Journal: Nature Communications

Article Title: Myelin-reactive B cells exacerbate CD4 + T cell-driven CNS autoimmunity in an IL-23-dependent manner

doi: 10.1038/s41467-024-49259-0

Figure Lengend Snippet: a Male WT ( n = 6) and IgH [MOG] ( n = 6) mice were immunized with MOG [35-55] and B cells were isolated from CNS tissues at endpoints. Expression of IL-23 heterodimer was assessed by intracellular flow cytometry. Data are presented from one of four immunizations. Two-tailed t -test was used. b IF detection of IL-23 (green), B220 (red), CD4 (aqua), DAPI (blue) in the meninges of female MOG [35-55] -immunized WT or IgH [MOG] mice. Representative of n = 3 mice of each genotype from a single immunization. c Female IgH [MOG] mice were immunized with MOG [35-55] and were treated on day (-1) and day 6 with 1 mg anti-p19 ( n = 5) or rIgG1 isotype ( n = 5). Disease curves representative of one of seven experiments. Two-tailed t -test was used. d Cerebellar meningeal sections from MOG [35-55] -immunized female IgH [MOG] mice, treated with isotype ( n = 6) or anti-p19 ( n = 6), were assessed for lymphocyte aggregation. Data are from two immunizations. Two-tailed t -test was used. e CNS-infiltrating mature, transitional and class-switched B cells were assessed by flow cytometry from anti-p19 ( n = 4) or rIgG1 isotype ( n = 4)-treated immunized female IgH [MOG] mice. Gated on live B220 + events. Data are from a single immunization. Two-tailed t -test was used. f CD4 + T cells were isolated from p19 ( n = 5) or rIgG1 isotype ( n = 5)-treated immunized female IgH [MOG] mice and the indicated cytokines were assessed by flow cytometry. Data are representative of one of two immunizations. Two-tailed t -test was used. g IL23A was assessed from CSF immune cells of MS-affected individuals (see individual information on pwMS in Supplementary Table ) by qPCR and was correlated to the frequency of CD19 + B cells within the CD45 + CSF leukocyte population of each patient. Linear regression analysis was applied to calculate Spearman r and p -value. In all violin plots, horizontal lines represent medians. Each datapoint represents a biological replicate. Exact p -values can be found in the Source Data file. Linked to Supplementary Fig. .

Article Snippet: For p19 blockade, mice were administered anti-p19 (clone G23-8) or isotype (rIgG1, clone HRPM; both BioXcell), 1 mg, on d -1 and d +6 post-immunization .

Techniques: Isolation, Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Myelin-reactive B cells exacerbate CD4 + T cell-driven CNS autoimmunity in an IL-23-dependent manner

doi: 10.1038/s41467-024-49259-0

Figure Lengend Snippet: a Male WT ( n = 5) and IgH [MOG] ( n = 5) mice were immunized with MOG [35-55] and T cells were isolated from CNS at endpoints. Expression of PD-1 and CXCR5 was assessed by flow cytometry. Data are representative of seven experiments. Two-tailed t -test was used. b Male WT or IgH [MOG] mice were immunized and sacrificed at onset ( n = 4 each), peak ( n = 3 each), recovery ( n = 3 each). CNS-infiltrating CD4 + T cells were isolated from these groups, as well as from mice that did not develop disease ( n = 4 each) and the frequency of PD-1 + CXCR5 - cells was assessed. Data are from one of two immunizations. Tukey’s post-hoc test after one-way ANOVA was used. c Expression of MHC class II (I–Ag 7 ), IL-21, ICOS and CXCL13 were assessed from CNS-infiltrating CD4 + T cells taken from immunized male WT ( n = 5) and IgH [MOG] ( n = 5) mice, and expression between PD-1 - and PD-1 + CD4 + subpopulations was compared. Data are representative of two immunizations. Representative plots are presented in Supplementary Fig. . Sidak’s multiple comparisons test after repeated measures two-way ANOVA was used. d Expression of IFNγ and IL-17 was assessed from PD-1 - and PD-1 + CD4 + T cells from immunized male WT or IgH [MOG] mice ( n = 3 each). Data are representative of two immunizations. Sidak’s multiple comparisons test after repeated measures two-way ANOVA was used. e Male WT ( n = 5) and IgH [MOG] ( n = 5) mice were immunized with MOG [35-55] and T cells were isolated from the dura mater and subdural meninges (SDM) at endpoints. Expression of PD-1 and CXCR5 was assessed by flow cytometry. Data are representative of two experiments for dura mater and one immunization for SDM. Two-tailed t -test was used. f T cells were isolated from spleen and CNS at endpoints from anti-p19 ( n = 5) or isotype-treated ( n = 4) immunized male IgH [MOG] mice. Expression of PD-1 and CXCR5 was assessed by flow cytometry. Data are representative of three experiments. Two-tailed t -test was used. In all violin plots, horizontal lines represent medians. Each datapoint represents an individual mouse. Datapoints connected by a line represent paired observations from the same mouse. Exact p -values can be found in the Source Data file. Linked to Supplementary Figs. , .

Article Snippet: For p19 blockade, mice were administered anti-p19 (clone G23-8) or isotype (rIgG1, clone HRPM; both BioXcell), 1 mg, on d -1 and d +6 post-immunization .

Techniques: Isolation, Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Myelin-reactive B cells exacerbate CD4 + T cell-driven CNS autoimmunity in an IL-23-dependent manner

doi: 10.1038/s41467-024-49259-0

Figure Lengend Snippet: a GO term analysis of the transcriptome of CNS-infiltrating B cells and Tph cells from female IgH [MOG] ( n = 3) vs WT ( n = 3) from a single immunization. b Normalized enrichment scores (NES) for “pathways of neurodegeneration” (mmu05022) and “oxidative phosphorylation” (mmu00190) from B cell and Tph cell transcriptomes from the same samples as in ( a) . The weighted Kolmogorov–Smirnov statistical test with the Benjamini-Hochberg method was used to adjust for the false discovery rate. Uptake of ROS indicator CM-H2DCFDA from B cells ( c , e ) or Tph cells ( d , f ) from immunized female IgH [MOG] vs WT mice ( c , d ; n = 5 each) or from immunized and anti-p19 ( n = 5) vs isotype-treated ( n = 6) immunized female IgH [MOG] mice. Data in c , d are representative of two immunizations. Data in e , f are representative of two immunizations. Two-tailed t -test was used. In all violin plots, horizontal lines represent medians. Each datapoint represents an individual mouse. Exact p -values can be found in the Source Data file. Linked to Supplementary Fig. .

Article Snippet: For p19 blockade, mice were administered anti-p19 (clone G23-8) or isotype (rIgG1, clone HRPM; both BioXcell), 1 mg, on d -1 and d +6 post-immunization .

Techniques: Phospho-proteomics, Two Tailed Test