Supplementary Figure 3: Reduced survival and increased TCR signal strength in SFR-KO iNKT cells.
From: SLAM receptors foster iNKT cell development by reducing TCR signal strength after positive selection
(a) Flow cytometry analyses showing 7-AAD staining in total, NK1.1- or NK1.1+ thymic and splenic iNKT cells from WT and SFR KO mice. n = 12. (b) Flow cytometry analyses of 7-AAD staining in total, NK1.1- or NK1.1+ thymic iNKT cells from WT and SFR KO mice after 20 h in vitro culture in growth medium. n = 10. (c) Flow cytometry gating strategies for stage 0 iNKT cells from the negative selection-enriched thymocytes (CD11b-CD11c-Gr-1-Ter-119-CD19-CD8α-CD62L-TCR γ/δ- cells; boxed in red). (d) Flow cytometry analyses showing expression of Nur77-GFP in T-bet+ (iNKT1) and T-bet- (iNKT2 plus iNKT17) iNKT cells from thymus and spleen of WT (lacking Nur77-GFP), Nur77GFP and SFR KO-Nur77GFP mice. n = 5. (e-h) Flow cytometry analyses showing expression of Egr2 (e, f) and PLZF (g, h) in thymic and splenic iNKT cells (e, g) and various stages of thymic iNKT cells (f, h) from WT and SFR KO mice. n = 8 (e, f); n = 9 (g, h). (i) Flow cytometry analyses showing expression of Egr2 and PLZF on stage 0 iNKT cells from WT, SFR KO and SAP KO mice. n = 9 [Egr2 (WT)], n = 6 [Egr2 (SFR KO and SAP KO)], 6 samples of Egr2 (WT) and Egr2 (SFR KO) were repeated from f; n = 6 [PLZF (WT and SAP KO)], n = 4 [PLZF (SFR KO)], 4 samples of PLZF (WT) and PLZF (SFR KO) were repeated from h. Numbers in histograms indicate the MFI of Nur77-GFP (d), Egr2 (e, f, i), and PLZF (g, h, i). (j) Flow cytometry analysis showing CD48 expression on thymocytes from a mixed Nur77GFP + SFR KO-Nur77GFP BM chimera. In (a,d-i), a representative experiment is shown on the left, while data from multiple mice are depicted on the right. In (b), a representative experiment is shown at the top, while data from multiple mice are depicted at the bottom. Each symbol (a, b, d-i) represents an individual mouse; error bars represent mean with s.d.. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns P > 0.05 (two-sided unpaired t-test; two-sided paired t-test for e, f). Data are representative of 6 (a), 5 (b), 3 (c, d), 4 (e-h, j), and 4 [Egr2 (WT and SAP KO)], 3 [Egr2 (SFR KO); PLZF (WT and SAP KO), 2 [PLZF (SFR KO)] (i) independent experiments.
