Extended Data Fig. 4: Experimental design and reagents for testing whether ΔFAS protects against TCR-mediated rejection of allogeneic lymphocytes.
From: CAR-engineered lymphocyte persistence is governed by a FAS ligand–FAS autoregulatory circuit
(a) Graphical overview of an experimental design to test whether ΔFAS coexpression protects CAR-T cells from elimination by allo-reactive T cells. CD8+ T cells from a non-HLA-A*03 donor were transduced either with control HLA-A*03:01-restricted TCRs or TCR (RG4382-5), an allo-reactive TCR that recognizes HLA-A*03+ cells in a peptide agnostic manner. TCR transduced T cells were co-cultured with a ~ 1:1 mixture of HLA-A*03:01+ T cells transduced with tLNGFR-1928ζ or tEGFR-1928ζ-ΔFAS at a 10:1 effector-to-target-ratio. (b) Table listing the TRAV, TRAJ, TRBV, TRBJ, and CDR3 sequences for a TCR (RG4382-5) that confers allogeneic recognition of HLA-A*03:01+ cells. (c) Representative FACS plots and (d) summary bar graphs of CD107a and TNFα expression by non-HLA-A*03 CD8+ T cells transduced with the RG4382-5 TCR and co-cultured with the indicated cells lines. The HLA-A haplotype of each cell line is listed as a table below. Numbers within each FACS plot and bar graph indicate the frequency of CD107a or TNFα producing T cells after pre-gating on live+mTCR+CD8+ cells. Bar graphs displayed as mean ± SEM (n = 3 biologically independent samples).
