Supplementary Figure 5: Activation-marker expression by mouse B cell subsets and leukocyte characterization after continuous neutrophil depletion during IL-18-induced inflammation.
From: Neutrophils license iNKT cells to regulate self-reactive mouse B cell responses
(a) Flow cytometry data showing frequencies of B cell subsets in wild-type (C57BL/6) compared to CD1d1–/– mice following injections with vehicle or IL-18, day 12. (b) ELISA measures of serum amounts of total IgM (left panel) and IgG (right panel) in WT and CD1d1–/– mice injected with vehicle or IL-18, day 12. (c-e) Flow cytometric analysis of activation marker expression by indicated B cell subset in the spleens of wild-type (C57BL/6) or CD1d1–/– mice following treatment with vehicle or IL-18, day 12. (f) Representative flow cytometry plots of splenic neutrophils on day 12 following vehicle or IL-18 treatment in combination with doubled neutrophil depletion strategy (Depl) using 500 μg of 1A8 antibody 24 h before IL-18 and on days 3, 6, and 9 in wild-type (C57BL/6) mice. Numbers below outlined areas indicate percent Ly6G+ CD11b+ neutrophils. (g) Summary of flow cytometry assessment of frequency of splenic Ly6G+ CD11b+ neutrophils from (g). (h) Left panel: Flow cytometry assessment of frequency of splenic CD138+ B220lo PCs day 12 after vehicle or IL-18 treatment in combination with neutrophil depletion according to (f). Center panel: Flow cytometry assessment of frequency of splenic B220+ CD95+ GL7+ GC B cells day 12 after vehicle or IL-18 treatment in combination with neutrophil depletion according to (f). Right panel: ELISA measure of serum concentrations of anti-DNA IgG antibodies in mice day 12 after vehicle or IL-18 treatment in combination with neutrophil depletion according to (f). (i) Intracellular flow cytometry assessment of expression in iNKT cells of the nuclear transcription factor GATA3 day 12 after vehicle or IL-18 treatment in combination with neutrophil depletion according to (f). *P < 0.05 (two-tailed unpaired Student’s t-test; wild-type (C57BL/6) IL-18 vs CD1d1–/– IL18 groups) (a-e; n = 5 mice per group) or (1way ANOVA with Tukey correction) (g-i; n = 4 per group). Data pooled from two experiments (a-e) or from one experiment (f-i). Box height indicates group mean + s.e.m.
