Fig. 1: ChTCRs reproduce canonical TCR structure, synapse formation and proximal signaling in T cells.
a, CD19-specific ChTCRs and CARs. Left to right—Split ChTCR, VhCα:FMC63 variable heavy chain-TCRα constant chain; VlCβ:FMC63 variable light chain-TCRβ constant chain. Full ChTCR, FMC63 VlVh-Cα:Cβ TCR. The ChTCRs are shown associated with CD3 subunits. 28z CAR, CD28ζ CAR with FMC63 VlVh scFv linked to the CD28 hinge/transmembrane (H/T) and costimulatory domain and CD3ζ. BBz CAR, BBζ CAR with FMC63 VlVh scFv linked to the IgG4 hinge, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3ζ. b, Flow plots of CD8+ T cells stained with anti-TCRαβ antibody and rCD19 protein after transduction and endogenous TCRαβ KO. c, Geometric mean ± s.d. of rCD19-APC binding to T cells expressing the split or full ChTCR with and without KO of TRAC, TRBC or both chains (TRAC + TRBC) (n = 5 independent samples). d, Representative TCRαβ expression on unedited and TCRαβKO ChTCR+ and CAR+ T cells. e, Left: representative CD3ε expression on unedited and TCRαβKO ChTCR+ and CAR+ T cells. Right: geometric mean ± s.d. of CD3ε-BUV395 (n = 4 independent donors). f, Left: TIRF images of ChTCR+ and CAR+ T cells interacting with a lipid bilayer functionalized with ICAM-1-AF488 (green) and CD19-AF647 (blue) and stained with an anti-CD45-AF555 antibody. Scale bars, 10 μm. g, Normalized mean intensity of ICAM-1-AF488 (green) and CD19-AF647 (blue) staining across cell radiuses in synapses; dots represent the mean at each position, with a solid trend line (n = 100 cells). h, Left: calcium flux after antigen cross-linking of T cells expressing each specified receptor. Arrows indicate the cross-linking of receptors and the addition of phorbol myristate acetate/ionomycin (PMA/iono). Right: area under the curve of calcium flux over 300 s, mean ± s.d. (n = 4 independent experiments). i, Left: western blot of LAT pTyr220, actin and LAT after antigen activation of T cells expressing the indicated receptor. Separate blots were run for phosphorylated and total protein analytes. Membranes were cut and processed to detect analytes of different molecular weights in parallel. Actin loading controls are displayed adjacent to the corresponding analyte. Right: heat map of the mean band intensity of LAT pTyr220 normalized to actin control (n = 3 independent experiments; FC, fold change). j, Left: western blot of ZAP70 pTyr319, actin and ZAP70 after antigen activation of T cells expressing the indicated receptor. Right: heat map of the mean band intensity of ZAP70 pTyr319 normalized to actin control (n = 3 independent experiments). P values, where shown, were calculated using a two-way analysis of variance (ANOVA). Panel a created using BioRender.com.
