Extended Data Fig. 5: Functional analysis of LTBR overexpression in T cells.

a, LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v8⁷⁵ (n = 948 donors). Box shows 25–75 percentile with a line at the median. b, LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from 2 donors⁷⁶. Cell types indicated are derived from Harmony tSNE clustering of single-cell transcriptomes. c, Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified in single-cell or bulk RNA-seq. d, e, TCF1 expression in LTBR or tNGFR transduced T cells. d, Representative histograms of TCF1 expression and the gate for TCF1+ cells (dashed line) are shown, as well as e, quantification of TCF1+ cells (n = 3 biological replicates). f–h, ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histograms (f), quantification (g) in n = 3 donors (CD8+) or n = 4 donors (CD4+) and time course (h) of expression in LTBR and tNGFR cells after CD3/CD28 stimulation (n = 3 biological replicates). i, Differentiation phenotype of NGFR and LTBR transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: Central memory. EM: Effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naïve: CD45RO^neg CCR7⁺, CM: CD45RO⁺ CCR7⁺, EM: CD45RO⁺ CCR7^neg, effector CD45RO^neg CCR7^neg). j, Representative dot plots of T cell viability after CD3/CD28 stimulation. Viable cells are in the lower left quadrant. k, Cell viability of CD4+ T cells transduced with LTBR or tNGFR lentivirus, either restimulated with CD3/CD28 for four days or left unstimulated (n = 2 donors with 3 biological replicates each). l, m, LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3/CD28 beads every three days for up to three rounds of stimulation. Following repeated stimulation, expression of TIM-3 and LAG-3 (l) was measured in resting cells, and secretion of IFNγ and IL2 (m) was measured in restimulated cells (n = 3 biological replicates). Statistical significance for panels e, i, and k: two-sided unpaired t-test; for panel g: two-sided paired t-test. Error bars indicate SEM.