Anti cd4

Total CD4⁺ T cells from infected and total CD4⁺ and CD8⁺ T cells from naïve Foxp3-GFP.KI mice were pre-purified from splenocytes and LNs using anti-CD4 or Pan T cell beads (Miltenyi), respectively. CD4⁺ and CD8⁺ effector T cells were flow sorted as CD4⁺GFP⁻ and CD8⁺ T cells, Treg cells from naïve mice as CD4⁺GFP⁺ and from days 12–14 LCMV-infected mice as CD4⁺GFP⁺CD85k⁺ or CD4⁺GFP⁺CXCR3⁻CD85k⁺ Treg cells. 1 × 10⁵ Treg cells and 5 × 10⁵ effector T cells each were adoptively transferred i.v. into Rag1^−/− recipient mice.
Expansion and activation of effector T cells was assessed 10 days post transfer in the presence or absence of the indicated Treg subsets.

Monoclonal antibodies were purchased from Thermo Fisher Scientific eBioscience or BD Bioscience. Clones used were: anti-CD4 (RM4-5, dilution 1:500), anti-CD8α (53–6.7 dilution 1:500), anti-TCRβ (H57-597, dilution 1:250), anti-HSA (M1/69, dilution 1:500), anti-CD69 (H1.2F3, dilution 1:250), anti-CD44 (IM7, dilution 1:500), anti-CD62L (MEL-14, dilution 1:500), anti-CD19 (ID3, dilution 1:300), anti-CD127 (SB/199, dilution 1:250), anti-Caspase-3 (C92-605, 10 μl of Ab per test). Cell proliferation dye E450 was obtained from Thermo Fisher Scientific eBioscience. After staining with antibodies and DAPI (Thermo Fisher Scientific), cells were analyzed with an LSRII flow cytometer (BD Biosciences) or sorted with an Aria II (BD Biosciences). Post sort sample purity was >98%. In most cases, anti-CD4, anti-CD8, anti-PE, and anti-B220 magnetic beads (Miltenyi) were used for enrichment and depletion on MACS columns (Miltenyi) before sorting. Flow cytometry data were analyzed using Flowjo software (Tree Star).

Human BLS cell lines and in vitro generated B cell mutants have been described and are established human cell lines [1 (link),30 (link)]. T cells were enriched using anti-CD4 and/or anti-CD8 magnetic beads (Miltenyi Biotec). For all flow cytometric analyses, gating on living cells and exclusion of doublets was performed. Enriched TEC suspensions [2 (link)] were washed in PBS, 2% FCS, 5mM EDTA and stained for flow cytometry using death exclusion markers (either DAPI or 7AAD), UEA1 (Sigma) and the following mAb-conjugated mix: α-CD45 (30F11, BioLegend) and α-BP1 (6C3; BioLegend), α-MHCII (M5/114.15.2; eBioscience) and α-EpCAM (G8.8, Developmental Studies Hybridoma Bank, Iowa), α-H2-Db (B22/24g) and α-H2-Kb (B8.24.3). Splenocytes were preincubated with anti-CD16/32 (2.4G2) to block FcRs and stained using Abs against CD8a (Ly-2), CD3e (145-2C11), CD4 (L3T4), CD11b (M1/70), CD11c (N418), CD19 (1D3), H2-Db (28-14-8), H2-Kb (AF6–88.5.5.3), MHCII (M5/114.15.2), NK1.1 (PK136), B220 (RA3-6B2), Qa-2 (69H1-9-9) (all from eBioscience). Streptavidin conjugated to different fluorophores was from eBioscience. Stainings were performed with appropriate combinations of fluorophores. Data was acquired with a FACSCanto flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star).

For isolation of NK cells and T cells from buffy coats, peripheral blood mononuclear cell (PBMC) preparation was performed by standard density-gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Resting NK cells were enriched from PBMCs by depleting the non-NK cell population using the NK cell isolation kit for human cells (Miltenyi Biotec). Where indicated, NK cell subsets were prepared using CX3CR1 expression as a marker. Cells were stained with an APC conjugated antibody targeting CX3CR1-, followed by labeling with APC MicroBeads and magnetic separation (Miltenyi Biotec). T cells were isolated from PBMC using microbeads conjugated with either anti-CD4 or anti-CD8 antibodies (Miltenyi Biotec).