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.
This in vitro assay can be effectively used to quantify cytokine levels restored by the blockade of inhibitory pathways to investigate a broad panel of effector molecules and also to evaluate of the impact of cytokines produced by HIV specific C for T-cells on other cell subsets. Megan Hart, a research technician from our laboratory, will now demonstrate this procedure, Deplete the CD eight positive T cells by adding the human CD eight positive depletion cocktail at 50 microliters per milliliter of whole blood mix well and incubate for 20 minutes. At room temperature, mix the whole blood with hank's buffer.
Now carefully layer the blood over histo pack, then centrifuge the gradient at 540 RCF for 30 minutes. Collect the layer of peripheral blood mononuclear cells. Transfer the cells to a new 50 milliliter conical tube and wash twice with 45 milliliters of complete RPMI 1640.
Re suspend the washed cells in complete RPMI 1640. Then aliquot 500 microliters of cell suspension per fax tube. Depending on the pathway investigated, add the blocking antibodies.
Also add the isotype controls to the appropriate tubes. Incubate the cells for 15 minutes in the cell culture incubator. Next, stimulate the cells with an HIV one gag peptide pool.
Also include a no stimulation control for each blocking condition. Continue to culture cells for 48 hours. Pellet the cells by centrifugation then without disturbing the pellet.
Transfer twice 225 microliters of supernatant into EOR tubes for the luminex speed arrays to inactivate the virus at 25 microliters of 0.5%PBS tween. Then measure the desired cytokine concentrations using the Millipore luminex kit according to the manufacturer's protocol. After washing the cells in three milliliters of PBS stain for viability, then resuspend the cells in 100 microliters of PBS containing 1%FBS if staining for monocytes or other antigen presenting cells at 1.4 microliters of FCR blocking reagent vortex to mix and incubate for 10 minutes at four degrees celsius.
Next, add the surface antibodies vortex and incubate for 20 minutes at four degrees Celsius in the dark. After one wash at 200 microliters of 4%para formaldehyde and incubate at room temperature for 20 minutes in the dark. After one wash, we suspend the cells in 250 microliters of 1%FBS for analysis on a multi laser flow cytometer for quantitative R-T-P-C-R lyce the cells in 300 microliters of kyogen buffer, RLT containing 1%Beamer keto ethanol isolate RNA, using an RNA isolation kit following the manufacturer's protocol.
Now synthesize CD NA from the RNA using a CD NA synthesis kit and following the manufacturer's protocol. Next, create a primer mix for each primer, including the housekeeping genes by adding primers to nuclease free water and keep on ice. For each amplification master mix, add 10.5 microliters of nuclease free water, 12.5 microliters of cyber green and one microliter of primer mix per plate.
Well now pipette 24 microliters of master mix into each reaction. Well also add one microliter of CD NA to each, well according to the template. Cap the wells and centrifuge the plate for three minutes at 1500 RCF.
Then run the plate on A QRT PCR R machine for cell surface staining. Add the felden and monin stains about six hours prior to intracellular cytokine staining. Then wash the cells with 1%FBS in PBS and stain for intracellular cytokines using a fixation permeation solution kit and protocol after washing the cells.
Once we suspend the pellets in 250 microliters of 1%FBS run the samples on a multi laser flow cytometer 16 hours after stimulation stain for viability using the live dead fixable dead cell stain kit. According to the manufacturer's protocol, after one PBS wash, we suspend the cells in 100 microliters of 1%FBS at the surface antibodies vortex to mix and incubate cells on ice in the dark for 20 minutes. Wash the cells once and suspend the cells in 500 microliters of cold complemented RPMI 1640.
Media then filter the cells through a cell strainer cap into a five milliliter polystyrene round bottom tube. Next at 200 microliters of complemented RPMI 1640 media to each collection tube. Keeping all tubes on ice during the sort live sort the cell subsets of interest on an instrument located in a facility equipped for biohazardous material culture.
The sorted cells for the desired amount of time. Following with luminex and PCR analyses analysis in this experiment, CD eight depleted pbmc stimulation with an HIV V one gag peptide pool results in a median of 800 fold increase for interferon gamma production. The level of interferon gamma transcripts were increased a median of tenfold after stimulation.
In this, in vitro analysis of HIV specific CD four T-cell function results indicate a strong correlation between interferon gamma protein secretion and interferon gamma mRNA levels. Interestingly, stimulation by the HIV gag peptide pool results in greater interferon gamma secretion than the bacterial antigens of tetanus. Toxoid further results with the IL 13 secretion indicate the different cytokine profiles of HIV and tetanus specific CD four T cell responses.
Blockade of the PD one pathway generally does not have a significant impact on cytokine secretion without any antigen stimulation, but enhances interferon gamma and IL two secretion. When cells are stimulated with HIV one gag peptide pools. When CD three cells are depleted from the pbmc, there is no interferon gamma or IL two produced in response to antigen stimulation.
These data indicate that secretion of interferon gamma and IL two is induced in response to HIV specific CD four T-cell and antigen specific stimulation. This experiment uses the demonstrated workflow using fact sorted CD four T cells to examine the impact of PD one blockade on interferon gamma and IL two. A key advantage of this method is the capacity to evaluate how immunoregulatory molecules regulate the interplay of HIV specific CD four T cells with antigen presenting cells.
Here the protocol was adapted to perform intracellular cytokine staining at 48 hours after stimulation. Results show that monocytes are primary source of IL 12 after stimulation with HIV one gag peptide pools. This in vitro experimental approach enables researchers in the field of T-cell immunology to explore the impact of immunoregulatory pathways in regulating effect effective function of Addison specific tcells in human samples.
