Extended Data Fig. 1: T1 and T3 cells maintain a predominantly naïve/CM phenotype following expansion.
From: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes
(a) Gating strategy to analyze TCR transduced T cells (percentage of parental gate is shown). Top four panels: gating on FSC/SSChigh, Live/Dead Fixable Near-IRneg, CD8+ singlets. Lower four panels: cells staining positively for APC and PE-labeled pMHC multimers complexed with either peptide-3 (left two plots: T3-transduced cells) or peptide-1 (right two plots: T1-transduced cells) and anti-mouse TCR-β antibody. Although >90% of T1 cells stain positively for the pMHC-PE multimer, a lower % was positive for the corresponding APC multimer due to lower fluorescence intensity of APC relative to PE. (b) Expansion of PB T cells after transduction with T1, T3 or 1G4 at indicated days after retroviral transduction. Data shown as mean ± s.e.m. of n = 3 (d3 and d7) or n = 2 (d5) HLA-A2pos donors. (c) FACS plots showing phenotyping of T cells transduced with T1 on day 7 after spinoculation, gating on FSC/SSChigh, Live/Dead Fixable Near-IRneg, CD3+ singlets, that are either CD4+ or CD8+. Naïve, CM (central memory) and EM (effector memory) T cells. Bar graph in the lower right corner shows proportion of naïve, CM and EM CD4+ or CD8+ T cells on d7 after retroviral transduction with T1, T3 or control TCR. Inset numbers in the top right plot represent mean ± s.e.m. of all groups analyzed together due to similar proportions of CD8+ and CD4+ T cells in experimental groups. Data shown as mean ± s.e.m. of n = 3 donors. (d) Top: The region of gDNA in exon 10 which ends with the first 7 amino acids of the A2-presented epitope, selected as binding site for guide RNA. Middle: sequencing of gDNA from wild type NALM-6 cells, in which the TdT sequence corresponds to the sequence deposited in the database. Bottom: after cloning of CRISPR/Cas9-modified NALM-6 cultures, the selected clone shows a clean deletion in both alleles of the TdT gene, including the first 7 amino acids of the A2-presented epitope. (e) HLA-A2 expression on NALM-6 cells with TdT knock out (right) is unaffected relative to parental cell line (left).
