Percp cy5.5 anti human cd3

Foxp3 is a specific marker of Treg. PBMC were separated from collected blood samples and Treg were detected by the expression of CD25 and Foxp3. We used the following fluorochrome-conjugated antibodies to stain the cell surface antigens: PerCp/Cy5.5 anti-human CD3, Brilliant Violet 421 anti-human CD4, APC anti-human CD25 (BioLegend, San Diego, CA, USA), and Ghost Rad780 Viability dye (Tonbo Bioscience, San Diego, CA, USA). We permeabilized cells using a Human Foxp3 buffer set kit (BD Bioscience, San Diego, CA USA), and stained Foxp3 using PE anti-human Foxp3 (BioLegend, San Diego, CA, USA). FCM was performed using BD FACS CantoII(Ver1.1, Diva6.1, BD Bioscience, San Diego, CA USA) and BD FACSDiva9, FlowJo (BD Bioscience, San Diego, CA USA) was used for data analysis. The gating strategy for the isolation of Treg involved the following steps: (1) isolation of live cells, (2) gating to isolate CD3- and CD4-positive cells, and (3) isolation of CD25- and Foxp3-positive cells (Supplementary Fig. 1).

Fresh human PBMCs were stained with monoclonal antibodies, such as PercP-Cy5.5-anti-human CD3, APC/Cy7-anti-human CD4, PE-anti-human CD25, and Brilliant Violet 510-anti-human CD127 (all purchased from BioLegend, San Diego, CA, USA), in 0.5% BSA + 2 mM EDTA in PBS. Splenocytes were obtained from the spleens of mice by mechanical disruption and through a 200-gauge steel mesh. Splenocytes were lysed with 1_X red blood cells (RBCs) lysis buffer to remove RBCs. First, magnetic microbeads (Miltenyi Biotec) bead-based enrichment of CD4⁺T cells was performed. Then, cells were stained with monoclonal antibodies, such as PE-anti-mice CD4, APC/Cyanine7-anti-mice CD25, and Alexa-Fluor647-anti-mice CCR6 (all purchased from BioLegend), in 0.5% BSA + 2 mM EDTA in PBS. Human Treg cells and murine Treg cells were sorted using a SH800S cell sorter (SONY, Japan) and analyzed with the SH800 software (SONY, Japan). Human Treg cells were gated as CD3⁺CD4⁺CD25⁺CD127^- within the lymphocyte gate, which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). Mice Treg cells were gated as CD4⁺CD25⁺ within the lymphocyte gate which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). The purity of the sorted cell population was above 95% for human and 91% for mice, which was verified using post-sorting flow cytometry.

CD4⁺ T cell activation profile was assessed as previously described [26 (link)]. Briefly, 10⁶ PBMCs in RPMI 1640 medium containing 5% FCS, 1% penicillin-streptomycin were stimulated with 10 μg/ml of S. Typhi OmpC/F or 50 μg/ml of tetanus toxoid for 24h at 37°C. After in vitro stimulation, surface staining was performed and the frequency of CD4⁺ T cells and intracellular expression of CD40L, IFN- γ and TNF was assessed by flow cytometry using the following antibodies: PerCP/Cy5.5 anti-human CD3, PE/Cy7 anti-human CD4 and FITC anti-human CD154, PE anti-human IFN- γ and APC anti-human TNF (all from Biolegend); the fixable viability stain 780 (e-Biosciences) was used to discriminate dead cells. Samples were analyzed using a FACS Canto flow cytometer (Becton Dickinson), and data were analyzed using FlowJo software version 10 (Tree Star, USA).