Tetracysteine tagged OVA (323–339) peptide as a novel tool for visualizing peptide-MHCII complexes in primary mouse cells

Abstract Professional APCs present peptides to CD4+ T cells in the context of MHCII. To understand the dynamics of peptide-MHCII complexes in T cell-APC interaction, real-time visualization of these complexes is crucial. However, current reagents such as peptide specific MHCII antibodies do not allow one to probe peptide-MHCII dynamics during live interactions in vivo, as they also block T cell-APC interactions. Fluorescent tags such as green fluorescent protein (GFP) consist of more than 200 amino acids and have the potential of disrupting the functionality of the antigenic peptides. Therefore, we constructed an OVA(323–339) peptide containing a pro-fluorescent tetracysteine tag (CCPGCC) at the C-terminus with aminocaproic acid linker (OVACACA). First, we tested whether the addition of tag had an impact on T cell stimulation and found that OVACACA pulsed APCs could activate OTII cells to same degree as OVA(323–339). Secondly, we treated the APCs with an organoarsenic compound to switch on the fluorescence and measured the signal by flow cytometry. We showed that the fluorescent signal specifically was derived from OVACACA-MHCII, as neither MHCII−/− APCs pulsed with OVACACA, nor MHCII+/+ APCs pulsed with OVA(323–339) produced the same signal. We also tracked the fluorescent signal in OTII-DC cultures in vitro and detected that it was concentrated at the synapse over time. Altogether our findings suggest that tetracysteine tagged peptides can be used for tracking peptide-MHCII complexes during the interactions of primary mouse T cells and APCs and have the potential to be an alternative to MHCII tetramers to detect antigen specific T cells.