Supplementary Figure 1: Defective iNKT cell development in SFR-KO mice.
From: SLAM receptors foster iNKT cell development by reducing TCR signal strength after positive selection
(a) Flow cytometry analyses showing numbers and frequencies in various stages of splenic iNKT cells from WT and SFR KO mice. n = 11. CD44+NK1.1- stage 2 cells (St 2) and CD44+NK1.1+ stage 3 cells (St 3). (b,c) Flow cytometry analyses showing various subpopulations of conventional T cells in thymus (b) and spleen (c) of WT and SFR KO mice. n = 10. Subpopulations are demarcated by boxes or ellipses. (d) Flow cytometry analysis showing CD48 expression on thymocytes of a mixed WT + SFR KO BM chimera. (e–g) Flow cytometry analyses showing various subsets of thymic and splenic iNKT cells from WT and SFR KO mice. n = 8. T-bet+PLZFlo iNKT1 cells (e), GATA3+PLZFhi iNKT2 cells (f) and RORγt+PLZFint iNKT17 cells (g). (h) Flow cytometry analyses showing expression of various TCR Vβ on thymic and splenic iNKT cells from WT and SFR KO mice. n = 5 (TCR Vβ7), n = 8 (TCR Vβ8.1, 8.2), n = 6 (TCR Vβ8.3). (i) Quantitative RT-PCR analysis showing TCR Vα14- and Vα10-encoding RNA (Trav11 or Trav13) expression in splenic iNKT cells from WT and SFR KO mice. n = 7. Results for SFR KO are normalized to WT (top), while results for TCR Vα10 are normalized to TCR Vα14 (bottom). (j) Sequences of the TCR Vα14-Jα18 rearrangements in splenic iNKT cells from WT and SFR KO mice. n = 7. M, nucleotide A or C; S, nucleotide C or G. In a previous report24, it was shown that, in some mutant mice, one of the nucleotides (boxed) of the codon for aspartate (D) 94 can be replaced by a nucleotide encoding an alternative amino-acid. This mutation was not seen in SFR KO mice. In (a), a representative experiment is shown at the top, while data from multiple mice are depicted at the bottom. In (b,c,e-h), a representative experiment is shown on the left, while data from multiple mice are depicted on the right. Each symbol (a-c, e-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). Data are representative of 6 (a); 3 (b,c,e-g); 4 (d); 4 (TCR Vβ7 and TCR Vβ8.1, 8.2) and 3 (TCR V8.3) (h); or 5 independent experiments (i,j).
