Fig. 5. Upregulation of Foxp3 in late GCs by adoptively transferred naïve T cells.

(A) Experimental setup. (B) Sorted RFP⁺ GFP(Foxp3)^– CD4⁺ T cells were transferred into P25 TCR-tg recipients, which were immunized with NP-OVA in alum and analyzed by multiphoton microscopy at days 10 (left) or 19–20 (right) post-immunization, as in fig. S3D. Images show single optical slices of GCs. GC cross-section (dotted white line) was defined based on in vivo FDC (CD35) labeling. RFP⁺GFP⁺ cells are indicated as yellow circles and magnified in the inset panels. Rendering shows full GC volumes with RFP⁺ cells shown as smaller red spheres and RFP⁺GFP⁺ cells shown as larger green spheres. (C) Quantification of data as in (F). (D) Representative flow cytometry plot from a late GC generated as in (A). (E and F) Longitudinal imaging of transferred RFP⁺ GFP(Foxp3)^– T cells. Details as in (A). iLN window was mounted on day 8 post-immunization. Data is for a single experiment. All scale bars are 50 μm. (G) Quantification of data in (E and F). P-values are for Student’s t test. In (C), each dot represents one GC from 3 mice in 3 independent experiments (early) and 5 mice in 3 independent experiments (late). Bar indicates the median. Data in (E and F) are for a single experiment.