Fig 2: Blimp1 Deletion in TFR Cells after Immunization Impairs TFR Suppressive Activity(A) Schematic diagram of experimental protocol. Donor CD45.2+ Prdm1fl/fliCre− (WT), Prdm1fl/fliCre+ (Blimp1-deleted; del), and CD45.1 + mice were immunized with NP-OVA in CFA for 7 days; tamoxifen was administered to WT and Del mice on day 6. On day 7, donors were euthanized and sorted CD45.2+ TFR (CD4+CD3+PD-1+CXCR5+GITR+) along with CD45.1+ TFH cells (CD4+CD3+PD-1+CXCR5+GITR−) were transferred into Tcra−/− hosts followed by immunization with NP-OVA in CFA and tamoxifen administration daily from days 7 to 9. Spleens from euthanized hosts were analyzed on day 18.(B) Histogram (upper) and MFI (bottom) of Blimp1 and Bcl6 expression in donor CD45.2+ TFR cells after tamoxifen injection. Ctrl, TFR cells from Prdm1fl/flFoxP3Cre (left), or Bcl6fl/flFoxP3Cre mice (right).(C) Frequency of CD45.2+ TFR (CD45.2+CD4+PD-1+CXCR5+FoxP3+), CD45.1+ TFH (CD45.2−CD4+Bcl6+CXCR5+), and GC B cells (B220+CD19+GL7+Fas+).(D and E) Expression (D) and quantification (E) of IL-17A, IFNγ, and IL-10 by donor CD45.2+ TFR and ex-TFR cells.(F) WT and KO mice were immunized with NP-OVA in CFA. Seven days later, TFR (WT versus KO: CD4+CD3+PD-1+CXCR5+YFP+) and non-TFR (WT versus KO: CD4+CD3+CXCR5+YFP+) were sorted and transferred (105/mouse) into Tcra−/− hosts followed by immunization with NP-OVA in CFA; hosts were further challenged with NP-OVA in IFA at day 13. Spleens or mLNs were analyzed on day 20 (n = 3/group). Splenic (upper) or mLN (center) TFR, TFH, intracellular expression of IL-17A by donor TFR and non-TFR cells. At right, serum anti-NP30 IgG, and anti-NP4 IgG levels.In (A)–(E), the data are representative of two independent experiments (B–E: n = 3–4/group). N.S., no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.See also Figure S3.

Fig 3: Tregs deficient in Nox2 are more suppressive than WT Tregs.(A and B) In vitro suppression assay using Tregs purified from spleen and lymph nodes of Nox2-deficient mice (Nox2–/y) and WT littermate controls. Cells were stimulated with APCs and anti-CD3ε Ab. Representative plots in B show proliferation of Teffs after 3 days of stimulation; numbers at the top are the ratio of Tregs to Teffs. Mean data are shown in A. The “IC50” is the ratio of Tregs/Teffs at which there was 50% suppression of Teff proliferation. (C) Levels of IL-10 in culture supernatants determined by cytometric bead array. The cell combinations that were cocultured are shown at the bottom. (D–G) Baseline levels of mRNA for CTLA-4, CD39, CD73, and GITR in Tregs purified from spleen and lymph nodes. *P < 0.05 for highlighted comparisons, by 2-way ANOVA (A), 1-way ANOVA followed by Tukey’s post-test (C), or unpaired t test (D–G); n = 3–6 per group.

Fig 4: Lycorine treatment inhibits T cell infiltration and function in cardiac allografts. On day 7 posttransplant, allograft‐infiltrated leukocytes were isolated from PBS‐treated mice (Vehicle) or lycorine‐treated mice (5 mg kg−1, b.i.d. Lycorine) were isolated and analyzed by flow cytometry. A) Counts of allograft‐infiltrated cells and CD45+ cells in each group (n = 6). B) Numbers of allograft‐infiltrated CD4+ T cells, CD8+ T cells, B cells and NK cells (n = 6). C) The expression of membrane CD69, CD25, OX40, and GITR on the surface of allograft‐infiltrated CD4+ T cells and CD8+ T cells was measured (n = 5). Values for mean fluorescence intensity (MFI) are shown. D) Bar graphs depict MFI values for PD1, CTLA4, LAG3, and TIM3 on T‐cell subsets (n = 5). E) Allograft‐infiltrated leukocytes were stimulated with a Cell Stimulation Cocktail (plus protein transport inhibitors) and intracellular staining was performed. Percentages of IFN‐γ + and TNF‐α + T‐cell subsets are shown (n = 5). F) Levels of CD107a expression on allograft‐infiltrating CD4+ T cells and CD8+ T cells from each group (n = 5). The graphs show the means ± SEM of three independent experiments. *P < 0.05, and **P < 0.01, Student's t‐test.

Fig 5: Expanded TFR Cells Express an Abnormal Phenotype and Altered GC Distribution after Immunization of Prdm1fl/flFoxP3Cre Mice(A and B) Flow cytometry (left) and numbers (right) of splenic (A) or mLN (B) Treg (FoxP3+CD4+) (upper), TFR (CD4+CD3+PD-1+CXCR5+FoxP3+), and non-TFR (CD4+CD3+CXCR5−FoxP3+) (bottom) from WT and KO mice (7–9 weeks old) 10 days post-immunization with NP-KLH in CFA.(C) Histogram of Ki67, annexin V, and CD69 expression in mLN TFR cells in (B) (upper) and quantification of mean fluorescence intensity (MFI) in mLN TFR and CXCR5− Treg cells (bottom).(D) Expression of TFR-associated gene products by splenic TFR from (A) and quantification of MFI (right).(E) IL-17A, IFNγ, and IL-4 expression (left) and frequency (right) by splenic TFR (CD4+CD3+PD-1+BTLA+FoxP3+) cells from WT or KO mice (6–8 weeks old) at day 7 post-immunization with NP-OVA in CFA.(F) Frequency of CD25lo (left), CD25int (center), and CD25hi (right) splenic TFR from WT and KO mice (6–8 weeks old) 10 days post-immunization with NP-OVA in CFA. (Bottom) FoxP3 and CTLA-4 expression in the CD25-expressing TFR subsets.(G) MFI of FoxP3, CTLA-4, GITR, and RORγt expression and frequencies of IL-17A+ cells in YFP+ Blimp1-deficient (KO) compared to YFP− Blimp1-sufficient (WT) TFR cells from the spleens of female Prdm1fl/flFoxP3Cre/+ mice (6–8 weeks old) 10 days post-immunization with NP-OVA in CFA.(H and I) CXCR5 (H) and CCR7 (I) expression in the splenic TFR cells in (A) and mLN TFR cells in (B).(J) Histology of 7-week-old WT and KO mice 14 days post-immunization with NP-OVA in CFA. TFR cells (CD3+FoxP3+, white arrows) and TFH cells (CD3+FoxP3−) in the GC area (Ki67+) within the B cell follicle (IgD+). Insets indicate the B cell follicles. (Bottom) Numbers of TFR and TFH cells in the GC.In (A)–(I), the data are representative of four independent experiments (A–F, n = 4/group; G, n = 5/group; H and I, n = 4–5/group). In (J), the data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.See also Figure S2.