Functional assays identify PD1 as a monogenic driver of TCR-dependent phenotypes. (A) Hypothesized effects of knockout of CTCL tumor suppressors and oncogenes in nontransformed T cells. (B) Log₂-fold change of single-guide RNAs (sgRNAs) targeting putative CTCL driver genes in primary TCR-stimulated T cells.²⁷ P < .0001, by 2-sided, unpaired Student t test. (C) Volcano plot of z-score and log₂-fold change of sgRNAs in primary T cells.²⁷ Putative oncogenes (red) and tumor suppressors (blue) with log₁₀P > 2 are highlighted. (D) Functional assays performed using ex vivo culture of CTCL cells. Malignant cells were isolated via FACS sorting from peripheral blood mononuclear cells and the indicated assays performed. (E) Representative proliferation assays of CTCL cells ex vivo under 5 stimuli. CD4⁺ T-cell subsets were isolated from patients with CTCL or healthy controls. The cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and treated with the indicated stimuli for 7 days. Dilution of the dyes represent cell division. (F) The approach to identifying regulation of heterogenous ex vivo proliferation. (G) Volcano plot of transcripts upregulated (red) or downregulated (blue) in high proliferative compared with nonproliferative CTCL cells. P_adj<.05. (H) PD1 protein expression on high (n = 6) and nonproliferative cells (n = 5) measured by flow cytometry. Flow cytometry data represent frequency of PD1 positivity ± standard error of the mean. P value determined by 2-tailed, unpaired Student t test. (I) Frequency of deletions in PDCD1 in high and nonproliferative CTCLs. P value calculated by Fisher’s exact test.