Figure 1. Characterization and distribution of T_FR in SIV-uninfected macaques.

(A) Double positive CD3⁺ (red) and Foxp3⁺ cells (green) are present in B-follicles (CD20 in grey) in lymph nodes from a naïve macaque (blue =DAPI; scale bar: 20μm). (B) Representative flow cytometry plots showing the gating strategy for T_FR, CCR7⁺-T_REG and T_FH cells in lymph nodes. All the subsets were gated on singlet/live/CD3⁺ CD4⁺ CD95⁺. T_FR and CCR7⁺-T_REG were identified as Foxp3⁺ CD25⁺ and CXCR5⁺ CCR7⁻ or CXCR5⁻ CCR7⁺, respectively; T_FH cells as Foxp3⁻ CXCR5⁺ PD-1^high. (C) Geometric mean (MFI) of Foxp3 and (D) CD25 expression. (E) Cell surface expression of PD-1, Bcl-6 ICOS and α4β7 in T_FR (red), T_REG (blue) and T_FH cells (green). Isotype controls are in grey. Frequency of (F) CXCR5⁺ & CCR7⁻ cells and (G) CXCR5⁻ & CCR7⁺ cells within Foxp3⁺ CD25⁺ CD4⁺ T (upper panel) or within memory CD4⁺ T cells (corresponding lower panels) in blood, peripheral lymph nodes and in the GALT (colon, jejunum and rectal mucosa) of naïve animals. The median is shown. (H) T_FH frequency on Foxp3 negative (upper panel) and memory CD4⁺ T cells (lower panel) in different tissues.