Fig 2: Cxcr5fl/flCd4cre/+ Mice Have an Intact GC Response after Influenza InfectionAnalysis of the GC response in Cxcr5fl/flCd4cre/+ mice 14 days after influenza A virus (HKx31) infection.(A) Representative histogram of CXCR5 expression in Foxp3+CD4+ Treg cells and B220+ B cells.(B) Quantification of the MFI (geometric mean) of CXCR5 in Foxp3+CD4+ Treg cells.(C) Representative histogram of CXCR5 expression in Foxp3−CD4+ T cells and B220+ B cells.(D) Quantification of the MFI (geometric mean) of CXCR5 in Foxp3−CD4+ T cells.(E) Flow cytometry contour plots of GC B cells, gated as Bcl6+Ki67+ cells of B220+ cells.(F and G) Quantification of the (F) frequency and (G) absolute number of Bcl6+Ki67+ B cells.(H and I) Quantification of the (H) percentage and (I) number of Bcl6+PD-1+Foxp3−CD4+ Tfh cells.(J and K) Quantification of the (J) percentage and (K) number of Bcl6+PD-1+Foxp3+CD4+ Tfr cells.(L) Cryosections were stained for Foxp3 (magenta), Ki67 (blue), CD3 (green), and IgD (orange). Scale bar, 100 μm. Representative confocal image of the GC, with Tfr cells and Tfh cells indicated by the arrows.(M) Quantification of the median number of Tfr cells, defined as CD3+Foxp3+ cells, per 5,000 μm2.(N) Quantification of the median number of Tfh cells, defined as CD3+Foxp3− cells, per 5,000 μm2.Each symbol represents one mouse, the horizontal bars represent mean values, and the error bars show the SD. The p values were determined using a Mann-Whitney U test. For the quantification of confocal images, 4–10 GCs were imaged per mouse. Data represent two independent experiments.

Fig 3: Fewer Tfr Cells Are Present in the GC of Cxcr5fl/flFoxp3cre-ERT2 Mice(A–C) Histogram (A), quantification (B). and dot plots (C) of CXCR5 expression on Foxp3+CD4+ Treg cells in mesenteric lymph nodes three weeks after initiating the tamoxifen diet, before immunization, in Cxcr5fl/flFoxp3cre-ERT2 and Cxcr5+/+Foxp3cre-ERT2 mice. A fluorescence minus one (FMO) control serves as a negative control, and B220+ B cells serve as a CXCR5-positive population.(D–M) Mice were immunized with NP-KLH/alum s.c., and the GC response was analyzed in draining lymph nodes 14 days after immunization.(D) Representative flow cytometry contour plots of PD-1+Bcl6+ cells within Foxp3+CD4+ cells (Tfr cells).(E and F) Quantification of the (E) percentage and (F) absolute number of Bcl6+PD-1+ Tfr cells.(G) Cryosections from iLNs were stained for Foxp3 (magenta), Ki67 (blue), CD3 (green), and IgD (orange). Scale bar, 100 µm. Representative confocal image of the GC, with Tfr cells and Tfh cells indicated by the arrows.(H) Quantification of the median number of Tfr cells, defined as CD3+Foxp3+, per 5,000 µm2 of GC area.(I) Quantification of confocal images of the median number of CD3+Foxp3+ Tfr cells per GC per mouse.(J) Quantification of the median number of Tfr cells per 10 Tfh cells, defined as Foxp3-CD3+.(K) Quantification of the median number of Treg cells, defined as CD3+Foxp3+, per 5,000 µm2 of IgD+ B cell follicle area.(L and M) CXCR4 MFI (L) and CCR7 MFI (M) (subtracting FMO control) on Bcl6+PD-1+Foxp3+CD4+ Tfr cells from mice of the indicated genotypes.Each symbol represents one mouse, the horizontal bars represent mean values, and the error bars show the SD. The p values were determined using a Mann-Whitney U test. For the quantification of confocal images, 4–10 GCs or B cell follicles were imaged per mouse. Data represent two or more independent experiments.

Fig 4: RBD-HR/trimer vaccine induced strong T cell immune response in vivo.a The levels of IFN-? and IL-4 in the supernatants of spleen lymphocytes from immunized mice were detected by ELISA after stimulation with RBD (10 µg/ml) for three days. b, c The number of RBD-specific IL-4 or INF-?-producing memory T cells in spleen were analyzed by flow cytometry after stimulation with RBD. Memory T cells were gated on CD44high B220-MHC-II- CD4+ or CD8+. d The percentage of effector memory (CD44+CD62L-) of CD4 or CD8 T cells in spleen. e The percentage of central memory (CD44+CD62L+) of CD4 or CD8 T cells in spleen. f The frequency of T follicular helper cells (CD4+CXCR5+PD-1+) and germinal center B cells (CD19+GL7+CD95+) in spleen were analyzed by flow cytometry. n = 5 mice each group in a–f. Spleen samples in a–f were collected on day 49 after the first vaccination, and isolation of lymphocytes was performed as described in Methods. Data are presented as mean values ± SEM. P values were conducted by one-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test in a–f. Source data are provided as a Source Data file.

Fig 5: Tfr Cells Are Present at Reduced Numbers in Cxcr5fl/flFoxp3cre-yfp MiceMice were immunized with NP-KLH/alum i.p., and the GC response was analyzed 14 days after immunization.(A) Histogram of CXCR5 expression in Foxp3+CD4+ Treg cells, naive T cells as a CXCR5-negative control population, and wild-type B cells as a CXCR5-positive population.(B) CXCR5 mean fluorescence intensity (MFI; geometric mean) in Foxp3+CD4+ Treg cells from mice of the indicated genotypes.(C) Analysis of Tfr and Tfh cells 14 days after influenza A virus (HKx31) infection in Cxcr5fl/flFoxp3cre mice and Cxcr5+/+Foxp3cre controls. Representative confocal images of splenic cryosections stained for Foxp3 (magenta), Ki67 (blue), CD3 (green), and IgD (orange); Foxp3+ cells are indicated by arrows. Scale bar, 40 µm.(D) Average GC size in square micrometers measured as the IgD-Ki67+ area. Each dot represents the average size of 2–6 GCs per mouse.(E) Quantification of the average number of Tfr cells per mouse, defined as CD3+Foxp3+ cells within the GC, per 5,000 µm2. Each dot represents the average number of Tfr cells per 5,000 µm2 of GC area per mouse, from 2–6 GCs.(F) Representative flow cytometry contour plots of CXCR5+PD-1+ Tfh cells from Foxp3-CD4+ cells.(G and H) Quantification of the (G) frequency and (H) absolute number of CXCR5+PD-1+ Tfh cells.(I) Representative flow cytometry contour plots of Bcl6+PD-1+ Tfh cells of Foxp3-CD4+ cells.(J and K) Quantification of the (J) percentage and (K) absolute number of Bcl6+PD-1+Foxp3-CD4+ Tfh cells.Each symbol represents one mouse, the horizontal bars represent mean values, and the error bars show the SD. The p values were determined using a Mann-Whitney U test. Data represent two independent experiments.