SIG35α predominantly pairs with TRBV2, 5–1 and 27 TCRβ chains with highly heterogeneous and unique CDR3β regions to recognize A2/MART1 in CD4+ T cells.
(a) SIG35α/ΔNGFR-transduced peripheral CD8+ (top) and CD4+ (bottom) T cells from HLA–A2+ and A2− donors were stimulated with aAPC pulsed with A2/MART1 peptide twice and stained with anti-NGFR mAb, A2/MART1 multimer, mAbs for TCR Vβ subtypes and anti-CD8 or CD4 mAb. The percentage of SIG35α+ A2/MART1 multimer+ CD8+ (top) and CD4+ (bottom) T cells expressing each Vβ subtype is shown. The data shown are gated on ΔNGFR+ A2/MART1 multimer+ cells and representative of two independent experiments. The percentage of overall SIG35α-transduced T cells expressing each Vβ subfamily is shown in Supplementary Fig. 2. (b) SIG35α/ΔNGFR-transduced CD4+ T cells from the 2 donors were stimulated with aAPC pulsed with A2/MART1 peptide. ΔNGFR+ A2/MART1 multimer+ CD4+ T cells were collected by fluorescence-activated cell sorting (>99% purity) and their TRBV2, 5–1 and 27 CDR3β regions were amplified by PCR and sequenced after cloning. The number of unique CDR3β sequences (top), the relative usage of Jβ gene segments (middle) and the CDR3β amino acid lengths (bottom) are depicted separately for TRBV2 (left), TRBV5–1 (center) and TRBV27 (right).