Fig 2: B cell analysis in the epididymis of Foxp3-DTR mice 2 weeks after Treg depletion.A Immunolabeling of CD19 (red) and CD20 (green) in the IS, caput, and cauda of DT-injected WT and Foxp3-DTR mice. n = 3. t-SNE and FlowSOM analysis of proximal (B) and distal (C) from WT and Foxp3-DTR epididymides. The most relevant populations that relatively increased in the Foxp3-DTR samples are highlighted: i. Cyan:CD20; magenta:CD8; purple:CD4; green:CD20/CD8. ii. Light green:CD8; brown:CD4; dark green:CD20/CD62L/CD23; magenta:CD27/FAS/CD21/CD38. n = 3. D Flow cytometry gating strategy and absolute count (#) of total B cells (CD3−B220+, CD3−CD19+, and CD3−CD20+ (E–J) per gram of proximal/distal epididymis. n = 12 (WT), n = 10 (Foxp3-DTR). K–P Tissue-resident B cells per gram of proximal and distal epididymis. n = 10 (WT and Foxp3-DTR: B220, CD20, CD19 prox.), n = 9 (WT and Foxp3-DTR: CD19 dist.). Q Flow cytometry gating strategy and relative abundance of total (R, S) and tissue-resident (T, U) Plasma B cells (CD3⁻CD19⁺IgD⁻CD138⁺) in the proximal/distal epididymides. n = 12 (WT total), n = 10 (Foxp3-DTR total and resident). V Flow cytometry gating strategy and relative abundance of total (W, X) and tissue-resident (Y, Z) Germinal center (GC) B cells (CD3⁻B220⁺IgD⁻GL7⁺) in the proximal/distal epididymides. n = 13 (WT total), n = 10 (Foxp3-DTR total and resident), n = 11 (WT resident). Aa Flow cytometry gating strategy and relative abundance of total (Bb, Cc) and tissue-resident (Dd, Ee) Regulatory B cells (CD3−CD19+CD5+CD62L−CD1dhi) in the proximal/distal epididymides. n = 13 (WT prox. total), n = 10 (Foxp3-DTR total and resident), n = 11 (WT dist. total, WT prox. resident), n = 9 (WT dist. resident). Ff–Hh Flow cytometry gating strategy and absolute count (#) of total follicular dendritic cells (DC, CD4−CD19−CD21+) in the proximal/distal epididymides. n = 10 (WT prox.), n = 7 (Foxp3-DTR), n = 8 (WT dist.). Flow cytometry gating strategy and absolute count (#) of total CD8+T cells (CD4−CD8+) (Ii–Kk) and activated CD8+T cells (CD4−CD8+CD44+) (Ll–Nn) per gram of proximal/distal epididymis. n = 13 (WT), n = 10 (Foxp3-DTR). DAPI (blue). Bars: 20 μm. Data are means ± SEM, and two-sided Student’s t-test (E, K, U, W, Gg), or Mann–Whitney test (F–J, L–P, R–T, X–Z, Bb–Ee, Hh–Nn) were performed. Each dot represents a pool of 2 organs. n = number of samples from different mice.