Phenotype analysis were conducted antibodies targeting human CD56 (NCAM16.2), CD3 (UCHT1), CD57 (NK-1), CD2 (RPA2.10), DNAM-1 (DX11), NKp44 (p44-8), NKp30 (p30-15), NKp46 (9E2), TNF-α (Mab11), Granzyme B (Gb11), and IFN-γ (B27) from Becton Dickinson (BD) Biosciences (CA, USA). Antibodies targeting human CXCR4 (12G5), IgG2a isotype (MOPC173), KIR2DL/DS/2/3 (DX27), NKG2D (1D11), KIR3DL/DS1 (DX9), 2B4 (C1.7), and CD107a (H4A3) were purchased from Biolegend (CA, USA). Human LIR-1 (HP-F1) antibody was purchased from Lifespan Biosciences Inc (WA, USA). Live/Dead Aqua marker was purchased from Invitrogen (CA, USA). Flow cytometry was performed with a BD LSRII Fortessa cytometer and the data were analyzed with the FlowJo software (Treestar Inc.).
Live dead aqua marker
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead Aqua marker is a fluorescent dye used to distinguish between live and dead cells in aqueous samples. It binds to DNA and emits fluorescence upon excitation, enabling the visualization and quantification of live and dead cells in a sample.
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Lab products found in correlation
Cd107a h4a3, by BioLegend (1 mentions)
Ucht1, by BD (1 mentions)
Cd25 apc cy7, by BD (1 mentions)
Cd127 pe cy7, by BD (1 mentions)
Anti human cd3 percp, by BD (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Anti cd8 sk1, by BD (1 mentions)
Anti cd14 m5e2, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
Magnetic microbead, by Miltenyi Biotec (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Cd3 brilliant violet 711 clone okt3, by BioLegend (1 mentions)
Cd45ra pe cf594 clone hi100, by BD (1 mentions)
Cd14 v500 clone m5e2, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
4 protocols using live dead aqua marker
1
Multiparametric Phenotypic Analysis of Immune Cells
2
Isolation and Characterization of Tr1 and iTreg Cells
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Cells were stained with relevant antibodies on ice for 30 minutes in PBS buffer containing 2% FCS and 0.1% sodium azide. Before staining for surface markers, cells were incubated with Fc Blocker (CD16/CD32) for 30 minutes to minimize non-specific staining. Cells were washed twice before being analysed by BD FACS ARIA II flow cytometer. Live cells were gated based on forward and side scatter profiles and based on exclusion with live/dead aqua marker (Invitrogen). The following antibodies were used for staining: anti-human CD3 PerCP, CD4 APC or CD4 PE-Texas red, CD25 APC-Cy7, CD127 PE-Cy7, GARP PE (All from BD Biosciences) and anti-human Latency associated peptide (LAP)-TGF-β1 Alexa Fluor 488 (R and D systems). Recombinant human LAP (rLAP) that associates with TGF-β1 was purchased from R and D systems. Analysis was performed using FlowJo software (Tree Star).
The Tr1 and iTreg cells were sorted on the BD FACS ARIAII Flow Cytometer aseptically for further experiments. 2.5 million PBMCs from each well of tissue culture plate were sorted in approximately 5 minutes. Sorted cells were collected in 12×75 mm polypropylene tubes pre-coated with human AB serum and containing complete RPMI-1640 with 10% human AB serum.
The Tr1 and iTreg cells were sorted on the BD FACS ARIAII Flow Cytometer aseptically for further experiments. 2.5 million PBMCs from each well of tissue culture plate were sorted in approximately 5 minutes. Sorted cells were collected in 12×75 mm polypropylene tubes pre-coated with human AB serum and containing complete RPMI-1640 with 10% human AB serum.
3
Tetramer-based Enrichment of Antigen-specific CD4+ T Cells
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Untouched CD4+ T cells were isolated from PBMCs using magnetic microbeads (Miltenyi Biotec). Tetramer staining and enrichment was performed as described previously (Su et al., 2013 (link)). In brief, cells were incubated for 30 min with live/dead Aqua marker (Invitrogen), washed, and then labeled with either gE or IE63 tetramers at room temperature for 45 min (14 µg/ml). Surface markers AF700-labeled anti-CD3 (UCHT3; BD), FITC-labeled anti-CD4 (SK3; BD), Pacific blue–labeled anti-CD45RA (MHCD45RA28; BD), and PE-cyanine 7 (PECy7)–labeled anti-CD56 (B159; BD), anti-CD14 (M5E2; BD), and anti-CD8 (SK1; BD) were incubated at room temperature for 15 min. Before tetramer enrichment, 1/10th staining volume was removed and added to TruCount tubes (BD) to give an absolute count of the starting number of CD4+ naive and memory T cells. The remaining staining volume was enriched for tetramer-positive cells using anti-PE microbeads (Miltenyi Biotec) and added to a separate TruCount tube. Samples were acquired using an LSR Fortessa (BD), and the frequency of tetramer-positive cells determined by dividing the absolute counts of tetramer positive cells by the starting number of CD4+ naive/memory T cells.
4
Longitudinal Isolation of CD4+ T-cell Subsets
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The CD4 + T-cell subsets were sorted from frozen aliquots of peripheral blood mononuclear cells (PBMCs) stored for 4–16 years which contained 50–100 × 106 cells that were collected from 5 of the participants at approximately years 3, 5, 10 on ART (Visit ID 1–3). At approximately 15 years of therapy, CD4+ T-cells were isolated from a leukapheresis for all 6 participants (Visit ID 4). Participant 2518 had a second leukapheresis sample collected for this study 2 years later at approximately 17 years on therapy (Visit ID 5). The cells were sorted using the following antibodies: CD3-Brilliant Violet 711 (clone OKT3, BioLegend), CD4-APC-eFluor 780 (clone OKT4, Thermo Fisher), CD14-V500 (clone M5E2, BD# 561391), LIVE/DEAD Aqua marker (Invitrogen# L34957), CD45RA-PECF594 (clone HI100, BD Biosciences), and HLA-DR− Brilliant Violet 421 (clone L243, BioLegend). CD3+CD4+ T-cells were gated on memory cells defined as CD45RA negative. HLA-DR± cells were then sorted on a BD FACS ARIA-II (Supplementary Figure 1 ). The HLD-DR± populations were sorted to >99% purity. Following sorting, the cell subsets were processed at 4°C and stored as a dry cell pellet at −80°C until analysis.
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