DualColorELISPOTAssayfortheSimultaneousDetection1

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

INTRODUCTION

The enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells inresponse 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 secretionintensity 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 ReagentProgram 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 sterileconditions.

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 amaximum 10⁷ (or 5 mL) cells per 50-mL PP conical tube