Cd45ro apc h7

The following fluorochrome conjugated antihuman monoclonal antibodies (MoAbs) were used for flow cytometry studies: ICOSBV421, ICOSAlexa488, CD40LBV605, CD69BV650, HLA-DRFITC, CD38APCCy7, and TNF-αAPCCy7 from BioLegend (San Diego, CA); CD3BUV395, CD4PerCPCy5.5, CD8Alexa-Fluor700, CCR7PECF594, IL-2BV711, CXCR5Alexa647, IFNγPE-Cy7, PD1BV650, CD45ROAPCH7, CD21PECy5, CD27PerCPCy5.5, IgDFITC, CD10PECy7, and CD20Alexa700 from BD Bioscience (San Jose, CA); IL-21PE, CD27PECy5, and IL-17Alexa488 from e-Biosciences (San Diego, CA); and CD45ROPE-TexasRed from Beckman Coulter (Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit and CellTrace Violet Cell Proliferation Kit were from ThermoFisher (Boston, MA). Recombinant human IL-21 (Cat #8879-IL-010) from R&D systems, purified antihuman IL-2 (Cat #3440-ON-500) and antihuman-IL-21 (Cat #MT216G/21.3m) from Mabtech, and antihumanTNF-α (Cat #502922) from BioLegend were used. Samples were acquired on a BD LSRFortessa (BD Biosciences, CA) flow cytometer and analyzed by FlowJo V10 (TreeStar, Inc).

For immunofluorescence staining of whole blood and PBMC, antibodies included CD45-ECD (Beckman Coulter, Indianapolis IN), CD3-e780, CD4-e450, CD8-AF700, CD15-FITC (eBioscience, San Diego, CA), CD45-FITC, CD4-PerCP-Cy5-5, CD14-PECy7, HLA-DR-APC, HLA-DR-BV421, CD8-APC-H7, CCR7-BV605, CD25-BV605, CD45RA-PECy7, CD45RO-APC-H7, CCR4-PECy7, CD38-APC, CD127-BV650 (BD Bioscience, San Jose, CA), CD14-AF700, and CD3-AF700 (Biolegend, San Diego, CA). For phosphoSTAT staining, antibodies included CD3-BV785 (Biolegend), CD4-BV605, CD8-BV510, CD14-PECy7, CD19-BV421, CD16-BV650, pSTAT3-647, pSTAT5-PE (BD Biosciences), and pSTAT1-488 (Cell Signaling Technologies, Danvers, MA). Cell types were identified according to the criteria of the Human Immunology Project [19 (link)], and a full list of antibodies used to identify individual cell types including those not reported here can be found in Additional file 2: Table S1. Hematological toxicity was graded according to NCI CTCAE v4.0 guidelines and is reported in Additional files 3 and 4: Tables S2 and S3.

After 5 days of culture, PBMC co-cultured with mature DC (mDC), VitD3-tolDC (100% VitD3-tolDC) and 50% VitD3-tolDC (50% VitD3-tolDC + 50% mDC) in the presence and absence of IFN-beta were harvested and stained using CD3-V450, CD4-PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Bioscience), CD183 AF488, CD196 BV605 and CD45 AF700 (Biolegend, San Diego, CA, USA) for T cell analysis; and CD4 PerCP-Cy5.5, CD25 PE, CCR4 PE-Cy7, CD127 AF647, CD45RO APC-H7, CD3 V450, HLA-DR V500 (BD Biosciences) and CD45 AF700 (Biolegend) for Treg analysis. Monoclonal antibodies were incubated for 20 min at room temperature and protected from the light. Samples were washed and a total of 50,000 CD3+ events were acquired on an LSR Fortessa flow cytometer (BD Biosciences). Both panels were analyzed using FACSDiva software (BD Biosciences). The gating strategy used to analyze the desired T cell subpopulations was previously reported [28 (link)].