Foxp3 buffer set

PBMCs were retrieved, thawed and washed. The viability was measured by the trypan blue dye exclusion method and cells with viability > 95% were used in the assay. All the antibodies were purchased from BD except anti-human FOXP3 fluorochrome-conjugated antibody (Biolegend). After washing, the cells were extracellularly stained with the following antibodies: anti-CD3-(APC H7), anti-CD4 (BUV 395), anti-CD8 (PerCP Cy5.5), anti-CD28 (APCR700), anti-CD57 (FITC) and anti-PD-1 (BUV737) on ice for 30 min. The cells were washed, fixed and permeabilized using FOXP3 buffer set (BD) according to manufacturer's instructions and intracellularly stained for FOXP3 (PE) and CTLA-4 (APC) on ice for 40 min. We gated for T cells by CD3, CD4, and CD8 lineage markers. Gates for inhibitory and senescence markers were defined using fluorescence minus one controls (Figure S1). Cells were acquired on a BD LSR Fortessa II-X20 cytometer. Data were compensated and analyzed using Flowjo V10 software (Tree Star, San Carlos, CA).

Multiparametric flow cytometry was used for ex vivo analysis of B cells. For subset gating, combinations of the following mAbs were used: CD19-BV786, CD27-BUV395, CD20–Alexa Fluor 700, CD10-BV605, CD3-BV711, CD21-BV421, and CD45-BUV05. Full details of mAbs used, including those used for phenotypic characterization, are given in Supplemental Table 1.
Cells were stained with Fixable LIVE/DEAD Dye (Life Technologies, Thermo Fisher Scientific) before incubation with saturating concentrations of surface mAbs diluted in 50% Brilliant Violet Buffer (BD Biosciences) and 50% PBS for 30 minutes at 4°C. In all instances, cells were stained in the presence of FcR-blocking reagent (Miltenyi Biotec). Cells were fixed and permeabilized for further functional assessment with either Cytofix/Cytoperm (BD Biosciences) or FoxP3 Buffer Set (BD Biosciences) according to the manufacturer’s instructions for intracellular or intranculear staining respectively. Saturating concentrations of mAbs for 30 minutes at 4°C were diluted in 0.1% saponin (Sigma-Aldrich) for the detection of intracellular proteins or in 1× PBS for the detection of intranuclear proteins. All samples were acquired on a Fortessa-X20 (BD Biosciences) and analyzed using FlowJo (Tree Star).

For evaluating the frequency of Treg cells, splenocytes were first surface stained with PE anti-mouse CD4 (Biolegend) and FITC anti-mouse CD25 (Biolegend). After permeabilization with Foxp3 buffer set (BD Biosciences), cells were incubated with PerCP/Cy5.5 anti-mouse Foxp3 for intracellular staining. Finally, Data were acquired and analyzed with FACSCalibur flow and flowJo software (Version 10).

Multiparametric flow cytometry was used for phenotypic and functional analysis of PBMCs and IHLs. Cells were stained with a fixable Live/Dead dye (Invitrogen) before incubation with saturating concentrations of surface mAbs diluted in 50% Brilliant violet buffer (BD) and 50% PBS for 30 min at 4°C. See also Table S1 for full details regarding antibodies used. Cells were fixed and permeabilized for further functional assessment with either Cytofix/Cytoperm (BD) or FoxP3 Buffer Set (BD) according to the manufacturer’s instructions. Saturated concentrations of mAbs for 30 min at 4°C were diluted in 0.1% saponin (Sigma-Aldrich) for the detection of intracellular proteins or in 1× PBS for the detection of intranuclear proteins. All samples were acquired on either an LSRII or X20 flow cytometer (BD) and analyzed using FlowJo (Tree Star).

IFN-γ and TNF-α secreting, and FoxP3+, CD4+ T-cells in infant PBMC were identified by ICS (intracellular cytokine staining). PBMC were washed with media, and then left unstimulated or stimulated for 4 h with PMA/Ionomycin (BD Biosciences). The stimulations were done in the presence of 1 μl Brefeldin A (BD Biosciences). Cells were first stained with surface Ab to CD45RO (clone UCHL1), fixed and permeabilized with the FoxP3 buffer set (BD Biosciences), and then stained with Abs to CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone SK1), IFN-γ (clone B27), TNF-α (clone 6401.1111), and FoxP3 (clone 259D/C7) (all Abs from BD Biosciences). Cells were analyzed using a FACSAria flow cytometer (BD Biosciences). LIVE/DEAD^® Fixable Dead Cell Stain Kit (LDA) (Invitrogen) was used to exclude nonviable cells from analysis. Relevant cells were identified as LDA−/CD3+/CD4+/CD8−/CD45RO+ or CD45RO−/IFN-γ+ or TNF-α+ or FoxP3+ cells (Supplementary Fig. 1). Data was analyzed using FlowJo^® software (Treestar).