Extended Data Fig. 2: Overexpression of select ORFs in screen-independent donors.

a, Histograms of selected ORF expression in T cells after puromycin selection. b, Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was complete after 7 days post transduction. To maintain T cells in culture, they were restimulated with CD3/CD28 on days 21 and 42. c, Correlation between ORF sizes and changes in proliferation relative to tNGFR. Mean log₂ fold-changes are shown. d, Proliferation of restimulated CD8+ or e, CD4+ T cells relative to tNGFR in individual donors (n = 3 biological replicates). Mean and SEM are shown. f, g, Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Fig. 2c) measured by dilution of CellTrace Yellow. Representative CellTrace Yellow histograms and fitted distributions (f) as well as quantifications of the proliferation index (g) are shown (n = 3 biological replicates). P values: < 0.0001, 0.0008, < 0.0001, 0.011, 0.0031, 0.0007, < 0.0001, 0.28, 0.004, < 0.0001, 0.58, 0.01, 0.0003, < 0.0001, 0.036, 0.0049 (left to right). h, Viability of ORF-transduced T cells 4 days after CD3/CD28 restimulation. Representative data from one donor (out of 4 donors tested) are shown (n = 3 biological replicates). i, j, Cell cycle analysis of T cells stimulated with CD3/CD28 for 24 h. Gating was performed based on isotype and fluorescence minus one controls. Representative gating (i) as well as (j) quantification of cells in the S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right). Statistical significance for panels g and j: one way ANOVA with Dunnett’s multiple comparisons test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars indicate SEM.