Fig 2: Generation of CLK21 human BCR knock-in mouse model Table shows the total number of pups, the number or frequency of human HC-, human LC-, and human BCR KI pups after One-step CRISPR/Cas9 microinjection of CLK21.CLK21 KI mice F0 and F1 generations. Squares represent male mice and circles represent female mice. Upper halves of squares or circles represent Ig ?, and the lower halves represent Ig H, as shown in the schematic. F0 generation mice genotyping results showing 7, 9 and 14 are HCLK21/WT?WT/WT, 1 and 4 are HWT/WT?CLK21/WT, and 2, 3, 6, and 12 are HCLK21/WT?CLK21/WT. Mouse 3 was crossed with WT to obtain 11 F1 progeny: Five F1 mice are HWT/WT ?CLK21/WT, four are HCLK21/WT?CLK21/WT and two are WT.Single-cell sequencing for naïve B cells from heterozygous CLK21 double KI mice. Left column shows the gating strategy for sorting naïve B cells (upper) and eOD-GT8-specific naïve B cells (lower). Right pie charts show the frequency of paired HC and LC sequences among total naïve B cells (upper) and eOD-GT8-specific naïve B cells (lower).Germline-targeting eOD-GT8 binding activity of B cells from WT, HCLK21/WT?WT/WT, HWT/WT ?CLK21/WT), and HCLK21/WT, ?CLK21/WT KI mice. 8-week-old mice were detected by FACS. X and Y axes represent that BCR were stained with eOD-GT8 tetramer conjugated with Alexa Fluor™ 488 and Alexa Fluor™ 647, respectively. Representative dots were gated from Scatter/Singlet/Live (SSL), B220+ IgM+ IgD+.Quantification of eOD-GT8 binding in CLK21 KI mice. X-axis represents F0 (n = 1) and F1 (n = 3) KI animals, and the Y-axis represents the percentage of eOD-GT8 targeting binders in mature (IgM+ IgD+) B cells. Bars indicate mean ± SD from mice in each group.

Fig 3: High-fat diet feeding promotes TLT formation in kidneys. (A) Experimental protocol. Wild-type (WT) 8-month-old female mice fed a control or high-fat diet (HFD) from 28 days prior to ischemic reperfusion injury (IRI). Diet was fed for 30 days after IRI. (B) Periodic acid–Schiff (PAS) staining of kidneys 30 days after 35 min IRI under control diet and under HFD. Arrows indicate TLTs. (C) PAS staining and immunofluorescence of TLTs under control diet and HFD. B220, CD3, Ki67, and DAPI were stained on the same section. CD21 and p75NTR were stained inside TLTs. Yellow arrows indicate TLT. (D) Cumulative TLT size per cortex (control diet n = 10, HFD n = 8), total TLT number per section (n = 10), and advanced stage (stage II or III) TLT number per section (n = 10). (E) Percentage of advanced stage TLTs detected in each sample (n = 10). (F) Relative mRNA expressions of Cxcl13, Ccl19, Ifng, and Tnfa. Expressions are normalized with Gapdh expression and values are presented relative to control kidneys (control n = 4, HFD n = 5). (G) Quantification of Picrosirius red-positive fibrotic area comparing control-fed and HFD-fed mice after IRI (n = 12). Values are mean ± S.E.M. Data were analyzed using student’s t-test, * p < 0.05, ** p < 0.01. Scale bars: (B) 500 µm, (C) 100 µm. TLT stages were defined as follows. Stage I: TLT lacking either CD21+ follicular dendritic cells (FDC) or germinal center, stage II: TLT containing FDC but lacking germinal center, stage III: TLT containing both FDC and germinal center, based on the criteria described in [12].

Fig 4: Ectopic livers formed after i.p. transplantation in FRGN mice with restored abdominal FALCs. (A) Macroscopic appearance of GFP+ bone marrow cells. Bone marrow isolated from GFP+ C57BL/6 mice was injected i.v. and i.p. into FRGN mice. GFP+ FALCs were formed in the omentum, perisplenic area, periportal area, mesentery, and gonadal area 3 weeks after transplantation. (B) Immunofluorescent staining of frozen sections shows that CD45+ cell clusters (red) are missing in the omentum of FRGN mice but are now present after bone marrow cell transplantation. Nuclei were counterstained with Hoechst (blue). (C) CD45, CD3, or B220 immunofluorescent staining (red) of FALCs formed in the omentum of FRGN mice after GFP+ bone marrow cell transplantation. Nuclei were counterstained with Hoechst (blue). (D) (Left image) Macroscopic appearance of dissected omenta from FRGN mice with or without restored FALCs 12 weeks after i.p. hepatocyte transplantation. (Middle panel) Fluorescence images of whole-mount omenta showing the engraftment of GFP+ hepatocytes in the two conditions. (Right panel) Fluorescence images of omental frozen sections. GFP+ hepatocytes (green) formed hepatic nodules in FRGN mice with restored abdominal FALCs. FALCs are characterized by a dense cluster of lymphoid cells (blue). In FRGN mice, there was only limited growth of hepatocytes. Nuclei were counterstained with Hoechst (blue). (E) Macroscopic appearance of dissected omentum, perisplenic area, periportal area, and mesentery of FRGN mouse with restored abdominal FALCs 23 weeks after hepatocyte transplantation. Ectopic hepatic nodules are clearly visible. (F) Frozen sections with CD105 immunofluorescence staining (red) and GFP+ hepatocytes (green) in the omentum, perisplenic area, and periportal area of FRGN mouse with restored abdominal FALCs after bone marrow transplantation. See Figure 5 for control without bone marrow transplantation. Scale bars, 250 µm. BM, bone marrow

Fig 5: Increased TFH-B-Ab response in Prdm1fl/flFoxP3YFP-Cre EAE mice. a Flow cytometry of each indicated subset in mice at day 20 post-EAE induction, as in Fig. 1a. b–d Serum IgG and IgE at day 8 (b) or day 20 (c) or anti-MOG IgG and anti-MOG IgE at day 8 (d). e The relationship of serum IgE titers and EAE scores for mice in c. Each dot represents one mouse. f Immunofluorescence analysis of CD3, B220 (left) or B220, IgE and DAPI (right) in the spinal cords. Magnification, × 100. WT, FoxP3YFP-Cre, KO, Prdm1fl/flFoxP3YFP-Cre. Data represent one of at least three experiments (b–d n = 5/group). *P < 0.05 and ****P < 0.0001 (unpaired two-tailed Student’s t test). In e, Spearman’s r, ***P = 0.0010. Bars, mean ± SEM