Fig 2: HPβCD attenuates the chronic inflammatory response to stroke in aged mice. A, Experimental design, 18-month-old mice received subcutaneous injections of 4 g/kg HPβCD or vehicle 3 times per week, beginning 1 week after DH stroke. Brains were dissected at 7 weeks after stroke and processed for immunohistochemistry or RNA-Seq. B, Representative 40× images of infarcts in vehicle- or HPβCD-treated aged mice stained for B lymphocytes (B220), T lymphocytes (CD3ε), and antibody-producing plasma cells (IgA), followed by representative 60× images of infarcts stained for lipid droplets (ORO) and 20× images of infarcts stained for sulfatides (Alcian blue). Quantification of images is shown to the right of each photomicrograph (n = 4-10). *p < 0.05; **p < 0.01; ***p < 0.001; unpaired t tests. Scale bars: ORO, 50 μm; B220, CD3ε, IgA, Alcian blue, 125 μm. Data are mean ± SEM.

Fig 3: SAP-Mediated T Cell Help Is Not Required for Generation of IgA+ GC B Cells in PPs(A) Representative flow cytometry plots showing IgG1 and IgA isotype frequency gated from GC B cells derived from WT or SAP-deficient mice.(B) Quantification of GC isotype frequencies, as shown in (A). Data are pooled from four independent experiments; each dot represents a single mouse; line represents the mean.(C) Levels of IgG1 in the sera of WT and SAPKO mice as estimated by standard ELISA.(D) Quantification of IgA antibodies in the intestinal contents of WT and SAPKO mice as measured by ELISA.(E) qPCR for IL21R, IL4R, STAT3, and STAT6 transcripts in sorted GC B cells (GL7+ FAS+) derived from PPs of WT and SAPKO mice.Data are pooled from two independent experiments with two mice in each experiment. Each dot represents a single mouse, line represents the mean. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, two-tailed Student’s t test. ns, not significant.

Fig 4: IgA + PCs appear in the infarct independently of T-lymphocytes.A.) Representative images of anti-IgA immunostaining of brains sections from WT, MHCII−/−, and CD4−/− mice at 7 weeks post-DH stroke. Images are taken at 10x and insets are 40x. Scale bar, 500 μm 10x and 150 μm 40x. B.) Quantification of the number of IgA + cells per mm2 in each of the three strains of mice indicates that the number of PCs present within the infarct of each strain is similar. Data represent mean ± SEM. Not significant by one-way ANOVA. n = 5–7 mice per group.

Fig 5: PCs take multiple weeks to appear within the stroke infarct.A.) A schematic representation of the infarcted brain, with gray indicating the area of damage and, thus, the area of quantification B&C.) Representative images of immunofluorescent staining with anti-IgA (green), anti-CD138 (purple), and DAPI (blue) showing colocalization of CD138, a plasma cell marker, with IgA at 4 (B) and 7 weeks post-stroke (C). Scale bar, 25 μm. n = 3. D.) Representative images of anti-IgA immunostaining of brains sections from naïve mice and mice 24 hours/, 1 week, 2 weeks, 4 weeks, and 7 weeks following DH stroke. Images are taken at 10x and insets are 40x. Scale bar, 500 μm 10x and 25 μm 40x. E.) Quantification of the number of IgA + cells per mm2 of stroke infarct at each time point post-stroke. IgA + plasma cells enter the stroke infarct between 2 and 4 weeks post-stroke and persist for at least 7 weeks post-stroke. Data represent mean ± S.E.M. *p < 0.05, ****p < 0.0001 by one-way ANOVA with comparisons against all other groups, with post-hoc Tukey’s multiple comparisons test. n = 5–7.