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. Author manuscript; available in PMC: 2007 Dec 19.
Published in final edited form as: Cancer Res. 2006 Sep 1;66(17):8878–8886. doi: 10.1158/0008-5472.CAN-06-1450

Figure 2.

Figure 2

A, comparison of the surface expression of the original TCRs and their hybrids. OKT-3-stimulated PBLs were electroporated with mRNA encoding the different TCR chains and assessed by FACS. Twenty-four hours after electroporation, we assessed p53 pentamer binding for p53-MM or p53-MH and MART-1 tetramer binding for MART-HM or MART-HH. Percentage of positive cells as well as the relative MFI (inside parentheses). B to D, recognition of peptide pulsed cells by original and hybrid TCRs. B, human PBLs were electroporated with the p53-MM, the p53-MH, the α chain of the p53-MM-Mα + the β chain of the p53-MH-Hβ (Mα/Hβ), or the opposite noted as Mβ/Hα. The electroporated cells were then cocultured for 16 hours with T2 cells that were pulsed at 1 μmol/L with specific peptide (p53264-272) or nonspecific peptides (control; data not shown). The concentration of IFN-γ secreted in the medium was measured using an ELISA procedure. C, PBLs were electroporated with the MART-HH, MART-HM, MART-Hα/Mβ, and MART-Hβ/Mα and then cocultured with cultured for 16 hours with T2 cells that were pulsed at 1 μmol/L with specific peptide (MART-1/27L26-35) or nonspecific peptides (control; data not shown). IFN-γ secretion in cultures with T2 pulsed with control peptides (gp100-209, + gp100-280, p53149-157, and HBVc) was ≤200 pg/mL (data not shown). D, CD4 enriched cells were electroporated with mRNA encoding the α and β chains of NY2-HH, a HLA-DP4-restricted NY-ESO-1 TCR, or its mouse hybrid, NY2-HM. NY-ESO-1161-180 peptide-pulsed HLA-DP4+ EBV-B line (DK-EBV-B) as well as nonpulsed cells as a control were cocultured overnight with TCR RNA electroporated CD4 T cells. IFN-γ secretion was detected by ELISA.