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Mononuclear cells isolated from LN were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-CD95, anti-CCR7, anti-PD-1 and anti-CD200. Sorting of CD4+ Tfh (PD1+CD200+) was performed using a FACS AriaΙΙ (BD Biosciences) in samples collected before and during the FTY720 treatment. Post-sorting FACS analysis determined that sorted CD4+ T cell subsets were on average >96% pure.

Splenocytes (1 × 10⁶) from GFP transgenic mice were isolated and injected into the tail vein of Dach1 fl/fl mice and male Tie2-Cre⁺ Dach1 fl/fl mice. Single-cell suspensions from spleen, inguinal lymph nodes (ILN), and MLNs were prepared from the recipient mice 1 h after transfer. The percentage of GFP⁺ cells in each tissue was determined by flow-cytometric analysis using a FACS Aria ΙΙ (BD Biosciences) and the FlowJo software (BD Biosciences). The transferred GFP⁺ cells in ILNs or MLNs were standardized by the number of GFP⁺ splenocytes in the same individual. For whole-mount analyses of LNs, mice were intravenously injected with GFP⁺ splenic cells (1.5 × 10⁷ cells/mouse). One hour after cell administration, mice were injected with 4 μg of eFluor660-labeled CD34 mAb and sacrificed 15 min later, and ILNs and pLNs were harvested. GFP⁺ lymphocytes and CD34⁺ blood vessels were detected by confocal microscopy. For pLNs, Z-plane images of the eFluor660 dye were acquired every 5 μm (approximately 150 μm thickness) and were analyzed by Metamorph software (Molecular Devices Company).

Mononuclear cells isolated from LN were stained with anti-CD3-APC-Cy7 (clone SP34-2, 5 μL, cat #557757), anti-CD28-PE-CF594 (clone CD28.2, 5 μL, cat #562296), and anti-CD95-PECy5 (clone DX2, 10 μL, cat #559773) from BD Biosciences; anti-CD4-BV650 (clone OKT4, 2 μL, cat #317436), anti-PD-1-BV421 (clone EH12.2H7, 5 μL, cat # 3299220), and anti-CD200-PE (clone OX-104, 5 μL, cat #329206) from Biolegend; anti-CD8-FITC (clone 3B5, 5 μL, cat #MHCD0801-4) from Thermo Fisher Scientific; and Aqua Live/Dead Fixable Aqua from Invitrogen (AmCyan, 2 μL of 1:20 PBS dilution, cat. L34957). Sorting of CD4+ Tfh (PD1+CD200hi) was performed using a FACS AriaΙΙ (BD Biosciences) in samples collected before, during FTY720 treatment, at ART interruption, and 29 days after ART interruption. Post-sorting FACS analysis determined that sorted CD4+ T cell subsets were on average >96% pure.