Extended Data Fig. 1: Pooled in vivo CRISPR–Cas9 screening and an in vivo dual transfer system to identify and validate potential regulators of TFH cells.
From: Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine
a, Diagram of the screening system. Naive Cas9-expressing SMARTA cells were transduced with lentiviral-derived sgRNA metabolic library, expanded in vitro and transferred into C57BL/6 mice, which were then infected with LCMV 24 h later. At day 7 after infection, splenic TFH (CXCR5+SLAM−) and TH1 (CXCR5−SLAM+) cells were purified and those gRNAs that were upregulated (corresponding to negative regulators of TFH responses) or downregulated (corresponding to positive regulators of TFH responses) in TFH versus TH1 cells (|log2(TFH/TH1)| > 0.5; adjusted P < 0.05) were determined for metabolism-related genes that establish TFH over TH1 cell differentiation. b, Diagram of in vivo dual transfer system. SMARTA cells transduced with sgRNA viral vectors expressing distinct fluorescent proteins were mixed and transferred into the same C57BL/6 mice, followed by LCMV infection and experimental analyses. c, SMARTA cells transduced with the indicated sgRNA viral vectors (Ametrine+) were mixed at a 2:1 ratio with sgNTC (mCherry+)-transduced SMARTA cells and transferred into C57BL/6 mice, followed by LCMV infection. Analyses of the proportion of donor-derived TFH (CXCR5+SLAM− or PSGL-1−Ly6C−) and TH1 (CXCR5−SLAM+ or PSGL-1+Ly6C+) cells and quantification of relative TFH cell percentage and number (lower right) in the spleen at day 7 (n = 4 mice). d, Insertion and deletion (indel) mutations after CRISPR–Cas9 targeted disruption in SMARTA cells transduced with sgNTC or sgPcyt2, through deep sequencing analysis of indels generated at the exonic target site of the Pcyt2 gene, including 74% of indel events in sgPcyt2-transduced cells compared to 0.6% in sgNTC-transduced cells. e, Immunoblot analyses of ETNK1 and PCYT2 in splenic SMARTA cells at day 3 after infection. Asterisk (*), non-specific band; arrow, the target band. Data are representative of two (d, e), or at least three (c) independent experiments. Data are mean ± s.e.m. P values are determined by one-way ANOVA (c). ***P < 0.001. Numbers in quadrants or gates indicate percentage of cells.
