Cytokine Profiling of HIV-specific CD8+ T cells

One million CD8 T cell-depleted PBMCs (Dynabeads CD8, StemCell) were incubated with an HIV Gag peptide pool (1μg/ml/peptide) or left unstimulated and incubated at 37°C in RPMI supplemented with 10% HAB and PenStrep. Cytokine secretion in supernatants was measured 48 hours after stimulation on a Luminex beads array platform, as previously described^{19 (link), 20 (link)}. Measurements were performed in duplicates using the Human High Sensitivity Cytokine Premixed Kit B (R&D Systems) for IL-2, IFN-γ, TNF-α, IL-17A, IL-22 and IL-31 or for IL-17F on a Bio-plex 200 array system (Bio-Rad Laboratories) per manufacturer’ s instructions. CXCL13 was measured separately with the Human CXCL13 Quantikine ELISA Kit (R&D Systems), as per manufacturer’s instructions.

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Morou A., Brunet-Ratnasingham E., Dubé M., Charlebois R., Mercier E., Darko S., Brassard N., Nganou-Makamdop K., Arumugam S., Gendron-Lepage G., Yang L., Niessl J., Baxter A.E., Billingsley J.M., Rajakumar P.A., Lefebvre F., Johnson R.P., Tremblay C., Routy J.P., Wyatt R.T., Finzi A., Douek D.C, & Kaufmann D.E. (2019). Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease. Nature immunology, 20(8), 1059-1070.

Publication 2019

Dynabeads CD8 Human High Sensitivity Cytokine Premixed Kit B Bio-plex 200 array system Human CXCL13 Quantikine ELISA Kit

Cd8 t cell Cxcl13 Cytokine Elisa Human Ifn γ Il 17a Il 17f Il 22 Il 31 Peptide Secretion Sensitivity Stemcell Tnf α

Corresponding Organization :

Other organizations : Centre Hospitalier de l’Université de Montréal, Ontario Genomics, Scripps Research Institute, Université de Montréal, Emory University, McGill University Health Centre, International AIDS Vaccine Initiative

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Variable analysis

independent variables

Stimulation with HIV Gag peptide pool (1μg/ml/peptide)

dependent variables

Cytokine secretion (IL-2, IFN-γ, TNF-α, IL-17A, IL-22, IL-31, IL-17F, CXCL13) in supernatants

control variables

One million CD8 T cell-depleted PBMCs
Incubation at 37°C in RPMI supplemented with 10% HAB and PenStrep
Incubation duration of 48 hours

controls

Unstimulated (negative control)

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