Extended Data Fig. 1: Optimisation of a genome-wide screen for regulators of TH2 cell differentiation. | Nature Immunology

Extended Data Fig. 1: Optimisation of a genome-wide screen for regulators of TH2 cell differentiation.

From: An αvβ3 integrin checkpoint is critical for efficient TH2 cell cytokine polarization and potentiation of antigen-specific immunity

Extended Data Fig. 1

Extended Data Fig. 1 (a) Schematic of the optimised TH2 cell culture protocol for CRISPR screening. (b) Flow cytometric analysis of IL-13Tom expression by TH2 cells transduced with NT or Gata3 sgRNAs using the optimised protocol. Data representative of 3 independent experiments. (c) RNA-sequencing analysis of Gata3-targeted versus non-targeted TH2 cells using the optimised screening protocol. (d) KEGG pathway analysis of genes downregulated in Gata3-targeted versus non-targeted TH2 cells. (e) and (f) Gene set enrichment analyses of genome-wide positive regulators of TH2 cell differentiation identified in the screen. (g) Selection of the top 1018 genes from the genome-wide screen for a secondary screen (fold change > 0.06 and p-value < 0.07). (h) Validation of novel regulators by individual confirmatory sgRNA knockdown. Data representative of 3 independent experiments; mean ± SD; one-way ANOVA with Dunnett’s post-hoc test. (i) Validation of novel regulators as in (h) with corresponding TH1 comparisons. (h) & (i) **** P < 0.0001, ***P = 0.0006 (Fermt3), **P = 0.0018 (Hsp90b1), 0.0053 (Tfap4), *P = 0.0102 (Fnta), 0.0169 (Smarcc1), 0.0212 (Apbb1ip), 0.0252 (Kmt2d).

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