Flow cytometry analysis was performed to quantify lymphocyte subsets on BD LSR II flow cytometer with BD FACS DIVA software (BD Biosciences, Heidelberg, Germany). The following antibodies were used for phenotypic analysis: CD45-FITC, CD19/20-PE, CD14-Cy-7, CD25-FITC, CD127-PE, CD45RA-APC, CD3-APC Cy-7, CD8-FITC, CD4-PE, CD4-APC Cy7, CD8-APC Cy7, CD62L-FITC, CCR7-PE, CD27-FITC, CD62L-PE, (Beckton Dickinson, Franklin Lakes, NJ, USA), CD3-ECD, CD56-APC, CD45RO-ECD (Beckman Coulter, Miami, FL, USA).30 (link)
Cd45ra apc
Manufactured by BD
Sourced in United States
CD45RA-APC is a fluorescently-labeled monoclonal antibody that binds to the CD45RA molecule, a isoform of the CD45 protein expressed on the surface of certain immune cells. It is commonly used in flow cytometry applications to identify and characterize different T cell subsets.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe, by BD (5 mentions)
Cd8 fitc, by BD (4 mentions)
Ccr7 pe, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Cd8 apc cy7, by BD (3 mentions)
Cd4 apc cy7, by BD (3 mentions)
Cd62l pe, by BD (3 mentions)
Cd3 apc cy7, by BD (3 mentions)
Cd27 fitc, by BD (3 mentions)
Cd3 fitc, by BD (3 mentions)
Cd25 fitc, by BD (2 mentions)
Cd127 pe, by BD (2 mentions)
Cd45 fitc, by BD (2 mentions)
Cd3 ecd, by Beckman Coulter (2 mentions)
Cd56 apc, by Beckman Coulter (2 mentions)
Cd19 20 pe, by BD (2 mentions)
Cd14 cy 7, by BD (2 mentions)
Cd62l fitc, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Cd45ro ecd, by Beckman Coulter (2 mentions)
16 protocols using cd45ra apc
1
Flow Cytometry Profiling of Lymphocyte Subsets
2
Sorting and Characterizing Naive and Memory T Cells
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Cryopreserved PBMC samples were labelled using CD4-PE, CD8-APC-Cy7, CD45RA-APC (all Becton Dickinson) and CD62L-ECD (Beckman Coulter) on ice, following overnight rest to restore CD62L expression (Figure A in S1 File ). CD4+ and CD8+ gated lymphocytes were sorted (Influx; Becton Dickinson) on the basis of CD45RA and CD62L expression [20 (link)]. Sort purities were regularly >98%. We confirmed naïve and memory populations by stimulating naïve or memory CD8+ T cells with cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza derived peptides (CEF peptides; Mabtech) and performing an IFN-γ ELISpot assay as described above. CEF peptides responses were only detected in stimulated memory phenotype T cells, but not naïve populations (Figure B in S1 File ).
3
Flow Cytometry Profiling of Lymphocyte Subsets
4
Profiling T-cell activation and pSTAT5 signaling
5
Immunophenotyping of Cryopreserved PBMCs
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Peripheral blood mononuclear cells (PBMC) were obtained from whole blood using Histopaque® and Ficoll (Sigma-Aldrich, Saint Louis, MO, USA) different density gradients. These cells were cryopreserved, and then thawed at the time of each assay. Then, was used a concentration of 2 × 105 viable cells/mL, and submitted to immunophenotyping assay with surface antibodies for 20 min at 2–8 °C. After, the cells were washed with phosphate buffer plus fetal bovine serum (FBS) (PBS pH 7.4 at 2% FBS), and centrifuged at 400× g for 5 min. After centrifugation, cells were fixed with 1% paraformaldehyde solution and subsequently acquired in a flow cytometer (LSR FortessaTM, BD Biosciences, Franklin Lakes, NJ, USA). The analysis was performed using Flow Jo software v10.6 (BD Biosciences).
The anti-human antibodies used in the immunophenotyping assay were: panel I (activation)-CD3-FITC, CD4-APCH7, CD8-BV605, CD38-PECy7, OX40-BV711 and panel II (memory)–CD3-APC-Cy7, CD4-BV421, CD8-BV605, CD45RA-APC, CCR7-BV510 (BD Biosciences).
The anti-human antibodies used in the immunophenotyping assay were: panel I (activation)-CD3-FITC, CD4-APCH7, CD8-BV605, CD38-PECy7, OX40-BV711 and panel II (memory)–CD3-APC-Cy7, CD4-BV421, CD8-BV605, CD45RA-APC, CCR7-BV510 (BD Biosciences).
6
Blocking Cytokines and Checkpoint Inhibitors in T-cell Assays
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Cells were thawed, washed and reconstituted in AIM V serum-free medium (Life Technologies, Oslo, Norway) containing 0.1% human serum albumin. After an overnight rest, cells were pulse-labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Life Technologies) at a concentration of 2 μM for 5 minutes. The following blocking antibodies were added to parallel culture wells at a final concentration of 10 μg/mL: anti-IL-10 (R&D Systems, MN, USA; clone 23738), anti-TGF-β (R&D; clone 1D11), anti-PD-L1 (eBioscience, CA, USA; clone MIH1), and anti-HVEM (R&D; clone 94801).
After a 30 minute incubation, cultures were stimulated with either Gag or Env 15-mer overlapping peptide panels (NIH AIDS Research and Reference Reagent Program, MD, USA) at a final concentration of 2 μg/mL/peptide. Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich, MO, USA) at a final concentration of 0.5 μg/mL was used as a positive control.
Cells were cultured at 37°C in 5% CO2 for 5 days, harvested, and stained with the following fluorochrome-conjugated antibodies: CD3 V450, CD8 APC-H7, HLA-DR BV605, CD45RA APC (all BD) and CD25 PE (Biolegend). 7-aminoactinomycin D (7-AAD, BD) was added for dead cell exclusion. Flow cytometry data were acquired on a BD FACS Canto II with BD Diva 6.1 software, and analyzed in FlowJo X (FlowJo LLC, OR, USA).
After a 30 minute incubation, cultures were stimulated with either Gag or Env 15-mer overlapping peptide panels (NIH AIDS Research and Reference Reagent Program, MD, USA) at a final concentration of 2 μg/mL/peptide. Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich, MO, USA) at a final concentration of 0.5 μg/mL was used as a positive control.
Cells were cultured at 37°C in 5% CO2 for 5 days, harvested, and stained with the following fluorochrome-conjugated antibodies: CD3 V450, CD8 APC-H7, HLA-DR BV605, CD45RA APC (all BD) and CD25 PE (Biolegend). 7-aminoactinomycin D (7-AAD, BD) was added for dead cell exclusion. Flow cytometry data were acquired on a BD FACS Canto II with BD Diva 6.1 software, and analyzed in FlowJo X (FlowJo LLC, OR, USA).
7
Multiparameter Flow Cytometry of PBMCs
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PBMCs were isolated from whole blood cells using Ficoll media purchased from Haoyang Bio-Manufacture CO., LTD (Tianjin, China). Staining fixable viability stain 780 (BD#565388) was added into the cell suspension at 1:1000, followed by an incubation with human Fcblock (BD#564220). Surface markers were stained using CD3-PerCP-Cy5.5 (BD#560835), CD4-PE-Cy7 (BD#560649), CD8-BV510 (BD#563256), CD25-PE (BD#557138), CD127-AF647 (BD#558598), CD45RA-APC (BD#550855) and CCR7-FITC (BD#560548). For intracellular staining, cells were fixed and permeablized using BD CytoFix/CytoPerm Kit (BD#554715) and then the cytokines were stained using IL-4-APC (BD#560671), IFN-γ-FITC (BD#554700), TNF-α-BV421 (BD#562783), and IL-17-PE (BD#560487), before the final flow cytometry assay using BD CantoII. Raw data of flow cytometry was analyzed by FlowJo software.
8
Phenotypic Analysis of CD8+ T Cells in HLH
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Standard of care immune workup was performed at the Clinical Laboratory Improvement Amendments (CLIA)-certified clinical laboratory of CHOA and at the diagnostic immunology laboratory at Cincinnati Children’s Hospital Medical Center. Immune investigations consisted of lymphocyte subset analysis, perforin and granzyme B expression, CD107a degranulation assay, plasma soluble interleukin-2 receptor (SIL2R) levels, NK cell activity, and cytotoxic T lymphocyte (CTL) function.
In a limited number of patients (three p-HLH and three T cell-EBV-HLH), additional T cell immunophenotyping for the purposes of research was performed. Peripheral blood mononuclear cells (PBMCs) were stained with CD3-PerCP/Cy5.5, CD8-BUV395, CD45RA-APC, CCR7-PE, HLA-DR-BV711, CD38-BUV496, and PD-1-BV421 antibodies (BD Biosciences and BioLegend). Live/dead fixable aqua dead cell stain (Thermo Fisher) was used to exclude dead cells in the analysis. Flow cytometry data was acquired on BD FACSymphony™ A5 and analyzed using FlowJo software v10. Effector memory (EM) population of CD8+ T cells was identified as CCR7− CD45RA− CD8+ T cells. Activated CD8+ T cells were identified as HLA-DR+CD38+ gated on CD8+ EM T cells or CD8+ T cells. Due to a lack of additional T-cell immunophenotypic data from infection-related HLH (iHLH) and MAS cohorts, our T cell activation analysis was restricted to p-HLH and T cell-EBV-HLH.
In a limited number of patients (three p-HLH and three T cell-EBV-HLH), additional T cell immunophenotyping for the purposes of research was performed. Peripheral blood mononuclear cells (PBMCs) were stained with CD3-PerCP/Cy5.5, CD8-BUV395, CD45RA-APC, CCR7-PE, HLA-DR-BV711, CD38-BUV496, and PD-1-BV421 antibodies (BD Biosciences and BioLegend). Live/dead fixable aqua dead cell stain (Thermo Fisher) was used to exclude dead cells in the analysis. Flow cytometry data was acquired on BD FACSymphony™ A5 and analyzed using FlowJo software v10. Effector memory (EM) population of CD8+ T cells was identified as CCR7− CD45RA− CD8+ T cells. Activated CD8+ T cells were identified as HLA-DR+CD38+ gated on CD8+ EM T cells or CD8+ T cells. Due to a lack of additional T-cell immunophenotypic data from infection-related HLH (iHLH) and MAS cohorts, our T cell activation analysis was restricted to p-HLH and T cell-EBV-HLH.
9
Isolation and Purification of CD4+ T-cell Subsets
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CD4+ Tmem cells and Tnaive were sorted with the MACS method, with the Memory CD4+ T-cell Isolation Kit (order no 130–091–893) and with the Naive Pan T-cell Isolation Kit (order no 130–097–095), respectively (both Miltenyi Biotech, Bergish Gladbach, Germany), according to the manufacturer’s protocols. Cells were thawed and rested overnight, then sorted the next day. Cells were sorted twice on a magnetic column to ensure high purity in the enriched cells. During Tnaive isolation, we added the optional anti-TCR γ/δ antibodies to ensure depletion of γ/δ T cells. We analyzed the purity of the enriched fractions by staining with Fixable Viability Stain 510, CD3-APC-H7, CD45RO-PE, and CD45RA-APC (all BD Biosciences, San José, CA, USA). Cells were analyzed on a FACS Canto II flow cytometer (BD Biosciences, San José, CA, USA) equipped with FACS Diva Software. Gating strategy for the identification of CD3+ T cells for the phenotypic analysis of memory cell markers is provided in Supplementary Material 2 .
10
CMV-Specific T-Cell Phenotyping
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The CMV status of donors was obtained by the overnight stimulation of fresh PBMC with CMV viral lysate and identification of IFN-γ production by CD4+ T cells [as described below; using CD4 conjugated with phycoerythrin-Cy7 (PE-Cy7), allophycocyanin (APC) or PE and IFN-γ conjugated with FITC or V450]. Previous data showed that there was total concordance between IFN-γ+ responses and seropositivity obtained from IgG serology obtained from the diagnostic laboratory of University College London Hospital. CD8+ T-cell phenotyping was achieved by incubating PBMC with CD8-peridinin chlorophyll protein (PerCP), CD8-FITC, CD27-FITC, CD27-PE, and CD45RA-APC (all from BD Biosciences, Oxford, UK). HLA typing was achieved by staining PBMC with HLA A2-PE (AbD Serotec, Kidlington, Oxfordshire, UK). Tetramer staining was conducted on HLA-matched CMV-responding donor PBMC for 20 min at 37° with HLA-A*0201 (NLVPMVATV) CMVpp65-specific tetramers, prior to antibody staining. Samples were acquired on a BD FACS Calibur of LSRII and analysed using Flowjo software.
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