In vitro assay to evaluate the impact of immunoregulatory pathways on HIV-specific CD4 T cell effector function.

TitleIn vitro assay to evaluate the impact of immunoregulatory pathways on HIV-specific CD4 T cell effector function.
Publication TypeJournal Article
Year of Publication2013
AuthorsPorichis F, Hart MG, Zupkosky J, Barblu L, Kaufmann DE
JournalJ Vis Exp
Issue80
Paginatione50821
Date Published10/15/2013
ISSN1940-087X
KeywordsCD4-Positive T-Lymphocytes, Cytokines, Epitopes, T-Lymphocyte, Flow Cytometry, gag Gene Products, Human Immunodeficiency Virus, HIV Infections, HIV-1, Humans
Abstract

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects. Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10Rα and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 °C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

DOI10.3791/50821
Alternate JournalJ Vis Exp
PubMed ID24193166
Grant ListP01 AI-080192 / AI / NIAID NIH HHS / United States
P01 AI080192 / AI / NIAID NIH HHS / United States
R01 HL-092565 / HL / NHLBI NIH HHS / United States
UM1 AI100663 / AI / NIAID NIH HHS / United States
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