Fig. 3: Endogenous TCR KO improves the expression and antigen sensitivity of HLA-independent receptors.

a, CD19-specific mutSTAR, split ChTCR and full ChTCR. b, Flow plots of mutSTAR and ChTCR T cells stained with TCRαβ-BV421 and rCD19-APC without TCR KO and after TRAC KO, TRBC KO or both TRAC and TRBC KO. c, Flow plots of mutSTAR T cells stained with mouse TCRβ-BV421 and rCD19-APC without or with KO of the indicated TCR chain. d, Geometric mean of mouse TCRβ-BV421 in T cells transduced with mutSTAR without or with KO of the indicated TCR chain. Data are shown as the mean ± s.d. for four independent experiments. e, Left: flow cytometry of TCR^KO T cells expressing the mutSTAR, split ChTCR and full ChTCR stained with CD3ε-BUV395 antibody. Right: geometric mean of CD3ε on TCR^KO T cells expressing the mutSTAR, split ChTCR and full ChTCR. Data are shown as the mean ± s.d. for three independent experiments. f,g, IL-2 (f) and IFNγ (g) concentrations in supernatants after coculture of T cells with Nalm-6 CD19^low target cells at an E/T of 1:1. Data are shown as the mean ± s.d. for three independent experiments. h, Schematic of CD19-specific HIT receptor and targeted knockin to the TRAC locus. i, Flow cytometry of TRAC knocked-in (Kin) HIT T cells stained with TCRαβ-BV421 and rCD19-APC with or without TRBC KO. j, Geometric mean of rCD19-APC binding to HIT-expressing T cells with or without TRBC KO. The mean ± s.d. for three independent experiments is shown. k, Flow plots of T cells expressing the HIT receptor, with TRAC Kin and TRBC KO, split ChTCR or full ChTCR, with TRAC/TRBC KO, stained with TCRαβ-BV421 and rCD19-APC. l, Geometric mean of TCRαβ-BV421 in TCR^KO T cells, KO as described in k, expressing the HIT receptor, split ChTCR or full ChTCR. The mean ± s.d. for three independent experiments is shown. m, Flow cytometry histograms (left) and geometric mean (right) of CD3ε-BUV395 in TCR^KO T cells, KO as described in k, expressing the HIT receptor, split ChTCR or full ChTCR. The mean ± s.d. for three independent experiments is shown. n,o, IFNγ (n) and IL-2 (o) concentrations in supernatants after coculture of T cells expressing the HIT receptor with or without TRBC KO with Nalm-6 CD19^low cells at an E/T of 1:1. The mean for three independent experiments is shown. p, Left: histograms of CTV dye dilution in T cells expressing the HIT receptor with and without TRBC KO after coculture with Nalm-6 CD19^low tumor cells. Right: percentage of divided cells (mean) for three independent experiments. q,r, IFNγ (q) and IL-2 (r) concentrations in supernatants after coculture of TCR^KO T cells expressing the HIT receptor, split ChTCR or full ChTCR with Nalm-6 CD19^low cells at an E/T of 1:1. The mean ± s.d. for three independent experiments is shown. P values, where shown, were calculated using a two-way ANOVA. Panels a,h created using BioRender.com.

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