Anti-CD3 mAbs OKT3 and teplizumab share a commonly induced transcriptomic signature in T-ALL. (A) Schematic representation of the experiment. NSG mice infused with 10⁶ leukemic cells from T-ALL case UPNT776, were monitored for leukemia expansion over time. Once leukemic, mice (n = 3 per group) were treated with 40 μg of either control mIgG2a, or OKT3 or teplizumab. Six hours later, mice were euthanized, hCD45⁺/hCD7⁺ leukemic cells sorted from the bone marrow by flow cytometry, and RNA extracted and analyzed using Affymetrix GeneChip Human Gene 2.0 ST array. (B) Volcano plot of probe sets differentially expressed between control and either OKT3- (top panel) or teplizumab (bottom panel) -treated leukemic cells described in panel A. Fold change (x-axis) is plotted against statistical significance (y-axis) for each probe set. Genes with a fold change of ≥1.5 and P < .05 are highlighted as red (OKT3) or orange (teplizumab) dots, respectively. (C) Top panel: Venn diagram of differentially expressed genes between control, OKT3-treated (red), and teplizumab-treated (grey) leukemic cells described in panel A. Commonly deregulated genes are highlighted in greyish red; lower panel: heat map representation of the 932 genes commonly deregulated by OKT3 and teplizumab as compared with the transcriptomic signature of control cells. (D) GSEA of the negative selection transcriptional signature of mouse thymocytes undergoing negative selection as published by Baldwin and Hogquist⁵ with the transcriptomic signature induced in vivo in T-ALL cells in response to OKT3 (left) or teplizumab (right), showing a strong and significant enrichment of this thymic signature in anti-CD3–treated samples. (E) GSEA of the “TNF⍺ signaling via NF-κB” signature with that induced in response to OKT3 (left) or teplizumab (right) in T-ALL cells, showing a strong and significant enrichment of this signature in anti-CD3–treated samples. ctl, control; FDR, false discovery rate; NES, normalized enrichment score; Tepli, teplizumab.