In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

The overall goal of this procedure is to analyze the cytokine secretion, transcription, and morphological impact of stimulated T cells. First, use a FI call gradient to isolate the peripheral CD eight depleted blood mononuclear cells from the HIV infected individuals incubate the cells with either the isotype control or the blocking antibody of interest. Then stimulate the cells with the antigen of interest.

Next, centrifuge the cell cultures in order to collect supernatants for cytokine measurements and cell pellets for phenotypic analysis or transcriptional analysis if desired. Adapt this protocol to include sorting the individual cell types by flow cytometry Results on cytokine secretion. Phenotypic or transcriptional analysis can show the impact of antibody blockade of inhibitory pathways on restoring the effector function of HIV specific CD four T cells.