Skip to main content
. Author manuscript; available in PMC: 2023 Feb 9.
Published in final edited form as: Nature. 2022 Mar 16;603(7902):728–735. doi: 10.1038/s41586-022-04494-7

Figure 4. LTBR overexpression improves T-cell function via canonical NF-κB activation.

Figure 4.

(a) Differential expression of genes in resting LTBR and tNGFR (negative control) T-cells. Genes highlighted in red are those with a 2-fold or greater change in expression and an adjusted p < 0.05. (b) Significantly enriched Gene Ontology biological processes in LTBR overexpressing T-cells (p < 0.05). (c) Cell viability of CD8+ T-cells transduced with LTBR or tNGFR lentivirus, either re-stimulated with CD3/CD28 for four days or left unstimulated (n = 2 donors with 3 biological replicates each). (d) PD-1 expression on resting LTBR or tNGFR T-cells stimulated with a 3:1 excess CD3/CD28 beads every 3 days, for up to 3 rounds of consecutive stimulation. (e) ICAM-1 expression (resting) and IL-2 secretion (activated) by T-cells transduced with FLAG-tagged LTBR mutants, normalised to wild-type LTBR (n = 6 replicates across two experiments). (f) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (g) Quantification of phospho-RelA in LTBR or tNGFR T-cells stimulated with CD3/CD28 antibodies for indicated periods of time. (h, i) Quantification of phosphorylated IκBα (h) or mature NF-κB2 (i) in resting or CD3/CD28-stimulated (15 minutes) LTBR or tNGFR cells. (j) IFNγ secretion by stimulated LTBR or tNGFR cells after CRISPR knockout of indicated genes (n = 18, 3 sgRNAs in 2 donors in 3 biological replicates). IFNγ quantities are normalised to corresponding non-targeting controls (either LTBR or tNGFR) to allow comparisons of relative impacts of gene knockout on T-cell activation. (k) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to non-targeting controls). Box shows 25-75 percentile with a line at the median while whiskers extend to 1.5x of interquartile range. Unpaired two-sided t test p values (panels c, g-k): ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 (exact p values are given in Supplementary Table 15). Error bars are SEM. N = 3 biological replicates, unless stated otherwise.