Pd 1 brilliant violet 421

MSCs were analyzed using the following antibodies: CD73-PE, CD105-PE, CD14-PE, CD45-FITC, CD90-APC (BD Biosciences), CD106-Pe-Cy5, CD34-Pe-Cy5 (BD Biosciences), HLA-DR-Qdot-605 (Invitrogen), mouse mAb specific to FAP (eBioscience) and PE-conjugated anti-mouse (BD Biosciences). Isotype-matched, fluorochrome-conjugated, mAb were used as negative controls.
Cultured SCS were stained, data acquired and analyzed as previously described using the following antibodies [11] (link); Surface: CD3-Qdot-605, CD4-Qdot-655 (Invitrogen), CXCR5-Alexa-488, CD25-APC-Cy7, PD-1-Brilliant Violet-421; Intracellular: Bcl-6-PE-CF594, active caspase-3-PE (BD Biosciences), and FoxP3-PE-Cy7 (eBioscience).
FL T-cell subsets were flow cytometrically sorted using the following markers; T-cell: DAPI⁻CD3⁺CD4⁺CD19⁻; TFH: Propidium iodide⁻CD3⁺CD4⁺CXCR5⁺PD-1⁺CD25⁻. Following the sort, an aliquot of TFH were permeabilized and stained with Bcl-6 and FoxP3 to confirm the TFH phenotype. TFH cells were defined as CD3⁺CD4⁺CXCR5⁺PD-1⁺CD25⁻Bcl-6⁺; TFR were defined as CD3⁺CD4⁺CXCR5⁺PD-1⁺CD25⁺Bcl-6⁺FoxP3⁺ and Tregs were defined as CD3⁺CD4⁺CD25⁺FoxP3⁺.

Cells were stained using fluorochrome-coupled monoclonal antibodies (mAbs). The following mAbs were used in this study. CD3-PECy7, CD3-FITC, CD4-AlexaFluor488, CD4-PerCP/Cy5.5, PD-1-BrilliantViolet421, CD8-APC CD8-PerCP/Cy5.5, CD45RO-PE, CCR7-PE, and CD45RA -AlexaFluor700 were all obtained from BD Bioscience. CD8-FITC, PD-1-PE, PD-1-APC, and CD3-APC were obtained from eBioscience. CD244-PE, CD4-PE/Cy5, CD8-PE/Cy5, CD3-FITC, CD19-FITC, CD19-AlexaFluor700 and CD19-BrilliantViolet421 were obtained from BioLegend. Flow cytometry analyses were performed on a LSRII flow cytometer (BD Biosciences) and subsequently analyzed using Flowjo, version 9.0.2 software (ThreeStar).