Fig 2: Peli1 deficiency aggravates the induction of lupus-like disease. a WT and Peli1-deficient (KO) mice were intraperitoneally injected with 7.5 million of CD4+ T cell from C57BL/6 mice (control) or from BM12 mice. Representative immunofluorescent images showing IgG deposits in kidney by staining with Alexa Fluor 488-labeled anti-mouse IgG. Scale bar, 100 µm. b Flow cytometric analysis of the percentages of CD19-CD138+ plasma cells and Fas+GL-7+ germinal center (GC) B cells in WT and KO immunized mice as described in (a). Data are presented as a representative plot (left panel) and summary graph (right panel). c Distinct anti-nuclear antibody (ANA) staining patterns of the Hep-2 cell line with serum from WT and KO mice 4 weeks after immunization as described in (a). Scale bar, 50 µm. d Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG in serum from WT and KO immunized mice as described in (a) at the indicate time point. e Immunoblot showing NIK and p52 protein levels in splenic B cells from control and BM12 CD4+ T cells immunized WT and KO mice as described in (a). Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t-tests were performed. *P < 0.05 and **P < 0.01

Fig 3: Peli1 deficiency promotes Tfh-mediated lupus-like pathology. A, B Flow cytometric analysis of the percentages of GFP-CXCR5+PD-1+ Tfh cells (A), GFP+Fas+GL-7+ germinal center (GC) B cells and GFP+CD138+CD19- plasma cells in spleens of bm12/GFP mice adoptively transferred with WT or Peli1-deficient CD4+ T cells. The data are presented as representative FACS plots (left panels) and summary graphs (right panels). C, D Immunofluorescence analysis of serum anti-nuclear antibody (ANA) in bm12/GFP mice that were left unimmunized or transferred with WT or Peli1-deficient CD4+ T cells was performed with Hep-2 cells. The results are presented as immunofluorescence images (C) and a quantitative bar graph (D). E ELISA of anti-dsDNA, anti-ssDNA, and anti-histone IgG in the serum of bm12/GFP mice that were left unimmunized or adoptively transferred with WT or Peli1-deficient CD4+ T cells

Fig 4: Peli1 deficiency specifically in B cells promotes autoimmunity in vivo. a Scheme showing how the µMT chimeric mice were constructed for immunization. b Enzyme-linked immunosorbent assay (ELISA) of NP-specific IgG1, IgG2a, IgG2b, and IgG3 in the serum of WT/µMT and Peli1-deficient (KO)/µMT chimeric mice that immunized intraperitoneally with vehicle (sham) or NP-KLH (KLH). c WT/µMT and KO/µMT chimeric mice were intraperitoneally injected with 7.5 million of CD4 + T cell from C57BL/6 mice (control) or from BM12 mice. Representative immunofluorescent images showing IgG deposits in kidney by staining with Alexa Fluor 488-labeled anti-mouse IgG. Scale bar, 100 µm. d, e Flow cytometric analysis of the percentages of Fas+GL-7+ germinal center (GC) B cells and CD19-CD138+ plasma cells in immunized WT/µMT and KO/µMT chimeric mice as described in (c). Data are presented as the representative FACS plots (d) and summary graphs (e). f Distinct anti-nuclear antibody (ANA) staining patterns of the Hep-2 cell line with serum from WT/µMT and KO/µMT chimeric mice 4 weeks after immunization as described in (c). Ctr represents control in the right panel bar graph. Scale bar, 50 µm. g Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG in serum from immunized WT/µMT and KO/µMT chimeric mice as described in (c) at the indicate time point. Data are shown as the mean ± SEM based on three independent experiments. Two-tailed Student’s t-tests were performed. *P < 0.05 and **P < 0.01

Fig 5: Peli1 intrinsically inhibits Tfh differentiation. A, B Flow cytometric analysis of the percentages of CXCR5+GL-7+ germinal center (GC) Tfh cells and CD138+CD19- plasma cells in spleens from Rag1-deficient mice (recipient, R) that were adoptively transferred with WT or Peli1-KO T cells and WT B cells (donor, D) and were then immunized with or without NP-KLH. The data are presented as representative FACS plots (A) and summary graphs (B). C Scheme showing the generation of WT/SJL and Peli1-KO/SJL chimeric mice for NP-KLH immunization. D, E Flow cytometric analysis of the percentages of CXCR5+PD-1+ Tfh cells, CXCR5+GL-7+ GC Tfh cells and Fas+GL-7+ GC B cells in spleens from immunized WT/SJL and Peli1-KO/SJL chimeric mice as described in (C). The data are presented as representative FACS plots (D) and summary graphs (E). F, G Flow cytometric analysis of the percentages of CXCR5+PD-1+ Tfh cells, CXCR5+GL-7+ GC Tfh cells and Fas+GL-7+ GC B cells in spleen from NP-OVA immunized WT-OT II/SJL or Peli1-KO-OT II/SJL chimeric mice that generated similar to the scheme in (C). The data are presented as representative FACS plots (F) and summary graphs (G). H ELISA of NP-specific IgM, IgG2a, and IgG3 in the serum of NP-OVA-immunized WT-OT II/SJL and Peli1-KO-OT II/SJL chimeric mice as described in (F). Data with error bars are presented as the mean ± SEM values. Each panel shows data for a representative experiment from at least three independent biological replicates. *p < 0.05, **p < 0.01 as determined by unpaired Student’s t test