PBMC were stained with monoclonal antibodies to CD4 (1:50, clone RPA-T4, Biolegend #300518), CD3 (1:50, clone OKT3, Biolegend #317332), CD45RO (1:40, clone UCHL1, Biolegend #304236) and CCR7 (1:40, clone G043H7, Biolegend #353216). Afterwards, cells were washed and CD45RO+ CCR7+ (central-memory) and CD45RO+ CCR7− (effector-memory) and CD3+ CD4+ (total) CD4+ T-cells were sorted in a specifically designated biosafety cabinet (Baker Hood), using a FACS Aria cell sorter (BD Biosciences) at 70 pounds per square inch. Cell sorting was performed by the Ragon Institute Imaging Core Facility at MGH and resulted in isolation of lymphocytes with the defined phenotypic characteristics of >95% purity. Data were analyzed using FlowJo software (Treestar).
Clone g043h7
Manufactured by BioLegend
Sourced in United States
Clone G043H7 is a primary antibody that recognizes a specific epitope on its target antigen. It is designed for research use and can be used in various immunoassays to detect the presence and/or abundance of the target antigen in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Clone rpa t4, by BioLegend (4 mentions)
Clone uchl1, by BioLegend (4 mentions)
Clone okt3, by BioLegend (2 mentions)
Clone hit3a, by BioLegend (2 mentions)
Fcgr block, by BD (2 mentions)
Clone o323, by BioLegend (2 mentions)
Moflo xdt cell sorter, by Beckman Coulter (2 mentions)
Cd3 clone ucth1, by BD (1 mentions)
Clone 2d1, by BD (1 mentions)
Clone ncam16, by BD (1 mentions)
Clone fn50, by BioLegend (1 mentions)
Brilliant violet 421, by BioLegend (1 mentions)
Zombie uv fixable viability kit, by BioLegend (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Clone eh12.2h7, by BioLegend (1 mentions)
Anti cd8 apc cy7, by BioLegend (1 mentions)
Fixable viability stain 450, by BD (1 mentions)
Anti cd4 pe vio770, by Miltenyi Biotec (1 mentions)
Clone rpa t8, by BioLegend (1 mentions)
Anti cd45ra apc, by BioLegend (1 mentions)
10 protocols using clone g043h7
1
Multiparametric Flow Cytometry Sorting of T-cell Subsets
2
Monitoring T cell Activation and Phenotype
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Purified T cells were stimulated as previously described and GM-CSF was measured using the GM-CSF Secretion Assay Enrichment and Detection Kit (PE, Miltenyi, 130-105-760). The manufacturer’s instructions were modified to a 96-well format with final volumes of 200 μl per well. Following the GM-CSF kit protocol, cells were additionally stained with CD4 (clone SK3, BD, BV480, 566104), CD8 (clone SK1, BD, BUV805, 564912), CD3 (clone UCTH1, BD, BB630, 624294), HLA-DR (clone G46-6, BD, BV786, 564041), CD16 (clone 3G8, BD, BV750, 624380), CD45 (clone 2D1, BD, BUV563, 624284), CD69 (clone FN50, BD, APC-H7, 560737), CCR7 (clone G043H7, Biolegend, BV650, 353134), CD45RA (cloneHI100, BD, PE, 561883), CD25 (clone 2A3, BD, PE-Cy7, 335789), CD14 (M5E2, BD, BV570, 624298), CD56 (clone NCAM16.2, BD, PE-Cy5, 624350) and live-dead dye FVS575V (BD, BV570, 565694). Subsequently, cells were fixed and permeabilized in Foxp3/transcription factor staining buffer as previously described, and stained with the above-mentioned metabolic antibodies, before acquiring on the X-30 FACSymphony.
3
Flow Cytometry for Cell Phenotyping
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Flow data were collected on an LSRII from BD Biosciences. Zombie UV Fixable Viability Kit (BioLegend) was used as live/dead stain for reactivation experiments. We had a range of 250 to 6000 events per data point and a minimum cut-off of 250 events in Fig 5B . The mean number of all events was 1364 and the median number was 678. All cells were washed and fixed in a final concentration of 2% paraformaldehyde prior to analysis. Cell sorting was performed on a MoFlo Astrios from Beckman Coulter. All flow experiments performed at Boston University School of Medicine Flow Cytometry Core Facility.
Cell activation and phenotypes were determined by CD69 expression (Brilliant Violet 421 anti-human CD69 antibody; Clone FN50, BioLegend) and CCR7 and CD45RA expression (Pe/Cy7 anti-human CCR7 antibody; Clone G043H7, BioLegend and PerCP/Cy5.5 anti-human CD45RA antibody; Clone HI100, BioLegend). We had a minimum of 1000 events to be included as a data point inFig 2 .
Cell activation and phenotypes were determined by CD69 expression (Brilliant Violet 421 anti-human CD69 antibody; Clone FN50, BioLegend) and CCR7 and CD45RA expression (Pe/Cy7 anti-human CCR7 antibody; Clone G043H7, BioLegend and PerCP/Cy5.5 anti-human CD45RA antibody; Clone HI100, BioLegend). We had a minimum of 1000 events to be included as a data point in
4
Isolation and Sorting of Resting CD4+ T Cell Subsets from HIV-1-Infected Donors
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Resting CD4+ T cells from HIV-1-infected donors on suppressive ART were isolated as described above. To sort resting memory subsets, we incubated cells with FcgR block (BD Pharmingen) for 10 minutes before staining with a FITC-labeled antibody to CD3 (Biolegend; Clone HIT3a), phycoerythrin (PE)-Cy7-labeled antibody to CD4 (Biolegend; Clone RPA-T4), allophycocyanin (APC)-labeled antibody to CD45RO (Biolegend; Clone UCHL1), BV421-labeled antibody to CD27 (Biolegend; Clone O323) and PE-labeled antibody to CCR7 (Biolegend; Clone G043H7). Dead cells were excluded using propidium iodide. PE Mouse IgG2aκ, BV421 Mouse IgG1κ, and APC IgG2aκ isotype antibodies were used in fluorescence-minus-one controls to set sorting gates. Memory cells were distinguished from naive cells by the presence or absence of CD45RO staining, respectively. Central memory cells were distinguished from effector and transitional memory subsets by the presence of CCR7 as described by Sallusto et al32 . CCR7- cells were subdivided into effector memory (Tem) (CD45RO+CCR7-) or transitional memory (Ttm) (CD45RO+CCR7-CD27+) as described by Chomont et al20 (link). Central, effector, and transitional resting memory subsets were sorted using a Beckman Coulter MoFlo XDT Cell sorter.
5
Phenotypic Characterization of Activated T Cells
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PBMC were purified as indicated above and activated with TransAct™ (1:100, Miltenyi-Biotec 130-111-160) and recombinant human IL2 (100 IU/ml; PeproTech). Then at days 0 and days 2-4-6 post-stimulation, the cells were stained with Fixable Viability Stain 450 (250 ng/mL; BD Biosciences), next with anti-CD3-PE-Cy5 (1/30; clone HIT3a; BD Biosciences), anti-CD4-PE-Vio770 (1/100; clone rea623; Miltenyi Biotec), anti-CD8-APC-Cy7 (1/30; clone RPA-T8; Biolegend), anti-CD25-Alexa fluor 488 (1/30; clone M-A251; Biolegend), anti-CD45RA-APC (1/30; clone HI100; Biolegend), anti-CCR7-PE (1/50; clone G043H7; Biolegend), anti-Granzyme-B-PE (1/50; clone GB12; Invitrogen), anti-PD1-Alexa 488 (1/50; clone EH12-2H7; Biolegend) and anti-KLRG1-APC (1/100; clone Rea261; Miltenyi Biotec). FACS analyses were performed on a MACSQuant Analyzer (Miltenyi Biotec) and data analyzed using FlowJo 10 software (FlowJo, LLC). All samples were gated on forward and side scatter, for singlets and for live cells.
6
Expansion and Immunophenotyping of T Cells
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To expand activated T cells, PBMCs were added to flasks pre-coated with anti-CD3 (1:500, Clone OKT-3, Invitrogen) and anti-CD28 (1:500, Clone CD28.2, Invitrogen) monoclonal antibodies and then incubated in serum-free KBM551 medium (Kohjin Bio) containing 200 IU/ml human recombinant interleukin-2 (rhIL-2) (R&D Systems) at 37 °C in a 5% CO2 atmosphere. Four days later, the cells were transferred into a culture bag (Kohjin Bio) and were cultured for a total of 10–14 days. Cultured T cells were harvested and analyzed by flow cytometry. The following monoclonal antibodies were used to stain cultured T cells: anti-CD45-BUV395 (1:200, Clone HI30, BD Biosciences), anti-CD3-PE-Cy7 (1:100, Clone SP34-2, BD Biosciences), anti-CD56-PE (1:100, Clone CMSSB, Invitrogen), anti-CD19-PE-CF594 (1:200, Clone HIB19, BD Biosciences), anti-CD14-PE-CF594 (1:200, Clone MQP9, BD Biosciences), anti-CD4-BV605 (1:100, Clone RPA-T4, BD Biosciences), anti-CD8-APC-R700 (1:100, Clone SK1, BD Biosciences), anti-CCR7-PerCP-Cy5.5 (1:100, Clone G043H7, Biolegend), and anti-CD45RA-APC-H7 (1:100, Clone HI100, BD Biosciences).
7
Multiparametric Flow Cytometry Sorting of T-cell Subsets
8
Comprehensive Immune Profiling of TILs
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The following antibodies were used, in this study, for flow cytometry: Pacific blue-CD3 (clone SK7, BioLegend, San Diego, CA, United States), PECy7 or FITC-CD8 (clone HIT8a, BioLegend), FITC-PD-1 (clone EH12.2H7, BioLegend), FITC-TIM-3 (clone F38-2E2, BioLegend), FITC-LAG-3 (clone 11C3C65, BioLegend) APC-CD25 (clone BC96, BioLegend), APC-CD28 (clone CD28.2, BioLegend), APC-vio770-CD45RA (clone HI100, BioLegend) and PerCP-CCR7 (clone G043H7, BioLegend). TIL cultures were washed and re-suspended in cell-staining buffer (BioLegend). Cells were incubated for 30 min with the antibodies on ice, washed in buffer and measured using MACSQuant flow cytometer (Miltenyi Biotech).
For localization and quantification of granzyme B, cells were fixed (BLG420801, Biolegend), permeabilized (BLG421002, Biolegend), and stained with granzyme B-specific antibodies (BLG515406, BioLegend). For carboxy fluorescein succinimidyl ester (CFSE) cell proliferation assays, TIL cultures were stained before seeding at day 11 of the REP with 5 μM CFSE (Thermo Fisher Scientific) for 20 min at 37°C, according to the manufacturer’s instructions. Four days later cells were taken for flow cytometry analysis and their CFSE fluorescence intensity was determined. Samples were analyzed using FlowJo software (FlowJo LLC, Ashland, OR).
For localization and quantification of granzyme B, cells were fixed (BLG420801, Biolegend), permeabilized (BLG421002, Biolegend), and stained with granzyme B-specific antibodies (BLG515406, BioLegend). For carboxy fluorescein succinimidyl ester (CFSE) cell proliferation assays, TIL cultures were stained before seeding at day 11 of the REP with 5 μM CFSE (Thermo Fisher Scientific) for 20 min at 37°C, according to the manufacturer’s instructions. Four days later cells were taken for flow cytometry analysis and their CFSE fluorescence intensity was determined. Samples were analyzed using FlowJo software (FlowJo LLC, Ashland, OR).
9
Isolation and Sorting of Resting CD4+ T Cell Subsets from HIV-1-Infected Donors
10
PBMC Isolation and Immunophenotyping
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Peripheral blood mononuclear cells (PBMC) from healthy subjects were isolated from heparinized whole blood by density gradient centrifugation and 1x106 PBMCs were stained with viable and dead cells LIVE/DEAD™ Fixable Aqua dead cell stain kit (Life Technologies) for 15 min followed by an extracellular staining. First CCR7 PerCp Cy 5.5 was stained (clone G043H7, Biolegend) for 15 min at 37 °C followed by CD3 AF700 (clone OKT3, Biolegend), CD4 BV605 (clone RPA-T4, BD Biosciences), CD8 PB (clone SK1, Biolegend), CD45 RA FITC (clone HI100, Biolegend), CD56 APC-Cy7 (clone HCD56, Biolegend), CD19 PE-Cy7 (clone HIB19, Biolegend), CD26 APC (clone BA5b, Biolegend) for 15 min at 4°C. Non-specific binding of Fc-receptors was blocked by 2% polyclonal IgG (Flebogamma). CD26 surface expression was measured with CytoFLEX LX (Beckman Coulter) and data was analyzed with FlowJo software 10.0.08. Gating strategies are shown in the Supplementary Information
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