Extended Data Fig. 7: Testosterone dependence of the GPR174-mediated migratory response to CCL21 does not involve modulation of GPR174 or CCR7 expression.
From: A GPR174–CCL21 module imparts sexual dimorphism to humoral immunity
a, CCL21-induced transwell migration of B cells from GPR174-sufficient (WT) or -deficient (KO) male littermate mice that were sham-operated (sham) or orchidectomized (ORX), allowed to recover for six weeks, and then treated with either vehicle or testosterone for two weeks. Data represent three biological replicates from one of two independent experiments with similar results. Blue asterisks, male sham wild type plus vehicle versus sham GPR174-knockout plus vehicle; red asterisks, male ORX wild type plus vehicle versus male ORX wild type plus testosterone; green asterisks, male sham wild type plus vehicle versus male ORX wild type plus testosterone. b, Relative Gpr174 mRNA levels normalized to Gapdh expression in naive and activated B cells from male or female mice that were either sham-operated or gonadectomized. Data (n = 3 biological replicates) represent three independent experiments with similar results. c, Surface CCR7 levels on naive or activated B cells isolated from mice of the indicated GPR174 genotypes, sexes and gonadectomies. Data shown are histogram overlays (coloured lines, CCR7; grey lines, isotype staining control) and CCR7 MFIs after isotype staining background subtraction. Each symbol in the MFI data represents one mouse, and data represent three independent experiments with similar results. d, e, Relative Gpr174 mRNA levels (d) and surface CCR7 levels (e) on activated B cells from male mice that were sham-operated or orchidectomized and then treated with either vehicle or testosterone. Data represent three mice per group from one of three independent experiments with similar results. f, g, Relative Gpr174 mRNA levels (f) and surface CCR7 levels (g) on activated B cells from male or female mice that were treated with either vehicle or testosterone. Data represent three mice per group from one of three independent experiments with similar results. h, Relative expression of Gpr174 mRNA in male or female dsRed-expressing MD4 B cells normalized against that in cotransferred male GFP-expressing MD4 B cells 90 h after activation by HEL–OVA immunization (as in Extended Data Fig. 1); three line-connected pairs indicate three independent experiments. Two-way ANOVA with Bonferroni’s multiple comparisons (a) or two-tailed unpaired (b–g) or paired (h) Student’s t-tests were used for statistical comparisons, with P values given in the graph.
