Figure 2.

Fewer Tfr Cells Are Present in the GC of Cxcr5^fl/flFoxp3^cre-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 Cxcr5^fl/flFoxp3^cre-ERT2 and Cxcr5^+/+Foxp3^cre-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.