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1

Multiparametric Phenotyping of T-cell Responses

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Cryo-preserved blood mononuclear cells were stained with blue viability dye (Life Technologies, 4°C for 20’), followed by incubation with appropriately titrated peptide-MHC class I multimer complexes at room temperature for 20 min in Ca²⁺-free media as described [10 (link)]. Cells were then washed and stained with antibodies against CD3, CD8, CD4, HLA-G at 4°C for 20 min. For intracellular cytokine staining, cells were stimulated overnight with optimal CD8 T cell peptides in presence of brefeldin A. Cells were then stained with blue viability dye (Life Technologies, 4°C for 20’), followed by incubation with appropriately titrated antibodies against CD3, CD4, CD8, HLA-G. After fixation and permeabilization for 20 min at 4°C using a commercial kit (Caltag), cells were stained intracellularly for IFN-γ, TNF-α, MIP-1β and IL-2. Cells were acquired on a Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo X (Tree star). Analysis and presentation of cell distributions were performed using Graph Pad prism (version 6) and SPICE version 5.32, downloaded from <http://exon.niaid.nih.gov/spice/>.

Viganò S., Negrón J.J., Tse S., Chowdhury F.Z., Lichterfeld M, & Yu X.G. (2017). HLA-G+ HIV-1-specific CD8 T cells are associated with HIV-1 immune control. AIDS (London, England), 31(2), 207-212.

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2

ADCC Assay for Antibody Potency

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ADCC was performed essentially as described by Bruel et al. (2016) (link). CEM-NKr-CCR5 target cells were infected with pseudotyped virus at an MOI ~0.5 infectious units per cell. At two days post-infection, CEM cells were stained with CellTrace far red (Invitrogen) and 5×10⁴ cells were added per well. Cells were washed and resuspended in diluted monoclonals for 5 minutes. After incubation, 1×10⁵ primary NK cells (a 2:1 effector: target ratio) were added per well, cells were spun for 1 minute at 300g to promote contacts, and the cells were incubated for 4 hours at 37°C. Cells were stained for viability (blue viability dye, Life Technologies) then fixed and permeabilized for p24 staining. ADCC scores are calculated as 100 × (%p24+FarRed+ cells _{no antibody} - %p24+FarRed+ cells _antibody)/(%p24+FarRed+ cells _{no antibody}), and any negative values are adjusted to zero. To assess optimal antibody concentration, we performed antibody titration experiments of bNAbs and a subset of HC mAbs, which showed maximal or near-maximal levels of ADCC at 25 μg/ml in our assays (data not shown).

Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.

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3

Comprehensive Fc Receptor Profiling

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Flow cytometric evaluation of Fc receptor expression was performed on freshly isolated cells from enzymatically digested colon and cervical tissues. 1x10⁶ colon or 1x10⁵ cervical cells were stained in two panels with anti-CD3 Alexa Fluor 700 (clone UCHT1), anti-CD56 PE-Cy7 (clone NCAM16.2), anti-CD64 FITC (clone 10.1), anti-CD89 PE (clone A59), anti-CD45 PE-Cy5 (clone HI30), anti-CD15 PacificBlue (clone W6D3), anti-CD16 BV510 (clone 3G8), anti-CD32 APC (clone FUN-2), anti-CD64 R-PE (clone 10.1, Dako), blue viability dye (Life Technologies). Cells were washed and fixed with Perm A buffer (Life Technologies). Intracellular staining with anti-CD68 FITC (clone KP1, Dako) was performed in the presence of permeabilization buffer (Perm B, Life Technologies). The data was acquired using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo and SPICE version 5.1.

Sips M., Krykbaeva M., Diefenbach T.J., Ghebremichael M., Bowman B.A., Dugast A.S., Boesch A.W., Streeck H., Kwon D.S., Ackerman M.E., Suscovich T.J., Brouckaert P., Schacker T.W, & Alter G. (2016). Fc Receptor-Mediated Phagocytosis in Tissues as a Potent Mechanism for Preventive and Therapeutic HIV Vaccine Strategies. Mucosal immunology, 9(6), 1584-1595.

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4

ADCC Assay for Antibody Potency

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ADCC was performed essentially as described by Bruel et al. (2016) (link). CEM-NKr-CCR5 target cells were infected with pseudotyped virus at an MOI ~0.5 infectious units per cell. At two days post-infection, CEM cells were stained with CellTrace far red (Invitrogen) and 5×10⁴ cells were added per well. Cells were washed and resuspended in diluted monoclonals for 5 minutes. After incubation, 1×10⁵ primary NK cells (a 2:1 effector: target ratio) were added per well, cells were spun for 1 minute at 300g to promote contacts, and the cells were incubated for 4 hours at 37°C. Cells were stained for viability (blue viability dye, Life Technologies) then fixed and permeabilized for p24 staining. ADCC scores are calculated as 100 × (%p24+FarRed+ cells _{no antibody} - %p24+FarRed+ cells _antibody)/(%p24+FarRed+ cells _{no antibody}), and any negative values are adjusted to zero. To assess optimal antibody concentration, we performed antibody titration experiments of bNAbs and a subset of HC mAbs, which showed maximal or near-maximal levels of ADCC at 25 μg/ml in our assays (data not shown).

Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.

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5

T-cell Subset Isolation and Flow Cytometry

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Cryo-preserved blood mononuclear cells were thawed and CD3⁺ T cell isolation was performed using immunomagnetic enrichment (Miltenyi Biotech). T cells were stained with blue viability dye (Life Technologies) at 4°C for 20min. Afterwards, cells were stained with antibodies directed against CD3, CD8, CD4, HLA-G, CD25, CD127. The following T-cell populations were live-sorted, after exclusion of CD25⁺ CD127⁻ CD4⁺ Tregs, using an Aria cell sorting device (Becton Dickinson): CD4⁺CD25^−/lowHLA-G⁻, CD4⁺CD25^−/lowHLA-G⁺, CD8⁺CD25^−/lowHLA-G⁻, CD8⁺CD25^−/lowHLA-G⁺.

Viganò S., Negrón J.J., Tse S., Chowdhury F.Z., Lichterfeld M, & Yu X.G. (2017). HLA-G+ HIV-1-specific CD8 T cells are associated with HIV-1 immune control. AIDS (London, England), 31(2), 207-212.

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6

Comprehensive Fc Receptor Profiling

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Flow cytometric evaluation of Fc receptor expression was performed on freshly isolated cells from enzymatically digested colon and cervical tissues. 1x10⁶ colon or 1x10⁵ cervical cells were stained in two panels with anti-CD3 Alexa Fluor 700 (clone UCHT1), anti-CD56 PE-Cy7 (clone NCAM16.2), anti-CD64 FITC (clone 10.1), anti-CD89 PE (clone A59), anti-CD45 PE-Cy5 (clone HI30), anti-CD15 PacificBlue (clone W6D3), anti-CD16 BV510 (clone 3G8), anti-CD32 APC (clone FUN-2), anti-CD64 R-PE (clone 10.1, Dako), blue viability dye (Life Technologies). Cells were washed and fixed with Perm A buffer (Life Technologies). Intracellular staining with anti-CD68 FITC (clone KP1, Dako) was performed in the presence of permeabilization buffer (Perm B, Life Technologies). The data was acquired using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo and SPICE version 5.1.

Sips M., Krykbaeva M., Diefenbach T.J., Ghebremichael M., Bowman B.A., Dugast A.S., Boesch A.W., Streeck H., Kwon D.S., Ackerman M.E., Suscovich T.J., Brouckaert P., Schacker T.W, & Alter G. (2016). Fc Receptor-Mediated Phagocytosis in Tissues as a Potent Mechanism for Preventive and Therapeutic HIV Vaccine Strategies. Mucosal immunology, 9(6), 1584-1595.

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7

HIV-1 ADCC Assay with Activated CD4+ T Cells

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For killing of primary target cells, PBMCs were isolated into buffy coats and split into two aliquots. Target cells were generated by activation with 3 μg/ml of PHA for 3 days, followed by CD4 isolation via negative selection (StemCell Technologies, Inc.) and spinoculation with 0.5 IU/cell of NL4–3 (1,200 × g for 2 hours). After spinoculation, they were washed three times, and cultured for 4 days in R10–50. Before addition of effectors, infected CD4s were CellTrace far red stained (Invitrogen) for discrimination of target cells. Effector PBMCs were cultured with R10–50 with IL-15 added (1 ng/ml), and added a ratio of 5:1 E:T for 18 hours before staining. Staining was carried out using Blue Viability Dye (Invitrogen), anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD Biosciences, clone HIT8a), followed by fixation and permeabilization for p24 staining. ADCC score was calculated as above.

Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.

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8

HIV-1 ADCC Assay with Activated CD4+ T Cells

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For killing of primary target cells, PBMCs were isolated into buffy coats and split into two aliquots. Target cells were generated by activation with 3 μg/ml of PHA for 3 days, followed by CD4 isolation via negative selection (StemCell Technologies, Inc.) and spinoculation with 0.5 IU/cell of NL4–3 (1,200 × g for 2 hours). After spinoculation, they were washed three times, and cultured for 4 days in R10–50. Before addition of effectors, infected CD4s were CellTrace far red stained (Invitrogen) for discrimination of target cells. Effector PBMCs were cultured with R10–50 with IL-15 added (1 ng/ml), and added a ratio of 5:1 E:T for 18 hours before staining. Staining was carried out using Blue Viability Dye (Invitrogen), anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD Biosciences, clone HIT8a), followed by fixation and permeabilization for p24 staining. ADCC score was calculated as above.

Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.

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Blue viability dye

Lab products found in correlation

8 protocols using blue viability dye

Multiparametric Phenotyping of T-cell Responses

ADCC Assay for Antibody Potency

Comprehensive Fc Receptor Profiling

ADCC Assay for Antibody Potency

T-cell Subset Isolation and Flow Cytometry

Comprehensive Fc Receptor Profiling

HIV-1 ADCC Assay with Activated CD4+ T Cells

HIV-1 ADCC Assay with Activated CD4+ T Cells

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