter Melan-A 26-35(A27L) analogue decapeptide, as determined in peptide titration assays ( Supplementary Fig. S2A-C).…”

“…4A and B). Attempts to promote peptide-specific proliferation of Melan-A [25][26][27][28][29][30][31][32][33][34][35][36] CD4 T cells in prevaccination PBMCs by either depleting CD4+CD25+ T cells or by adding several stimulating or inhibitory molecules in the culture wells [e.g., CpG-ODNs, CD40L, high doses IL-2, IL-6, IL-15, IL-7, anti-transforming growth factor (TGF)h, anti-IL-10 receptor] failed (data not shown).…”

“…The generation of HLA-DQB1*0602 multimers was performed as already described using the Melan-A [25][26][27][28][29][30][31][32][33][34][35][36] , Melan-A 26-35(A27L) , or the HA 57-75 peptides (12). Multimer labeling was performed for 1 h at 37jC before staining with fluorescent monoclonal antibodies (mAb) directed against surface molecules (20 min at 4jC).…”

“…We have shown that the natural Melan-A [26][27][28][29][30][31][32][33][34][35] decapeptide binds weakly to the HLA-A2 molecule because of the lack of a strong anchor residue at position P2. The replacement of Ala at position 27 for Leu results in enhanced binding to HLA-A2, in 2-to 3-log increase of its relative antigenicity and, more importantly, to an increased immunogenicity (17).…”

“…Moreover, HLA-DQ6/ Melan-A 25-36 multimers directly identified specific CD4 T cells by flow cytometry. Interestingly, this optimal CD4 T-cell epitope fully encompasses the HLA-A2-restricted decapeptide Melan-A [26][27][28][29][30][31][32][33][34][35] . In this report, we used HLA-DQ6/Melan-A peptide multimers to analyze in detail Melan-A-specific CD4 T-cell responses in healthy individuals (HD) and a cohort of HLA-A*0201 metastatic melanoma patients who shared expression of the HLA-DQB1*0602 allele.…”