Protocol

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

Research Institute of the McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada

¹Corresponding author (nicole.bernard{at}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- 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- 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- 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- 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- and IL-2 secretion for monitoring immune response. Darrah et al. (2007) demonstrate that immune responses secreting IFN- 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

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-)

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

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.

2. 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.

3. Add 100 µL of coating buffer (dual, IL-2, and IFN-) to the appropriate wells.

4. 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 x 10⁶ cells/mL in warm (37°C) R10 medium. Aliquot a maximum 10⁷ (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 x 10⁶ 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- 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- 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- 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- 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- 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- responding cells, single IL-2 responding cells, and IFN-/IL-2 dual cytokine responding cells.

iv. Always audit the counts obtained from the software well-by-well.
See Troubleshooting.

TROUBLESHOOTING

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 <70% after thawing (Step 8).

2. Avoid cell stress, such as prolonged exposure to concentrated dimethyl sulfoxide (DMSO), insufficient medium, insufficient CO₂, or prolonged exposure to cold temperatures.

3. Increase incubation time in Step 23 and/or Step 25.

Problem: Spot quality is poor.

[Step 28]

Solution: Spot quality can be affected in several ways:

1. A spot with a white center is the result of improper washing of the plate during Step 17; cells remain bound to the membrane and create a "ghost" image during development. Make sure plates are washed vigorously.

2. Double spots or spots with a cobweb appearance result from plate displacement occurring during Step 16. Ensure plates are not moved during incubation and that the incubator is not located in an active area or near vibrating equipment (e.g., a centrifuge).

3. Spots that are too dark with a black center make compensation with the counting instrument difficult. Reduce incubation time in Step 23 and/or Step 25.

Problem: The response to background stimuli is elevated.

[Step 28]

Solution: Consider the following:

1. Avoid cell stress (see solution above for the positive control being weak or negative).

2. Put cells on a shaker during Step 9 to avoid cell clumping.

3. In wash steps using R10 and R20 media, screen lots of fetal bovine serum (FBS) to identify ones supporting low background. Some lots result in increased background and it is recommended that these be tested prior to use. If the problem persists, replace FBS with human AB serum.

4. Check reagents for contaminants.

5. Filtration may remove precipitated material that could cause background.

DISCUSSION

Immune monitoring methods have become essential tools for understanding the correlates of protective immune responses. The dual-color ELISPOT assay described in this protocol allows for the simultaneous detection of antigen-specific T cells secreting IFN- and/or IL-2. The rationale for selecting cytokines to target in a dual-color ELISPOT assay may vary based on the experimental question being posed. IFN- secretion is an antiviral T cell function that is resistant to functional exhaustion in the course of infections characterized by high persistent antigen load and it is the cytokine most frequently assessed in standard ELISPOT assays used to monitor vaccine trials (Shiver et al. 2002; Wherry et al. 2003; Harari et al. 2005; Hill et al. 2007; Kester et al. 2008). IL-2 secretion, on the other hand, is sensitive to functional exhaustion in a setting of high persistent antigen load. However, it tends to identify responses that are more likely to be multifunctional, i.e., have the ability to proliferate and effectively control viral replication (Wherry et al. 2003; Harari et al. 2004; Zimmerli et al. 2005).

Intracellular cytokine staining (ICS) is also used to characterize antigen-specific immune responses. It has the advantage of allowing for extensive phenotyping and functional analyses of the responding cells. However, ICS usually requires a greater number of PBMCs per condition tested, and it is more expensive and less amenable to high-throughput screening than the ELISPOT assay. The ELISPOT assay is at least as sensitive (if not more so) than ICS for detecting antigen-specific cells (Sun et al. 2003; Streek et al. 2009). In resource-limited settings, comprehensive screening with the dual-color ELISPOT could be used to identify specificities that could later be further phenotypically and functionally characterized by multiparametric flow cytometry.

Although both CD4+ and CD8+ T cell responses can be detected with the dual-color ELISPOT, the use of optimal peptides or overlapping 15-mers usually favors the detection of antigen-specific CD8+ T cells (Boulet et al. 2007; Ndongala et al. 2009). However, the ELISPOT assay also allows for the stimulation of T cells with complex antigens such as whole proteins, cell lysates, or whole or apoptotic cells. In the case of complex antigens, these would need to be pre-incubated with antigen-presenting cells (APC) (Schmittel et al. 2000). In this case, antigens would be processed by the APC and appropriately presented to CD4+ and CD8+ T cells, with co-stimulatory signals, according to each cell’s specific requirements. In this context, CD4+ T cells may be more effectively stimulated and, therefore, their responses more accurately detected. In addition, stimulation of T cells with APCs previously exposed to the antigen may favor the detection of antigen-specific cells that were not previously primed in vivo. If the dual-color ELISPOT protocol is adapted to utilize antigen-pulsed APCs as a stimulus, then the APC-priming procedure and incubation time with the responder cells should be optimized.

Another caveat of this method is the decreased magnitude of IFN- responses, by ~15%, in wells where both IL-2 and IFN- are captured when compared to wells coated with IFN- alone (Boulet et al. 2007). This may be a consequence of the mAb-mediated sequestration of IL-2, which is a known inducer of IFN-. Depending on the type of study undertaken, it is therefore necessary to determine whether the information gained from simultaneously detecting both cytokines outweighs the cost of a decreased IFN- magnitude.

The dual-color ELISPOT assay has been used to demonstrate the importance of the link between the predominance of different T cell functional subsets in HIV-infected individuals with different clinical outcomes or at different stages of disease (Peretz et al. 2007; Ndongala et al. 2009).

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