PMA and Ionomycin were purchased from Sigma-Aldrich and used for non-specific stimulation and the positive controls. Mouse anti-human CD3-APC CY7 (clone SK7) and anti-IFNγ-APC were from BD Biosciences. Mouse anti-human CD8-PerCP (clone RPA-T8) was from BIOLEGEND. Mouse anti-human IL-2-allophycocyanin (clone 17-7029-71), anti-CD45RA-FITC (clone HI100), anti-CCR7-PE CY7 (clone 3D12), anti-TNFα-PE (clone MAB11) and anti-IL-17-PE (clone BL 168) were from eBioscience. Anti-human HLA-A2-PE (clone BB7.2) was from Abcam. CMV-EBV-Flu peptide pool was used for the study of antigen specific CD8 T cell responses as described previously (22 (link)). Peptides were synthesized by the GeneImmune (Suzhou, China), consisting of 23 HLA class I-restricted T cell epitopes from well-defined CMV, EBV and FLU viral proteins. The peptides were restricted by the most common HLA alleles including HLA-A1, A2, A3, A11, A24, A68, B7, B8, B27, B35, B44 (Supplementary Figure 4A ) and pooled into 12 groups as indicated in Supplementary Figure 4B . Function-graded mouse anti-human CD28 and anti-CD49d antibodies were from BD Biosciences.
Anti cd49d
Manufactured by BD
Sourced in United States, Belgium, United Kingdom
Anti-CD49d is a laboratory reagent used for the detection and analysis of CD49d, a cell surface protein expressed on various immune cells. It is a tool commonly used in flow cytometry and other immunological research applications to study cellular phenotypes and functions.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28, by BD (55 mentions)
Brefeldin a, by Merck Group (22 mentions)
Facs lysing solution, by BD (9 mentions)
Monensin, by BD (7 mentions)
Brefeldin a, by BD (6 mentions)
Staphylococcal enterotoxin b, by Merck Group (6 mentions)
Golgistop, by BD (6 mentions)
Perm wash buffer, by BD (6 mentions)
X vivo 15 medium, by Lonza (5 mentions)
Lsrii flow cytometer, by BD (5 mentions)
Facscanto 2, by BD (5 mentions)
Anti ifn γ alexa 700, by BD (4 mentions)
Anti tnfα pecy7, by BD (4 mentions)
Anti cd107a fitc, by BD (4 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Anti cd3, by BD (4 mentions)
Polypropylene tube, by BD (3 mentions)
Live dead cell dye, by Thermo Fisher Scientific (3 mentions)
Anti cd3 alexa 780, by Thermo Fisher Scientific (3 mentions)
67 protocols using anti cd49d
1
Comprehensive Characterization of Antigen-Specific T Cells
2
SARS-CoV-2 T Cell Immune Response Assay
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The assay was performed as described before (23 (link)). Briefly, ~2 × 106 PBMCs were cultured in 200-μl final volume in 5-ml polypropylene tubes (BD Biosciences, San Diego, CA, USA) in the presence of anti-CD28 (1 μg/ml) and anti-CD49d (1 μg/ml) (BD Biosciences) and the following conditions: (i) negative control with dimethyl sulfoxide only, (ii) whole spike (S) peptide pool 1 (n = 253 peptides, 15-mers with 10-residue overlap) (Weiskopf and Sette labs, LJI, La Jolla, CA) at a final concentration of 1 μg/ml, (iii) RBD peptide pool 2 (53 peptide pool, 15-mers with 11-residue overlap, JPT Peptide Technologies, Germany) at a final concentration of 1 μg/ml, (iv) N peptide pool (only prechallenge and postchallenge time points), and (v) phorbol 12-myristate 13-acetate/ionomycin. Brefeldin A was added to all tubes at 10 μg/ml (Sigma-Aldrich, St Louis, MO) and cells were cultured for 6 hours and transferred to 4° before staining for flow cytometry as detailed in the Supplementary Materials.
3
PBMC Stimulation with Mycobacterial Antigens
4
Regulatory T-cell Immunophenotyping and Cytokine Analysis
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Fresh peripheral blood mononuclear cells (PBMNCs) from patients 2 and 5 (Supplementary Table 3 ) and six healthy controls were used. For regulatory T-cell immunophenotyping, cells were stained using monoclonal antibodies (mAb) against the antigens CD3, CD4 and CD25 (BD Biosciences) and FOXP3 (clone259D, eBioscience) and data collected with 4- or 6-color flow cytometry. Regulatory T-cells were defined as CD3+CD4+CD25highFOXP3+. Evaluation of T-cell cytokine production is described in detail elsewhere23 (link). Briefly, fresh mononuclear cells were stimulated for 6 hours with anti-CD3, anti-CD28 and anti-CD49d (BD Biosciences). The cells were analyzed using a 6-color flow cytometry panel with mAbs against the antigens CD3, CD4, CD8, IFN-γ and TNF-α (BD Biosciences). The data were analyzed with FACSAria II flow cytometer and FACSDiva software (BD Biosciences).
5
Polyfunctional T-cell Profiling by Flow Cytometry
6
Polyfunctional T-cell Immune Response Analysis
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Flow cytometry based ICS assay was done as previously described [35 ,49 (link),50 (link)]. In brief, 106 PBMCs were pulsed with peptide at 10uM in the presence of co-stimulatory antibodies (anti-CD28 and anti-CD49D) and anti-CD107a-FITC (all from BD Biosciences) for 2 hrs at 37°C. Monensin and brefeldin A (both from BD Biosciences) were then added and the cultures incubated for an additional 12 hrs. Next day, the cells were labeled with LIVE/DEAD cell dye (Invitrogen) and surface stained with anti-CD3-Pac Blue, anti-CD8-V500, and anti-CD4-Alexa 780 (both from BD Biosciences). The cells were permeabilized and labeled with anti-IFN-γ-Alexa 700, anti-IL-2-APC, anti-TNFα-PECy7, and anti-Granzyme A-PE (all from BD Biosciences). CD3 events greater than 100,000 were acquired on an LSR II (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar). Polyfunctionality analysis was performed using Boolean gating and SPICE and Pestle software (version 5.1; NIAID).
7
Activation Induced Molecule Assay for HIV Gag
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Cryopreserved PBMC were thawed and allowed to rest for 2–3 hours prior to stimulation with HIV gag PTE peptides for a modified AIM (Activation Induced Molecule) assay [58 (link)]. Cells were cultured at 5 million per milliliter for 18–20 h in presence of 2 μg/ml GAG PTE peptides (AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health [NIH]: HIV-1 PTE Gag Peptide Pool from NIAID, Division of AIDS), 1 μg/ml of staphylococcal enterotoxin B (SEB; List Biological Laboratories) or medium (negative control). At the beginning of culture, the following reagents were added: 1 μg/ml costimulatory molecule anti-CD28 mAb (BD Biosciences), 1ug/mL of anti-CD49d (BD Biosciences) and 0.5ug/mL the degranulation marker (CD107a PerCP-Cy5.5).
Following the culture period, cells were stained for flow sorting using previously titrated mAb (CD3, CD4, CD8, OX40, and CD25). Sorting was performed using the gating strategy shown (S5 Fig
Following the culture period, cells were stained for flow sorting using previously titrated mAb (CD3, CD4, CD8, OX40, and CD25). Sorting was performed using the gating strategy shown (
8
PBMC Stimulation and Cytokine Analysis
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PBMC were isolated by Ficoll-Hypaque density gradient centrifugation (GE Health care), cryopreserved and stored until needed. Cryopreserved PBMC were thawed and rested in RPMI 1640 containing 10% heat-inactivated FCS prior to antigen stimulation. PBMC were stimulated using Mtb cell lysate (strain H37Rv, BEI resources) or early secretory antigenic target (ESAT)-6/culture filtrate protein (CFP)-10 peptide pool consisting of 17 and 16 peptides covering the entire 6-kDa ESAT-6 and 10-kDa CFP-10, respectively (2 µg/ml, Peptide Synthetics) or a pool of cytomegalovirus virus (CMV) peptides consisting of 138 peptides covering the entire pp65 protein (2 µg/ml). Brefeldin A (10 µg/ml, Sigma) was added 3 h after Mtb lysate stimulation or at the time of peptides addition. All stimulations were performed in the presence of co-stimulatory antibodies, anti-CD28 and anti-CD49d (both at 1 µg/ml; BD) for 16 h.
9
Cytokine Production Assay for PBMCs
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Freshly isolated PBMCs (1 × 106 cells) were incubated with the indicated stimulants (1 μg/mL CMV pp65 antigen, 0.33 μg/mL CMV lysate, 80 μL [8 U]/mL CMVIG) in the presence of costimulatory anti-CD28 (1 µg/mL) and anti-CD49d (1 µg/mL) antibodies (BD, Heidelberg, Germany) for 6 h (T-cell protocol; all cytokines except IL-10 and IL-12) or 16 h (APC protocol; IL-10 and IL-12 staining) at 37 °C. Samples incubated with AIM-V medium with the same amount of costimulatory anti-CD28 and anti-CD49d antibodies served as negative control. After incubating for 2 h (T-cell protocol) or 12 h (APC protocol) at 37 °C in a humidified atmosphere under 5% CO2, 10 mg/mL brefeldin A (Sigma-Aldrich, Munich, Germany) was added to prevent cytokine secretion and incubated for an additional 4 h. Stimulated cells were washed twice with phosphate-buffered saline before further cell surface and intracellular cytokine staining, as detailed in Supplemental Materials and Methods (SDC, http://links.lww.com/TXD/A374 ). The cytokine-producing cells were detected using a Canto II flow cytometer (BD) and the data were analyzed using Flow Jo Data Analysis Software (version 10). The gating strategy is shown in Figure S2 (SDC, http://links.lww.com/TXD/A374 ). Results are reported as percentage of the gated population producing the indicated cytokines.
10
CD107a Expression in T-cell Activation
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T-cells were incubated in 96-well plates (80,000 cells/well), together with an equal amount of THP-1 or KOx3 cells, in the presence or not of functional grade anti-CD3 (clone OKT3, Miltenyi Biotech, Köln, Germany) at a concentration of 7.5 µg/ml. Co-cultures were maintained in a final volume of 100 µl of X-Vivo-15 medium (Lonza, Basel, Switzerland) for 6 hours at 37 °C with 5% CO2. CD107a staining was done during cell stimulation, by the addition of an APC-conjugated anti-CD107a antibody (BD Biosciences, San Jose, California) at the beginning of the co-culture, together with 1 µg/ml of anti-CD49d (BD Biosciences, San Jose, California), 1 µg/ml of anti-CD28 (Miltenyi Biotech, Köln, Germany), and 1× Monensin solution (eBioscience, San Diego, California). After the 6 hours incubation period, cells were stained with a fixable viability dye (eBioscience, San Diego, California) and PE-conjugated anti-CD8 (Miltenyi Biotech, Köln, Germany) and analyzed by flow cytometry.
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