Extended Data Fig. 4: The CDP–ethanolamine pathway but not the CDP–choline pathway is required for TFH cell differentiation.
From: Metabolic control of TFH cells and humoral immunity by phosphatidylethanolamine
a, Diagram summary of the effects of single or combined deficiency of genes in the CDP–ethanolamine and CDP–choline pathways on TFH cell differentiation after LCMV infection. b, Immunoblot analyses of the indicated proteins in the indicated sgRNA-transduced SMARTA cells isolated from the spleen at 3–5 days after adoptive transfer and LCMV infection. Asterisk (*), non-specific band; arrow, the target band. c, Summary of the relative proportions of TFH (CXCR5+SLAM−) and TH1 (CXCR5−SLAM+) cells in donor-derived cells in spleen of mice receiving the indicated sgRNA-transduced SMARTA cells at day 7 after LCMV infection (n = 4 mice). d, Lipidomic analysis of the lipid content (PC and PE) in wild-type TFH (transduced with sgNTC), Pcyt1a-deficient TFH and Pcyt1a and Pcyt1b doubly deficient TFH cells (n = 3 samples, each pooled from multiple mice). e, Flow cytometry analysis (left) and summary of the proportion of wild-type and Pcyt2-deficient CXCR5+ SMARTA cells (middle) and their dilution of CellTrace Violet (CTV; right) at day 2 after LCMV infection (n = 3 mice). f, Summary of apoptotic wild-type and Pcyt2-deficient SMARTA cells as analysed by Annexin V and 7-AAD staining (or unstained) in freshly isolated splenocytes at day 2 after LCMV infection (n = 3 mice). g, Distribution of SMARTA cells in the follicle in the spleen at day 3 after infection. Scale bar, 50 μm (n = 50 sections). h, Diagram of TFH cell effector functional assay. sgNTC (mCherry+) and sgPcyt2 (Ametrine+)-transduced SMARTA cells (CD45.1+) were transferred (first transfer) into C57BL/6 mice (CD45.2+), followed by LCMV infection. Seven days later, the fully differentiated wild-type or Pcyt2-deficient CXCR5+SLAM− TFH cells (CD45.1+) were sorted and equal numbers of these cells were transferred (second transfer) into LCMV-infected mice (CD45.2+; this transfer occurred at day 1 after LCMV infection). GC B cell and plasma cell formation was analysed at day 5 after the second adoptive transfer. Data are representative of one (d) or at least two (b, c, e–g) independent experiments. Data are mean ± s.e.m. P values are determined by one-way ANOVA (c, d) or by two-tailed unpaired Student’s t-test (e, f). NS, not significant; **P < 0.01 and ***P < 0.001. Numbers in gates indicate percentage of cells.
