Counting Bovine IFN-γ-Secreting Cells using ELISPOT Protocols

Introduction of Counting Bovine IFN-γ-Secreting Cells

We are engaged in developing novel vaccines against bovine tuberculosis which is caused by infection with Mycobacterium bovis. In this disease, Th1 responses, and in particular IFN-γ production, are considered to be major contributors to protective immunity. Therefore, this chapter describes ELISPOT assays detecting bovine IFN-γ. An example of ELISPOT results observed in cattle after subunit vaccination with a mycobacterial antigen, delivered as DNA vaccine or as pool of synthetic peptides. The same assay principles apply to the detection of other cytokines or chemokines. However, due to the lack of bovine specific reagents, important cytokines assessed in human systems, like interleukin-2 (IL-2), cannot be probed by ELISPOT in cattle at present. Cytokine ELISPOT assays in their basic form detect effector T-cell responses since assays are initiated directly using ex vivo peripheral blood mononuclear cells (PBMCs). However, assessment of memory responses is also a vital parameter to be assessed following vaccination. This can also be addressed using a modification of the ex vivo ELISPOT, the so-called cultured ELISPOT system. The principle of the cultured ELISPOT is that the IFN-γ ELISPOT is not performed on ex vivo PBMC, but on cells that have been expanded and differentiated in vivo for around 2 weeks by initial stimulation with antigen and then regular feeds of IL-2. The majority of effector cells that are initially stimulated by this approach die of IFN-γ -induced cell apoptosis during this culture and expansion step, whereas memory cells differentiate into secondary effector cells that can be enumerated at the end of this culture step by IFN-γ ELISPOT. This assay was first applied in the human system and applied to diseases, such as malaria. A study by Godkin et al. has demonstrated that the CD4+ T-cell memory population probed with this assays consisted mainly of central memory cells expressing the chemokine receptor CCR7. We have adapted this system for use in cattle, and its protocol is also described in this chapter. Interestingly, in our hands, cultured ELISPOT responses were a direct predictor of vaccine efficacy against M. bovis challenge in cattle.

Figure 1. Flow chart of ex vivo and cultured ELISPOT assay.

Methods of Counting Bovine IFN-γ-Secreting Cells

ELISPOT Plate Preparation

1. Coat ELISPOT plates with 100 μl/well capture antibody (bIFNγ-1, at 7.5μg/mL) diluted in coating buffer. Wrap plates in cling film and store in refrigerator; plates can be kept up to 3 days in refrigerator until use (see Note 7).

Ex Vivo ELISPOT Preparation

Note: Steps 1–4 need to be performed in class 2 biosafety cabinet.

1. Discard coating antibody solution and wash plates twice with 200 μl/well RPMI-1640 using a multi-channel pipette to add wash fluid. Flick off fluid between and after washes. Then, block wells for 45–60 min with 200 μl/well of TCM at 37°C.
2. Flick off blocking solution and add 100 μl/well antigens in duplicate or triplicate diluted in TCM according to requirements of experiment (see Note 2).
3. Add 100 μl/well of PBMC suspended in TCM at 1–4 × 10⁶ /mL. Incubate for 20–24 h at 37°C and 5% CO₂. Ensure that plates are level so that cells are evenly distributed (see Note 8).
4. After this incubation period, shake plates on plate shaker (400– 500 rpm, 5–10 s) and flick off cells into container in a biosafety class 2 cabinet. Wash plates twice with H₂O_dd and then three times with PBST by adding 200 μl/well wash fluids using multi-channel pipette. Place on shaker each time for 10 s before flicking off wash fluid. Remove as much wash fluid as possible by vigorously tapping plate on paper towels.
5. Add 100 μl/well detection mAb (PAN-biotin, 0.25 μg/mL, diluted in PBST–BSA) and incubate for 2 h at room temperature (above 20°C).
6. Wash three to four times with PBST as in step 4. Remove as much wash fluid as possible by tapping plate on paper towels.
7. Add 100 μl/well streptavidin–peroxidase conjugate (diluted 1:100 in PBST/BSA) and incubate for 1 h at room temperature.
8. Wash plates six times with PBST and then add 100 μl/well AEC substrate prepared as per kit instructions (see Note 5).
9. Once spots have developed (ca. 10 min, but this should be closely monitored to avoid over-development), flick off substrate and wash with copious amounts of tap water (under tap will do), remove plastic cover, and wash back of wells. Allow plates to dry either in air or using a drying oven set at 30°C.
10. Keep plates in dark before counting using an automated ELISPOT reader. Plates can also be read manually using a dissecting microscope.
11. Result presentation: Conventionally, responses are expressed as mean spot-forming cell (SFC)/106 cells of duplicate or triplicate wells.

Cultured ELISPOT

1. PBMCs are stimulated in 24-well plates (2 × 10⁶ PBMC/mL, 1-mL aliquot in TCM) with antigens (e.g. recombinant proteins at 2–10 Mg/mL, see Note 2). PBMC should preferentially be freshly prepared, but can also be prepared from cryo-preserved stocks. Incubate in 37°C humidified CO₂ incubator. Set up at least two wells per cell line (see Note 9).
2. PBMC cultures to be fed on days 3 and 7 with recombinant human IL-2 to a final concentration of 10 U/mL in the following way; on day 3, add 0.5 mL of IL-2 (30 U/mL in TCM) directly to each 1 mL culture; on day 7, carefully remove and discard 0.5 mL of culture supernatant without disturbing cell layer at bottom of wells and replace with 0.5 mL of IL-2 (30 U/mL in TCM) (see Note 10).
3. On day 10, carefully remove half of the supernatant and replace with equal volume of TCM without IL-2. Do not disturb cell layer.
4. On day 12, remove 1–1.25 mL medium from each well, and replace with same volume of fresh TCM without IL-2.
5. The day before the assay (day 12), prepare ELISPOT plates by coating them overnight at 4°C with a bovine IFN-γ-specific mAb ELISPOT.
6. On the day of assay (day 13), prepare fresh PBMC to be used as antigen-presenting cells (APCs) and re-suspend PBMC at 2–4 × 10⁶ /mL in TCM.
7. To the pre-blocked ELISPOT plates, add APC (2 × 10⁵ PBMC/well) to required wells (see Note 11).
8. Incubate for 90–120 min at 37°C.
9. In the meantime, warm up TCM aliquot to 37°C.
10. After the 90–120-min incubation step (step 8), shake up plates vigorously (plate shaker) and flick off cells into container in cabinet.
11. Wash plates with warm TCM by addition of 200 μl TCM/ well, shake plates on orbital plate shaker (100–200 rpm, 10 s), and discard medium in container in cabinet. Repeat this step twice more.
12. After last wash, add antigen solutions in TCM in 100 μl/well in duplicate or triplicate and return plates to 37°C humidified CO₂ incubator. Add 100 μl TCM to "no antigen" control wells and add mitogen solution to positive control wells. Antigens can be recombinant proteins (assay concentrations 2–5 μg/mL) or synthetic peptides (assay concentrations 2–10 μg/mL) (see Note 2); positive control can be SEB (1–2 μg/mL) or other mitogens (see Note 3).
13. Pool cells from 24-well T-cell line culture plates and wash cultured cells four times with HBSS solution by centrifugation (300 × g, room temperature), count viable cells, and re-suspend at required cell titres (see next step).
14. Add between 5 × 10³ and 2 × 10⁴ cells to wells of ELISPOT plates containing the antigen solutions and APCs (see Note 11).
15. After 24 h of incubation in the presence of cells, develop spots as described for ex vivo ELISPOT.
16. Tabulation of results and interpretation criteria: There are a number of ways to tabulate the results. We advise to normalize results obtained (SFC/well) to the number of input cells by using the following formula: SFC/10⁶ input cells = [S × (N/E)]/IR (see Note 12).
    Input cell ratio (IR): Number of input cells divided by 10⁶ ; N: output cell number; E: number of cells/well during ELISPOT; S: SFC/well.

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