Ctla 4 apc

Flow cytometry measurements were performed using a CyAn ADP Analyzer (Beckman Coulter) and data were analyzed using version 9.8.2 of FlowJo software. Viable cells were detected by LIVE/DEAD (L/D) Fixable Aqua stain (Invitrogen) and the following antibodies were used for surface and intracellular staining: CD3e Pacific Blue, CD4 FITC, CD8 APC-Cy7, CD45.1 Pe-Cy7, CD25 PE, PD-1 PE, CTLA4 APC, IFNγ APC, FOXP3 PerCP-Cy5.5 (eBioscience). For staining of blood and splenocytes, cells were exposed for 5 min at RT to 0.155 NH₄Cl to lyse erythrocytes. The following staining steps were performed on ice. Cells were washed with PBS, stained for 15 min with Aqua L/D stain, resuspended in PBS + 2% FBS for surface staining for 15 min and finally fixed for 15 min in PBS + 2% PFA. IFNγ intracellular staining was performed in PBS + 2% FBS supplemented with 0.5% Saponin. Foxp3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit (ebioscience) was used for FOXP3 staining.

Fresh total cells from the respective tissues were directly stained with the following monoclonal antibodies (mAbs) at predetermined optimal dilutions for 20 min at 4°C: CD3-PE, CD8-Alexa700, CD4-HorizonV500, CD127-FITC, and CD25-PeCy7 (eBioscience). Intracellular detection of FOXP3 (FOXP3-E450, eBioscience), CTLA-4 (CTLA-4-APC, eBioscience), Ki-67 (Ki-67-FITC, BD Pharmingen), and Bcl-2 (Bcl-2-PE, BD Pharmingen) was performed on fixed and permeabilized cells using appropriate buffer (eBioscience) (incubation 30 min at 4°C). Cells were acquired on an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star, Inc.). Dead cells were excluded by forward/side scatter gating.

The following antibodies were used for flow cytometry analysis: CD11c APC, CD80 FITC, CD83 FITC, CD86 PE, HLA-DR FITC, CD1a FITC, CD14 FITC, CD4PE, CD4 PE-Cy7, CD45RA PE, CD45RO FITC, CD25 PE-Cy7, CTLA-4 APC, CD39 PerCP-eFluor710, FOXP3 Alexa Fluor 700, IL-10 PE, IFN-γ PE-Cy7, and IL-17 APC (all eBioscience). For surface staining, cells were incubated in PBS 10% FBS containing the respective antibodies for 30 min at 4°C, washed, and stored in IC fixation buffer until analysis (eBioscience). Intracellular FoxP3 was detected using FoxP3-staining kit (eBioscience) according to the manufacturer’s instructions. For intracellular cytokines detection, cells were treated with 50 ng/ml PMA, 1 μg/ml ionomycin, and 1 μl/ml brefeldin A for 5 h. After harvesting, cells were surface stained for CD4. Intracellular cytokine staining was performed in permeabilization buffer (eBioscience) before cells were washed and resuspended in FACS buffer for flow cytometry analysis. Cell viability was assessed by 7-AAD and annexin-V PE staining (eBioscience). Data were collected on FACSCalibur and FACSAria cytometers (Beckton Dickinson, San Diego, CA, USA) and analyzed with Weasel v3.0.2 software.

For research expansion samples on days 0, 14 and 21 as well as post-transplant immune monitoring, antibodies against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 (all Beckman-Coulter) and FOXP3-PC5 (eBioscience, San Diego, CA) were used. Chemokine and receptor characterizations were performed using CXCR3/GARP-Pacific Blue, CXCR4/CD62L-PerCP-Cy5.5, CCR7/CD45RO-APC-Cy7, CD45RA-Alexa700, and CTLA-4-APC (all from eBioscience, San Diego, CA).
For the GMP expanded Treg products 1–9, three separate panels were used consistently throughout Treg expansion and all antibodies were purchased from Miltenyi. Panel one: CD8-APC, CD20-FITC, CD14/15/56-PE, CD45-Pacific Blue and 7AAD-PerCP-Cy5.5. Panel two: CD25-APC, CD4-FITC, CD127-PE, CD45-Pacific Blue and 7AAD-PerCP-Cy5.5. Panel three: CD4-FITC, CD25-PE, Foxp3-APC and CD45-Pacific Blue. Research grade flow cytometry was performed on a Beckman-Coulter FC500 whereas clinical flow cytometry was done on a BD-LSR, as previously described^{37 (link),54 ,55 (link)}.
Patient post-transplant flow cytometric analyses were done on a Beckman-Coulter FC500 using antibodies (all Beckman-Coulter) against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 and FOXP3-PC5 (eBioscience); IL10-FITC, IgD/M-PE, CD19-ECD, CD27-PC5, CD24-PC7; Helios-FITC, CD3-ECD, CD28-PC5, CD8-PC7 and FOXP3-PE (eBioscience), among others.

Cell surface antigens were stained first. The dendritic cell antibody panel quantified the following: CD11c-APC-eFluor780, CD80-PE-Cy7, CD86-FITC, and CD40-eFluor450 (all mAbs are from eBioscience now Thermo Fisher, Waltham, MA). The CD4+ T_Reg cell panel included the following: CD3ε-PE, CD4-PE-Cy7, and CD25-PerCP-Cy5.5s (all mAbs are from eBioscience). For T_Reg intracellular staining, cells were fixed and permeabilized with the one step eBioscience Fix-Perm Foxp3 Buffer Staining Kit (eBioscience). The panel included Foxp3-eFluor450 and CTLA4-APC (mAbs are from eBioscience). Intracellular staining was also used to detect TGFβ-PE, IL12 p40-PE, IL10-APC, and IFNγ-APC production (mAbs are from eBioscience) after ex vivo nonspecific antigen stimulation with a Leukocyte Activation Cocktail with GolgiPlug (BD Biosciences) for flow cytometry. Mouse INFγ Single-Color ELISPOT to determine antigen-specific response of T cells was from Cellular Technology Limited (CTL), Cleveland, OH. For attempting to assess SIRT1 expression by flow cytometry, we used antibodies from Santa Cruz and Abcam.