Dual-Color ELISPOT Assay for the Simultaneous Detection of IL-2 and/or IFN-γ Secreting T Cells

Authors: Salix Boulet, Michel L. Ndongala and Nicole F. Bernard1 Corresponding author ([nicole.bernard@mcgill.ca](nicole.bernard@mcgill.ca)). ### INTRODUCTION The enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells in response to ex vivo antigenic stimulation, as well as the frequency of these responding cells. This assay is highly sensitive, quantitative, easy to use, and amenable to high-throughput screening. For these reasons, the ELISPOT assay is considered by many as a gold standard for monitoring cellular immune responses. Until recently, ELISPOT assays using chromophores to detect the T cell secretion of cytokines were limited to the characterization of a single effector molecule. Notably, studies evaluating the immune response to chronic viral infections often measured IFN-γ secretion by ELISPOT because of the known antiviral effects of this cytokine as well as its correlation to the cytotoxic capacity of T cells. However, maintenance of both IFN-γ and IL-2 secretion by pathogen-specific T cells has been linked to a more favorable clinical outcome in human immunodeficiency virus (HIV) and Leishmania infections. Therefore, an ELISPOT assay able to simultaneously characterize T cell responses in terms of IL-2 and IFN-γ secretion is potentially relevant for the monitoring of immune responses to certain infectious agents. In this protocol, we describe an ELISPOT assay for the simultaneous detection of IL-2 and IFN-γ upon stimulation with viral peptides. ### RELATED INFORMATION Early uses of the ELISPOT assay for studies of immune cell secretion intensity and frequency are described in Currier et al. (2002) and Czerkinsky et al. (1988). The principles of the ELISPOT assay are reviewed in Letsch and Scheibenbogen (2003), and its use in high-throughput screening is described in Hernandez-Fuentes et al. (2003). The pathogen studies by Harari et al. (2005) illustrate the potential utility of simultaneous detection of IFN-γ and IL-2 secretion for monitoring immune response. Darrah et al. (2007) demonstrate that immune responses secreting IFN-γ and IL-2 and other cytokines play a protective role in a Leishmania major infection model. The assay presented in this protocol has also been described in Boulet et al. (2007). ### MATERIALS **Reagents** 1. AEC buffer (freshly prepared) - *Immediately before use, activate AEC buffer using H2O2 (see Step 25)*. - Anti-CD3 monoclonal antibody (mAb) (Fisher Scientific) - Blocking buffer for ELISPOT assay - CEF peptide pool (available from the NIH AIDS Research and Reagent Program and other commercial sources) (for use as control; see Step 13.iv) - Coating buffer (dual, IL-2, and IFN-γ) - Detection buffer for ELISPOT assay - Developing buffer for ELISPOT assay - Erythrosine B dye - H2O2 (30%; Sigma) - Methanol (MeOH; 90%) (American Chemicals) - Phosphate-buffered saline (PBS) (10X stock solution from Roche) - PBS-T buffer - Peptides from infectious agent (see Step 13.v) - Peripheral blood mononuclear cells (PBMCs) - R10 medium (prewarmed to 37°C) - R20 medium (prewarmed to 37°C) - RPMI medium 1640 (GIBCO) - Vector Blue solution - Wash medium for ELISPOT assay (prewarmed to 37°C) ### Equipment 1. Centrifuge with swing-out bucket rotor for 15-mL polypropylene tubes - CTL-Immunospot Analyzer (Cellular Technology Ltd.) - Filters (0.45-μM) - Hemacytometer - Incubator (humidified atmosphere, preset to 37°C and 5% CO2) - Microscope - Multichannel pipette (Rainin) - MultiScreen IP white-walled sterile plates (Millipore) - Pipette tips - Pipettes (plastic transfer) - Pipettes (serological) - Plastic wrap - Trough (plastic, 50-mL) - Tubes (polypropylene [PP], 15-mL and 50-mL) - Vacuum plate washer (VPW) (Millipore) - Waterbath preset to 37°C ### METHOD *All procedures on Days 1, 2, and 3 should be done under sterile conditions*. **Day 1: Coating Plates and Thawing Cells** ***Coating Plates (30 min)*** 1. Permeabilize the MultiScreen IP white-walled ELISPOT plate by aliquoting 50 μL of 90% MeOH from a 50-mL plastic trough to each well using a multichannel pipette. Wait ~45 sec or until all the wells in the plate appear slightly translucent. - Immediately wash the plate with 1X PBS, adding 200 μL from a 50-mL plastic trough to each well using the multichannel pipette. Repeat four times. - *The goal of washing with ~200 μL of fluid is to remove specific reagents after each step. As such, the volume used for the washes does not have to be precisely 200 μL*. - Add 100 μL of coating buffer (dual, IL-2, and IFN-γ) to the appropriate wells. - Seal plate in plastic wrap and incubate overnight at 4°C. ***Thawing Cells (30 min)*** 5.Remove frozen PBMCs from the liquid nitrogen tank and thaw in a 37°C waterbath, shaking gently until the frozen aliquot is reduced to a small ice pellet. 6.Transfer cells into a 15-mL PP tube using a plastic transfer pipette and add ~10 mL of warm (37°C) wash medium drop by drop while gently mixing the contents of the tube. Centrifuge at 1500 rpm for 7 min. 7.Discard the supernatant by decanting, and resuspend the pellet in 10 mL of warm (37°C) wash medium. Centrifuge at 1500 rpm for 7 min. 8.Repeat Step 7, but remove a 30-μL aliquot prior to centrifugation in order to count cells under the microscope. To count cells, dilute in Erythrosine B dye to check viability and use a hemacytometer. 9.After the last centrifugation, discard the supernatant and resuspend PBMCs at 2 × 10e6 cells/mL in warm (37°C) R10 medium. Aliquot a maximum 10e7 (or 5 mL) cells per 50-mL PP conical tube and incubate overnight in a humidified atmosphere incubator preset to 37°C and 5% CO2. **Day 2: Blocking Plates, Cell Plating** ***Blocking Plates (90 min)*** 10.Retrieve coated plates from the refrigerator and remove coating buffers by washing plates three times with 1X PBS, adding 200 μL of 1X PBS to each well with a multichannel pipette. *See Troubleshooting*. 11.Add 200 μL of blocking buffer and incubate for at least 1 h at room temperature. 12.Wash plates five times with 200 μL/well of 1X PBS. Leave 200 μL per well of 1X PBS until ready to proceed to Step 13. ***Cell Plating (2 h)*** 13.Remove 1X PBS from ELISPOT plate and add stimuli in RPMI medium 1640 to appropriate wells in a volume of 50 μL (or 100 μL for the medium control). During this step, do not allow wells to dry. Once done, place plate in a humidified atmosphere incubator preset to 37°C and 5% CO2. Stimuli are usually plated in triplicates and may include the following: - i. Medium control: 100 μL R10 medium with no stimulus added. No cells are added to these wells in Step 15. - *No spots should be detected in Step 28. This controls for medium quality*. - ii. Negative control: 50 μL R10 medium with no stimulus added. - *Unstimulated cells should give no or few spots in Step 28*. - iii. Positive control: Anti-CD3 mAb at a final well concentration of 0.3 μg/mL. - iv. CEF control: Most individuals respond to this pool of peptides isolated from cytomegalovirus (C), Epstein-Barr virus (E), and influenza virus (F). - v. Peptides from infectious agent: Optimal peptides (8- to 11-mers) or 15-mers are usually recommended. Pools of peptides may be used. Final concentration of the individual peptides or each peptide within a pool should be ~4 μg/mL. 14.Retrieve cells from the incubator and pool into a single 50‐mL PP tube. Add warm (37°C) wash medium to fill the tube and remove a 30-μL aliquot to count PBMCs. 15.Centrifuge cells at 1500 rpm for 7 min. Discard the supernatant and resuspend the pellet in warm (37°C) R20 medium at 4 × 10e6 cells per mL. Retrieve plate from the incubator and add 50 μL of cells to appropriate wells. - *Do not add cells to the “medium control” wells (Step 13.i)*. 16.Incubate plate for 28 h in a humidified atmosphere incubator preset to 37°C and 5% CO2. **Day 3: Detection (30 min)** 17.Wash ELISPOT plate: - i. Three times with 1X PBS, 200 μL per well. - ii. Three times with PBS-T buffer, 200 μL per well. 18.Add 100 μL per well of detection buffer. 19.Seal plate in plastic wrap and refrigerate overnight at 4°C. **Day 4: Spot Color Development (3.5 h)** 20.Wash the ELISPOT plate: - i. Three times with PBS-T buffer using 200 μL per well. Between each wash, empty wash fluid into a container. - ii. Three times with PBS-T buffer using 200 μL per well and the VPW. 21.Add 100 μL of developing buffer to all wells using a multichannel pipette and incubate at room temperature for at least 2 h. 22.Wash the ELISPOT plate: - i. Twice with 200 μL per well of PBS-T buffer, pouring off the wash buffer between each wash step. - ii. Twice with PBS-T buffer using 200 μL per well and the VPW. - iii. Three times with 200 μL per well of 1X PBS, using the VPW. - iv. Once with 200 μL per well of filtered H2O. 23.Add 100 μL per well of Vector Blue solution and incubate for ~5 min at room temperature. 24.Remove Vector Blue solution and wash the ELISPOT plates: - i. Three times with 200 μL per well of 1X PBS, pouring off the wash buffer between each wash step. - ii. Three times with 200 μL of 1X PBS, using the VPW. 25.Activate AEC buffer by adding 4.8 μL of H2O2 to 10 mL of AEC buffer and filter through a 0.45-μm filter. Add 100 μL per well of activated AEC buffer to each well and incubate up to 5-10 min. 26.Wash the ELISPOT plate: - i. Twice with 200 μL per well of filtered H2O, using the VPW. - ii. One minute under running tap H2O. Remove the bottom of the ELISPOT plate and wash both sides of the membrane. 27.Air-dry the plates. 28.Take a picture of the wells and count spots using the CTL-Immunospot Analyzer as described by the manufacturer. If a picture of the plates cannot be taken and counted on the same day, keep the plates away from light until it can be done. - *For counting spots, sensitivity and compensation are set using wells coated with either anti-IL-2 or anti-IFN-γ coating buffer (Step 3), in which cells were stimulated with one of the positive control stimuli, i.e., CEF or anti-CD3 mAb (Step 13). Wells coated with anti-IL-2 coating buffer should contain only blue spots. Wells coated with anti-IFN-γ coating buffer should contain only red spots*. - i. Set sensitivity for each color separately, such that a maximum number of spots are detected in each of the anti-IL-2 or anti-IFN-γ coated wells. - ii. Using the software’s embedded algorithm, set compensation such that no blue spots are detected in the wells coated with anti-IFN-γ coating buffer and that no red spots are detected in the wells coated with anti-IL-2 coating buffer. - *Using these settings, a spot that represents the footprint of both IFN-γ and IL-2 secretion appears purple and is detected when the color of a spot meets the threshold at the intersection of both single colors*. - iii. Count the cells in each of the three types of antigen-specific cell populations: single IFN-γ responding cells, single IL-2 responding cells, and IFN-γ/IL-2 dual cytokine responding cells. - iv. Always audit the counts obtained from the software well-by-well. *See Troubleshooting*. ### TROUBLESHOOTING 1. **Problem**: Wells leak coating buffer. **[Step 10]** - **Solution**: Treatment with MeOH in Step 1 may be too long. Make sure exposure of the wells to MeOH is kept to a minimum. If the problem persists, try less concentrated MeOH solutions or replace MeOH with ethanol. - **Problem**: The positive control is weak or negative. **[Step 28]** - **Solution**: Consider the following: - 1. Avoid using cells for which viability is