Extended Data Fig. 2: The expression of S1PR1 mRNA in Tfh cells, and the controls of S1PR1 protein staining.
(a) Gating strategy of non-TH17/TFH, Rorγt+ T, Rorγt− TFH and Rorγt+ TFH cells in K/BxN mice. PP cells from SFB + K/BxN mouse were stained with Abs against CD4, TCRβ, Foxp3, Rorγt, CXCR5 and PD-1. (b) IL-17− TFH (eGFP−TdTom−CXCR5+PD-1+) and TFH17 (eGFP+TdTom+CXCR5+PD-1+) cells were sorted from PPs of Current-Ex TH17 fate-mapping mice expressing both TH17-fate reporter TdTom and IL-17 “current” reporter eGFP. S1pr1 transcripts were determined by qRT-PCR. Data were first normalized with the HPRT housekeeping gene, and then further normalized to the average expression of S1pr1 in IL-17− TFH cells (value set as 1) in each experiment. (n = 6 mice/ IL-17− TFH, and 5 mice/TFH17, data combined from 3 independent experiments). (c) Data show the specificity of anti-S1PR1 staining. (I) PP cells from SFB + K/BxN mice were treated in vitro with or without FTY720P for 1 hr at 37 °C and then stained for CD4, TCRβ, and S1PR1. Representative contour plots from the indicated FTY720P concentration are shown. (II) Thymocytes from WT and S1PR1 cKO (CD4CreS1PR1fl/fl) mice were stained with the Abs against CD4, CD8, TCRβ, and S1PR1. Representative plots show the percentage of S1PR1 on CD4 single-positive T cells from WT and S1PR1 cKO thymocytes.
