Supplementary Figure 2: Search for viral epitopes.
From: Deciphering CD4+ T cell specificity using novel MHC–TCR chimeric receptors
(a,b) CD4+ T cells from mice infected with LCMV were purified from the spleens 5 and 8 days p.i. and fused with α−β−BW5147 cells carrying an NFAT (GFP) reporter. Reactivity of the resulting hybridomas was determined by NFAT activation after co-culture with LCMV- or gp61-pulsed dendritic cells. Histograms (a) show examples of different types of reactivity (LCMV-open or gp61-filled dark gray) which are summarized in b. (c) A scheme of the cloning strategy for the construction of cDNA-derived peptide (CDP) libraries enriched for natural peptides. ORF – open reading frame dictating the natural protein sequence. (d) Cells transduced with the MCR2-CDP library were sorted into MCR2low, MCR2med and MCR2hi, and the length of the MCR-connected peptides was determined by PCR on the isolated DNA. No major difference in the length of the PCR products was observed, indicating that, in all fractions, mainly peptides between 15 and 50aa were present. (e) MCR2-LCMV+ 16.2c11 reporter cells were co-cultured with LCMV-reactive hybridomas (H2, H3, H14 and H30) of unknown peptide specificity. Activated reporter cells were sorted, expanded and co-cultured again with the corresponding hybridomas. Dot plots show example NFAT activation after two or three rounds of co-culture. (f) After the 3rd round of enrichment, single cells were sorted, expanded and their reactivity verified by co-culture with hybridomas (histogram shows example NFAT reactivity in reporter cells). The table on the right, shows frequencies of reactive clones. Peptide sequences from the MCR2 constructs, representing the dominant NP311 epitope and the new NP547 epitope are shown below. Figure represents data from one experiment.
