Fig 1. Design of the sTCR and its use as a labeling reagent.
(a) TCRα/β chains in full-length (upper part) and in soluble form (lower part). V: Variable domain, C: Constant domain, TM: transmembrane, CYTO: cytosolic domain. Interchain cystein bridges are indicated with S. (b) Design of the expression construct in which truncated TCRα and β chains are separated by a ribosome skipping sequence (2A) and tagged on their 3’-end. The tags used in this study are listed at their respective position. To increase interchain stability, a high affinity leucine zipper (LZ) was added to some sTCR constructs. (c) HLA-A2pos T2 cells were loaded O/N with 10 μM MART-1p (red, blue) or 10 μM of an irrelevant peptide (CD20p188–196) (filled grey). Ten μL of supernatant from HEK293 cells producing the DMF5 sTCR (red), or from mock transfected HEK 293 (blue), was added to the T2 cells and incubated for 15 minutes at RT followed by labeling with anti-His-647 antibodies and flow cytometric analysis (d) SupT1 cells expressing SCT-M1 (red) or SCT-CD20 (filled grey) were incubated with the DMF5 sTCR supernatant as described in (c) and stained using anti-His-647. The results in (c) and (d) are representative of at least two experiments and histograms are gated on viable cells, displayed as FSChi, SSChi events.