This protocol provides a platform for studying CD4T cell responses that are often weak and difficult to detect. This technique allows for the detection, enumeration, and isolation of CD4T cells without prior knowledge regarding antigen presentation. The main advantage is the analysis of CD4T cells that are quite often rare populations associated with autoimmune conditions such as type one diabetes.
Upon obtaining the peripheral blood sample from a healthy donor, dilute the blood in PBS in at least one to two ratio before layering 35 milliliters of blood over 15 milliliters of density gradient medium in a 50 milliliter tube. Separate the cells by density gradient centrifugation and remove approximately 20 milliliters of the resulting top plasma layer. Then collect the white blood cell layer taking care to avoid the red blood cell pellet and wash the cells three times in fresh PBS.
After counting, dilute the cells to a one times 10 to the sixth peripheral blood mononuclear cell or PBMC per milliliter of PBS concentration. Add 300 microliters of cells for each control into individual 10 milliliter tubes. After topping each tube with PBS, sediment the cells by centrifugation and resuspend the cells at a one times 10 to the sixth cells per milliliter of RP5 medium concentration.
Then plate 100 microliters of cells per control to each of three wells of a 96 well plate containing 100 microliters of RP5 medium per well. Place the plate in the cell culture incubator for seven days. Next, transfer the remaining PBMCs into a new 50 milliliter tube and add one microliter of CFSE working stock solution per one milliliter of cell suspension to the side of the tube.
Quickly invert the tube several times to mix and place the cells in a cell culture incubator for five minutes. At the end of the incubation, arrest the reaction with five milliliters of RP5 medium and collect the cells by centrifugation. Resuspend the pellet at one times 10 to the sixth PBMCs per milliliter of fresh RP5 medium and add one milliliter of cells to one 10 milliliter tube per antigen to be tested.
Then add 100 microliters of cells per antigen to each of three wells of a 96 well plate well containing 100 microliters of RP5 medium supplemented with the diluted antigen of interest per well. Place the plate in the cell culture incubator for seven days. To stain the CD4+T cell populations for flow cytometric analysis, at the end of the culture transfer the entire volume of cells from each well into individual fluorescence activated cell sorting or FACS tubes.
Wash each sample with one milliliter of PBS supplemented with 0.1%fetal calf serum or FCS. Resuspend the pellets in an appropriate anti-human CD4 antibody in 100 microliters of PBS plus FCS per tube. Incubate the cells at four degrees Celsius for 20 minutes protected from light.
At the end of the incubation, wash the samples in one milliliter of fresh PBS plus FCS per tube and resuspend the pellets in 100 microliters of fresh PBS plus FCS. Immediately before the analysis, add one microliter of propidium iodide to each tube to allow exclusion of dead cells. Gate the lymphocytes according to their forward and side scatter areas.
To exclude the apoptotic cells, gate the forward scatter area versus propidium iodide negative cells and use the unstained cells to set a voltage baseline for the non-fluorescent cells. Set the voltages for the CD4 antibody fluorophore and the CFSE signal so that the fluorescent signal is below 1000 for each. Use the single color control CFSE and CD4 samples to confirm a positive fluorescent signal for each color using the voltages set with the unstained cells.
As CFSE and propidium iodide have some spectral overlap, adjust the compensation to subtract the propidium iodide fluorescence from the CFSE fluorescence until the CFSE only sample does not yield a signal in the propidium iodide channel. When all of the samples have been analyzed, calculate the cell division index by dividing the number of divided cells by 5000 undivided cells from the antigen stimulated group by the mean number of divided cells per 5000 undivided cells from the cells cultured without antigen. Almost all donors demonstrate a strong T-cell response to Tetanus toxoid after in vitro stimulation because the donors have been vaccinated making Tetanus toxoid a useful positive control antigen.
The proliferation of CD4+T cells from unstimulated PBMCs is minimal however. After seven days of stimulation with human pro-insulin C-peptide, pro-insulin C-peptide specific CD4+T cells can be detected in the peripheral blood of an individual with type one diabetes. PBMC stimulated with influenza A virus matrix protein also demonstrate proliferation.
Taken together, these data demonstrate that the assay can be performed using full length proteins as well as short synthetic peptides. The FACS component of this method can be performed using a cell sorting flow cytometer. Using a cell sorter, the antigen-specific T-cells can be isolated at the single cell level for downstream analysis.
This method has allowed for the discovery of CD4T cell responses in individuals with early onset type one diabetes. Analyzing these rare T-cell populations utilizing other methods presents various technical challenges.
