Fig 2: Basal Ig isotypes sera concentrations of WT (n = 17) and C57-CD40L-/- (n = 19) mice. (a) Basal IgM, IgG, and IgA concentrations. Ig concentrations were determined by extrapolation from a standard curve. The total mean IgM concentration was similar among mice strains. Total IgG and IgA serum concentrations of C57-CD40L-/- were significantly lower than concentrations of WT mice. (b) Basal IgG subclasses sera concentrations. IgG3 levels were essentially identical between the two groups and all other subclasses concentrations were significantly higher in WT than in CD40L deficient mice. Mean comparisons were done by Mann-Whitney U-test. WT = wild type.

Fig 3: Btk-deficiency alters IgA plasma cell localization in the small intestine of K/BxN mice. Small intestine was collected from 8-11wk-old Btk+/y (black) and Btk-/y (red) K/BxN mice (n = 3-4 mice per group) as described in Materials and Methods. Immunostaining for IgA (red), EpCAM (green), and DAPI (blue) was performed to identify IgA+ cells in the small intestine villi and lamina propria. Representative images of non-overlapping areas of sections from each mouse are shown for (A) Btk+/y /K/BxN and (B) Btk-/y /K/BxN mouse S1 duodenal sections (Btk+/y S1 mean/min/max = 13/7/24 images per mouse; Btk-/y S1 mean/min/max = 9/6/12 images per mouse). Representative images for (C) Btk+/y /K/BxN and (D) Btk-/y /K/BxN mouse S4 ileal sections. (Btk+/y S4 mean/min/max = 17/10/23 images per mouse; Btk-/y S4 mean/min/max = 9/8/12 images per mouse). Scale bar represents 50 µm. (E, F) Analysis of IgA+ plasma cell frequency in (E) duodenal and ileal villi and lamina propria (LP) and (F) duodenal and ileal overall tissues as measured by IgA score on immunofluorescent sections. Each dot represents a single image and data are compiled from n = 3-4 mice per genotype. ****p < 0.0001 ANOVA with Bonferroni correction. (G, H) Average IgA scores for each mouse are plotted for (G) villi and lamina propria (LP) or (H) total duodenal (S1) or ileal (S4) sections. **p < 0.01, NS, not significant, two-tailed t test with Welch’s correction.

Fig 4: IgA+ plasma cells persist in the lamina propria of Btk-deficient K/BxN mice. Lamina propria cells were harvested from the entire small intestine of 8-9 wk-old K/BxN (black) and Btk-/y /K/BxN (white) littermates as in Methods and cells were counted; n = 6 mice per group, n = 5 independent experiments. Flow cytometry was used to identify the indicated subsets. (A) Representative plots depict total B cells (B220+ CD19+) among live singlet lymphocyte gated cells in K/BxN (left) and Btk-/y /K/BxN (right) mice (top) and IgA+ B cells among total B cells (bottom). (B) Total B cell frequencies (left) and numbers (right) and (C) IgA+ B cell frequencies (left) and numbers (right) are shown. (D, E) Plasma cells were identified among B220- CD19- intracellular Ig?+ CD138+ live singlet cells as surface BCR-positive (surface Ig?+), surface BCR-negative (surface Ig?-), surface BCR-positive IgA (surface Ig?+, surface IgA+), and surface BCR-negative IgA (surface Ig?-, intracellular IgA+). (D) Representative plots depict the indicated subsets in K/BxN (left) and Btk-/y /K/BxN (right). (E) Cell subset numbers are shown for surface BCR+ plasma cells (top left), surface BCR+ IgA+ plasma cells (top right), surface BCR- plasma cells (bottom left), and surface BCR- IgA+ plasma cells (bottom right). (F–H) CD138- cells with a plasma cell phenotype were further examined as follows. (F) Representative gating scheme shows B220+ CD19+ and B220- CD19- populations among live singlet lymphocytes (left). B220- CD19- cells (red) were further gated on intracellular Ig?+ cells (green, middle, left), followed by identification of CD138- (blue) and CD138+ (pink) cells (middle, right). Intracellular IgA expression was examined among CD138- cells (right, lavender), among which surface Ig? expression was used to delineate surface BCR-positive (orange) and surface BCR-negative (light blue) plasma cells. (G, H) Numbers of live singlet lymphocytes that were B220- CD19- intracellular Ig?+ CD138- cells are shown for (G) total cells or (H) IgA-switched cells. No significant differences were observed between K/BxN and Btk-/y /K/BxN groups, Mann-Whitney U test. NS, Not significant.

Fig 5: Btk absence leads to reduced numbers of GC and IgA class-switched B cells in the Peyer’s patches in K/BxN mice. Peyer’s patches were harvested from 8-9 wk-old K/BxN (black) and Btk -/y/K/BxN (white) littermates and cells isolated from all Peyer’s patches dissected from each mouse were counted; n = 6 mice per group, n = 5 independent experiments, as in Methods. Flow cytometry was used to identify the indicated subsets. (A-D) Representative plots for K/BxN (left) and Btk -/y/K/BxN (right) mice depict (A) B cells (B220+ CD19+) among live singlet lymphocytes, (B) IgA+ B cells among B220+ CD19+ live singlet lymphocytes, (C) GC B cells (GL7high FAShigh) among B220+ CD19+ live singlet lymphocytes, and (D) GC B cells among IgA+ B220+ CD19+ live lymphocytes. (E-H) Frequencies (left), numbers across all Peyer’s patches harvested per mouse (middle), and numbers divided by the number of Peyer’s patches harvested per mouse (right) are plotted for individual mice to show (E) total B cells, (F) IgA+ B cells, (G) GC B cells, and (H) IgA+ GC B cells. *p < 0.05, **p < 0.01, Mann-Whitney U test.