Fig 2: Peli1 is required for Klf2 expression to inhibit Tfh differentiation. A Immunoblot analysis of phosphorylated (p) and total PI3K and AKT, Peli1 and actin (loading control) in whole-cell lysates of WT and Peli1-deficient CD4+ T cells that were primed with anti-CD3 plus anti-CD28 antibodies for 24 h and were then left unstimulated or stimulated with anti-CD3 plus anti-ICOS antibodies at the indicated time points. B, C qPCR analysis of Klf2 and S1pr1 mRNA in WT and Peli1-deficient CD4+ T cells that were left untreated (NT) or treated with TCR stimulators (anti-CD3 plus anti-CD28 antibodies) (B) or were pretreated with DMSO (DM) or an AKT inhibitor (AKTi) and then stimulated with anti-CD3 plus anti-CD28 antibodies (C). D, E Flow cytometric analysis of the percentages of CXCR5+PD-1+ Tfh cells, CXCR5+GL-7+ GC Tfh cells, Fas+GL-7+ GC B cells and CD138+CD19- plasma cells in spleens from Rag1-deficient mice (recipient, R) that were adoptively transferred with WT B cells plus WT or Peli1-KO T cells transduced with empty vector (EV) or retroviral vectors encoding Klf2 (donor, D) and were then immunized with NP-KLH. The data are presented as representative FACS plots (D) and summary graphs (E). F Correlation analysis of PELI1 mRNA expression with KLF2 mRNA expression in PBMCs from human blood samples. 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

Fig 3: Peli1 deficiency does not affect Tfh cell migration. A, B Flow cytometric analysis of the percentages of CD45.2+CD45.1-CXCR5+PD-1+ Tfh cells (A) and ICOS expression in the spleens of bm12 and SJL mice immunized with CD45.2+ WT or Peli1-deficient CD4+ T cells for 2, 3, and 6 days. The data are presented as representative FACS plots (A) and summary graphs (B). C Immunohistochemical staining of draining lymph nodes from bm12 and SJL mice immunized with WT or Peli1-deficient CD4+ T cells. Cryosections of draining lymph nodes from immunized recipient mice were stained with CD45.2 (blue) and IgD (red) and analyzed by fluorescence microscopy. D Density of CD45.2+ cells in the whole area and the B-cell zone. Each dot represents an individual mouse. 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

Fig 4: Peli1 deficiency enhanced c-Rel-mediated ICOS expression. A Immunoblot of p52, p65, p50, c-Rel and actin (loading control) expression in splenic CD4+ T cells isolated from mice immunized with (+) or without (-) NP-KLH. ChIP-qPCR analysis of c-Rel binding activity at the Icos gene promoter (B) and qPCR analysis of Icos mRNA expression (C) in WT and Peli1-KO CD4+ T cells that were left untreated (NT) or treated with TCR stimulators (anti-CD3 plus anti-CD28 antibodies). D, E Flow cytometric analysis of ICOS expression on and Tfh differentiation of WT and Peli1-KO CD4+ T cells treated with PBS or pentoxifylline (PTXF) and then left untreated (NT) or treated with TCR stimulators (anti-CD3 plus anti-CD28 antibodies). F, G Flow cytometric analysis of the percentages of CXCR5+PD-1+ Tfh cells, CXCR5+GL-7+ GC Tfh cells, Fas+GL-7+ GC B 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 NP-KLH. The immunized mice were treated with PBS or PTXF daily on days 0–7 after immunization. The data are presented as representative FACS plots (F) and summary graphs (G). 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

Fig 5: Increased T cell-mediated immune responses by rAd5-Gn priming and Gn protein boosting. Six-week-old C57BL/6 mice (n = 5/group) are immunized intramuscularly with 1 × 108 infectious units of rAd5-Gn or alum and RNA-adjuvanted Gn protein at 0 and 2 weeks. Mice are sacrificed one week after boosting. (a, d, g), and (j) Representative flow cytometry plots showing (a) follicular helper T cells, (d) effector T cells, central memory t cells, (g) interferon ? cytokine-producing T cells, (j) activated T cells. Frequencies of (b) CXCR5, (c) PD-1 expression in CD4+ T cell, and ICOS expression in CD4 + PD-1 + populations from immunized mice spleen. Frequencies of (e) effector memory T cells and (f) central memory T cells in CD4+ T cells were analyzed using flow cytometry. IFN-?-producing (h) CD4+ and (i) CD8+ T cells in the spleen were analyzed using flow cytometry. (k) CD69 and (l) CD25 expression in CD4 + T cells in the spleen was analyzed using flow cytometry. Data are presented as mean values ± SD. P values are calculated using one-way ANOVA with Bonferroni multiple comparison test.