Supplementary Figure 7: Related to Fig. 7. Hyperactivation of Ikaros-deficient B cells upon TLR stimulation.
From: Ikaros prevents autoimmunity by controlling anergy and Toll-like receptor signaling in B cells
a, Expression of the different Tlr genes in naïve FO B cells of the indicated genotypes, as determined by RNA-seq. b, Time course analysis of B cell activation in response to LPS, R848 or CpG stimulation. CD43– FO B cells isolated from the spleen of Ikaros mutant (MD4 BCR-tg Ikzf1B–; dark purple line) and control (MD4 BCR-tg Ikzf1B+; light purple) mice were stained with the CellTrace Violet reagent prior to LPS, R848 or CpG stimulation for up to 4 days. The CellTrace Violet staining, cell size and expression of the activation marker CD69 were analyzed at the indicated time points by flow cytometry. Unstimulated ex vivo sorted FO B cells are shown as a reference. c, Schematic diagram of the stimulatory CpG-HEL immune complex (SCHIC), which was generated by mixing HEL-Bio1-2, CpG-Bio and streptavidin (SA) at a 1:1:2 molar ratio (see Online Methods). d, Survival of CD43– FO B cells of the indicated genotypes after stimulation with a HEL-Bio-SA complex (n = 10), CpG-Bio (n = 10) and the SCHIC reagent (n = 5) for 3 or 4 days, as determined by staining with the viability dye eF780. e, Activation of the CD43– FO B cells of the indicated genotypes with the SCHIC reagent for up to 4 days. The proliferation and survival of the FO B cells were measured by staining with CellTrace Violet and the viability dye eF780, respectively (upper panel). The viable FO B cells were further analyzed for their cell size and CD69 expression by flow cytometry (lower panel). Dead cells are indicated by blue shading. The RNA-seq data presented in a were obtained from 2 independent experiments and are shown as mean values with SEM. The data in b, d and e are representative of 3, 2 and 2 independent experiments. Statistical data in d are shown as box plots with minimal to maximal whiskers including all data points and were analyzed by multiple t-test (unpaired, two-tailed, Holm-Sidak); *P < 0.03.
