Fig 2: Roles of XBP-1s and the S729A mutation of IRE-1α in B cell activation, differentiation, and IgM production in vitro. B cells were isolated from WT, XBP-1KO, S729A, and XBP-1KO/S729A mice and stimulated with anti-IgM F(ab’)2 (10 mu;g/ml) and anti-CD40 (10 mu;g/ml) for 48 hrs. The expression of MHCII (A) and CD86 (B) were detected by flow cytometry. The percentages of germinal center B cells (FAS+GL-7+) were presented (C). (D, E) B cells were stimulated with PMA and Ionomycin for 4 hrs on day 4. Intracellular levels of IgM (D) and IgG1 (E) were detected by flow cytometry analysis. Cell culture media were also collected, and anti-dsDNA autoantibodies were detected using ELISA (F, G). Statistics were performed using two-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001 when compared to WT. #p < 0.05, ##p <0.005, ###p < 0.0005, and ####p < 0.0001 when compared to XBP-1KO group.

Fig 3: Effect of XBP-1s and IRE-1α on B cell activation and cGVHD pathogenicity. BALB/c mice were lethally irradiated and transplanted with 5 x 106 TCD-BM cells from WT, XBP-1KO, IRE-1αKO, and DKO mice on a B6 background with (n = 20) or without (n = 8) 0.35 - 0.5 x 106 splenocytes. GVHD scoring (A) and mouse weight (B) were monitored during the experiment. Subsets of recipient mice were euthanized on day 28, and spleens were dissected and processed into single-cell suspension. The percentages of B220+ B cells (C) and the expression levels of MHCII (D), CD86 (E), ICOSL (F), and CD40 (G) were detected by flow cytometry analysis. Intracellular expression of IgM (H, I) was also determined using flow cytometry. Data show the representative result from three independently repeated experiments. 28 recipient mice were used for each experiment. Statistics were performed using two-way ANOVA with Tukey’s multiple comparison test for (A, B) One-way ANOVA with Tukey’s multiple comparison test was used for others. *p < 0.05, **p < 0.005, and ****p < 0.0001 when compared to WT. #p < 0.05, ##p < 0.005, ###p < 0.0005, and ####p < 0.0001 when compared to XBP-1KO group.

Fig 4: Cell surface in CK‐666‐treated cells. Related to Fig 4 Phalloidin‐Alexa Fluor 555 staining of fixed and permeabilized Ramos B cells expressing EGFP‐CaaX and IgM‐BCR with or without 30 min 100 μM CK‐666 treatment. Scale bar = 5 μm.Node degree analysis of the skeletonized ridge network in untreated and 100 μM CK‐666‐treated cells for up to 30 min. Mean ± SD (n = 4 for untreated and n = 5 for CK‐666).Sum projections of nodes and skeletonized ridge network (excluding the microvilli) in an untreated and 100 μM CK‐666‐treated cell for 15 consecutive time frames (duration: 37.5 s). Scale bar = 5 μm. Source data are available online for this figure.

Fig 5: IgM‐BCR clusters are enriched on ridges and microvilli ATopographical features of the B cell surface. The cell body is separated from the microvilli (MV) protrusions using mathematical morphology. On the cell body, the ridge (R) network (yellow) is computed and its nodes (N; blue) are annotated. The remaining surface area is termed shallow invaginations (SI, dark green). Individual microvilli are split into their shaft (MS, purple) and tip (MT, red) and all features are combined in one image. Scale bar = 5 μm.BSegmented IgM‐BCR clusters (black) are superimposed onto the extracted surface features. Cluster‐surface feature overlay reveals the accumulation of IgM‐BCR clusters on ridges and their nodes. The zoomed‐in microvillus displays a cluster density at its root (corresponding to a node) and tip. Scale bar inset = 1 μm.CCluster density at surface features. IgM‐BCR cluster density within different topographical features quantified as cluster count per surface feature volume.DNormalized cluster density. Comparison of mean cluster densities computed in (C) as normalized to the value of shallow invaginations. The black squares indicate the SI‐normalized cluster density of randomly assigned cluster centroids on the cell surface. Bars and error bars represent mean ± SD.EIgM‐BCR intensity enrichment within the segmented clusters at different surface features, calculated for the cells quantified in (C). Intensity enrichment was measured by dividing the average voxel intensity in the IgM‐BCR clusters (μci) by the average voxel intensity of the non‐clustered IgM‐BCR residing within the surface feature mask (μdi). Data points and error bars represent mean ± SD.F–HContinuous analysis of IgM‐BCR cluster density using geodesic distance maps initiated from (F) microvilli tips towards microvilli roots, (G) centerline of ridges towards cell body surface, and (H) nodes towards cell body surface. Data points and the dotted lines represent mean ± SD. Scale bars = 5 μm. Data information: n = 7 cells (30‐time frames/cell) from four independent experiments were analyzed. Two‐tailed, paired Student t‐test was used in (C) and (E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison in (C) between N and MS, P = 0.0003; between N and MT, P = 0.0145. Source data are available online for this figure.