Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells

Lindenstrøm, Thomas; Andersen, Peter; Agger, Else Marie

doi:10.1007/978-1-60761-585-9_15

Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells

Thomas Lindenstrøm²,
Peter Andersen² &
Else Marie Agger²

Protocol
First Online: 28 December 2009

2908 Accesses
2 Citations

Part of the book series: Methods in Molecular Biology ((MIMB,volume 626))

Abstract

Adjuvants constitute a critical component in vaccine development in terms of both stimulating and directing immune responses of a suitable profile to promote protection against a diverse range of disease targets. In the past, the field of adjuvant research was mainly dominated by empirical testing and serendipity. However, there is a strong need to develop new generations of adjuvants based on rational design, as well as a requirement to characterise and comprehend their mechanism(s) of action. Adjuvant development can be characterised as an iterative process where potential candidates are repeatedly tested in vitro and in vivo for immunogenicity and optimised in terms of formulation and delivery. Novel lead candidates of adjuvants with a suitable immunological profile relative to specific disease targets are subsequently selected and evaluated in efficacy studies. A central aspect in such a development and selection process is to determine the adjuvant activity on T-cell function in vivo. Expanding our knowledge on these mechanisms will improve our chance of developing new successful vaccines designed to target specific diseases.

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1 Introduction

Innate immunity has been revitalised since the discovery of the Toll-like receptors more than a decade ago (1, 2). The ensuing years of research have thus greatly improved our understanding of how the innate immune system perceives pathogens and tissue injury/stressors through pattern recognition receptors (PRRs) (3). These basic findings have clearly invigorated the field of adjuvant research, not only in terms of developing new adjuvants but also in understanding their mode of action (4–9). It is becoming increasingly clear that innate immunity not only initiates adaptive responses but also plays a central role for shaping and modulating the subsequent adaptive responses by eliciting cytokines that influence the polarisation of T-helper cells into Th1, Th2 and Th17 T-cell subsets (10, 11) (Fig . 15.1). In vitro assays can be employed to determine adjuvant activity on T cells. This can be tremendously helpful in comprehending their mode of action and additionally gives clues to how they might promote differentiation and polarisation of T-helper cells. Still, such studies do not take into account the in vivo complexity or consider factors such as those related to route of administration and immunisation schedules. For characterising adjuvant activity on T-cell composition/polarisation, magnitude and quality of the induced T-cell responses as well as the ability for memory formation, in vivo assays thus remain one of the ultimate read-outs. The current section describes a number of techniques that will guide in characterising these aspects of T-cell function following vaccination with adjuvanted vaccines. The following part will thus describe the major steps involved in characterising the adjuvant activity on T-cell function in vivo from the initial isolation of cells, subsequent recall stimulation of vaccine-induced T cells to a thorough characterisation of the response in terms of T-cell polarisation by cytokine ELISAs, cytometric bead analysis (CBA) for multiplex analysis of secreted cytokines and for assessing the frequency of antigen-specific T cells by ELISPOT and intracellular flow cytometry assays. Finally, functional aspects of the response will be dealt with by assays measuring the proliferative potential of the vaccine-induced response by the CFSE dilution method and for the quality of the response in terms of measuring the degree of T-cell multifunctionality (capacity of cytokine co-expression).

2 Materials

2.1 Isolation of PBMCs

1.
0.9% NaCl; sterile.
2.
Lympholyte (Cedarlane CL5120), sterile.
3.
RPMI medium: RPMI-1640 supplemented with 5 × 10^–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco).
4.
Foetal calf serum (FCS) (Biochrome AG).

2.2 Isolation of Cells from Spleen and Lymph Nodes

1.
Cell strainer (BD Biosciences) or sterile metal grid.
2.
RPMI medium: RPMI-1640 supplemented with 5 × 10^–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco).
3.
Foetal calf serum (FCS) (Biochrome AG).

2.3 Cell Culture and Antigen Recall

1.
U-bottom 96-well tissue culture-treated microtitre plates (NUNC).
2.
RPMI medium: RPMI-1640 supplemented with 5 × 10^–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco).
3.
Foetal calf serum (FCS) (Biochrome AG).
4.
Relevant recall antigens (proteins, peptides).

2.4 Conventional Capture-ELISAs—A: IFN-γ ELISA; B: IL-17A ELISA

1.
F-bottom 96-well Maxisorp microtitre plates (NUNC).
2.
Coating buffer (carbonate buffer): 0.03 M Na₂CO₃, 0.056 M NaHCO₃ pH 9.6 in milliQ-water + phenol red 1 mL/L.
3.
Capture antibody (see Note 1)
1. (a)
  For IFN-γ ELISA: purified rat anti-mouse IFN-γ (clone R4-6A2; BD Pharmingen) diluted in coating buffer to a final concentration of 1 μg/mL.
2. (b)
  For IL-17A ELISA: purified rat anti-mouse IL-17A (clone: TC11-18H10.1; BioLegend) diluted in coating buffer to a final concentration of 1 μg/mL.
4.
Blocking buffer: 2% skimmed milk powder in PBS.
5.
Wash buffer: 0.2% Tween20 in PBS.
6.
Assay buffer: 2 or 1% bovine serum albumin (BSA) in PBS.
7.
ELISA standards
1. (a)
  For IFN-γ ELISA: IFN-γ standard (BD Pharmingen).
2. (b)
  For IL-17A ELISA: IL-17A recombinant standard (BioLegend).
8.
FCS (Biochrome AG).
9.
Biotinylated detection antibody (see Note 1).
1. (a)
  Biotinylated rat anti-mouse IFN-γ (clone XMG1.2, BD Pharmingen) diluted in 1% assay buffer to a final concentration of 0.1 μg/mL.
2. (b)
  Biotinylated rat anti-mouse IL-17A (clone: TC11-8H4; BioLegend) diluted in 1% assay buffer to a final concentration of 0.25 μg/mL.
10.
Horseradish peroxidase-conjugated streptavidin (SA-HRP) (Zymed) diluted in 1% assay buffer to a final concentration of 0.25 μg/mL.
11.
Substrate: TMB Plus ready-to-use substrate (Kem-En-Tec).
12.
Stop solution: 0.2 M H₂SO₄.

2.5 Cytometric Bead Analysis—Th1/Th2 Cytokine CBA

1.
Mouse Th1/Th2 Cytokine CBA Kit (BD Biosciences) (see Note 2) containing
1. (a)
  Recombinant mouse Th1/Th2 cytokine standards.
2. (b)
  Assay diluent.
3. (c)
  Capture Beads A1–A5 (A1: IL-2; A2: IL-4; A3: IL-5; A4: IFN-γ; A5: TNF-α).
4. (d)
  PE detection reagent.
5. (e)
  Wash buffer.
6. (f)
  Cytometer Setup Beads.
7. (g)
  FITC and PE positive control detector.

2.6 ELISPOT—A: IFN-γ ELISPOT; B: IL-5 ELISPOT

1.
MAHA S45 10 (cellulose ester) ELISPOT plates (Millipore).
2.
Sterile PBS.
3.
Capture antibody (see Note 1)
1. (a)
  For IFN-γ ELISPOT: rat anti-mouse IFN-γ (clone R4-6A2; BD Pharmingen) diluted in sterile PBS to a final concentration of 4 μg/mL.
2. (b)
  For IL-5 ELISPOT: rat anti-mouse/human IL-5 (clone TRFK5; BD Pharmingen) diluted in sterile PBS to a final concentration of 2 μg/mL.
4.
RPMI medium: RPMI-1640 supplemented with 5 × 10-5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco).
5.
Foetal calf serum (FCS) (Biochrome AG).
6.
Blocking buffer: 10% FCS in RPMI medium.
7.
Wash buffer: 0.05% Tween20 in PBS.
8.
Biotinylated detection antibody (see Note 1)
1. (a)
  For IFN-γ ELISPOT: biotinylated rat anti-mouse IFN-γ (clone XMG1.2, BD Pharmingen) diluted in PBS to a final concentration of 1.25 μg/mL.
2. (b)
  For IL-5 ELISPOT: biotinylated rat anti-mouse IL-5 (clone TRFK4; BD Pharmingen) diluted in PBS to a final concentration of 1 μg/mL.
9.
Alkaline phosphatase-conjugated streptavidin (SA-AP) (Jackson ImmunoResearch) diluted in PBS to a final concentration
1. (a)
  For IFN-γ ELISPOT: SA-AP 0.5 μg/mL.
2. (b)
  For IL-5 ELISPOT: SA-AP 2 μg/mL.
10.
Substrate: BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; Sigma-Aldrich).

2.7 Proliferation: CFSE Dilution Assay

1.
U-bottom 96-well tissue culture-treated microtitre plates (NUNC).
2.
RPMI medium: RPMI-1640 supplemented with 5 × 10⁻⁵ M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco).
3.
Foetal calf serum (FCS) (Biochrome AG).
4.
Relevant recall antigens (proteins, peptides).
5.
Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE/CFSE; Molecular Probes) diluted to a stock of 20 mM in 100% DMSO. Aliquot and keep in the dark (–20°C).
6.
FACS buffer: PBS + 1% FCS. For longer term storage at 4°C supplement with 0.05–0.1% NaN₃.

2.8 Intracellular FACS Staining for Multifunctional T Cells

1.
ThermostatPlus heatblock (Eppendorf).
2.
V-bottom 96-well cell culture-treated microtitre plates (Corning).
3.
RPMI medium: RPMI-1640 supplemented with 5 × 10^–5 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES (all Gibco).
4.
Foetal calf serum (FCS) (Biochrome AG).
5.
Co-stimulatory antibodies: anti-murine CD28 (clone 37.51) and anti-murine CD49d (clone 9C10(MFR4.B)) (both BD Pharmingen) (see Note 3).
6.
Brefeldin A 5 mg/mL in EtOH (Sigma-Aldrich).
7.
Monensin/“GolgiStop” (BD Pharmingen).
8.
FACS buffer: PBS + 1% FCS.
9.
Conjugated antibodies for surface markers and cytokines: anti-CD4:APC-Cy7 (clone GK1.5), anti-CD8:PerCp-Cy5.5 (clone 53-6.7), anti-CD44:FITC (clone IM7), anti-IFN-γ:PE-Cy7 (clone XMG1.2), anti-TNF-α: PE (MP6-XT22) and anti-IL-2:APC (clone JES6-5h4) (all BD Pharmingen, except anti- IFN-γ:PE-Cy7: eBiosciences).
10.
Fluorophore-conjugated anti-CD4 (clone GK1.5) or anti-CD8 (clone 53-6.7) for compensation (conjugates: FITC, PE, PerCp-Cy5.5; PE-Cy7, APC, APC-Cy7)—alternatively use compensation beads (e.g. CompBead; BD Pharmingen).
11.
Cytofix/Cytoperm kit (BD Pharmingen).

3 Methods

3.1 Isolation of PBMCs

1.
The lympholyte solution has to be at room temperature (rt) before usage. Remember to shake well. Work aseptically. Add 5 mL lympholyte solution to a 15 mL Falcon tube.
2.
Dilute the blood from an immunized animal (approximately 500–750 μL) with an equal volume of 0.9% NaCl before adding to the tube containing lympholyte. Carefully layer the diluted blood over the lympholyte solution.
3.
Centrifuge 20 min at 800 × g without brake.
4.
Carefully harvest the cells at the interface layer with a Pasteur pipette and transfer to a new centrifuge tube. Fill it up with RPMI medium and pellet the cells by centrifugation at 800 × g for 5 min.
5.
Remove the supernatant, and wash the cells one more time in RPMI medium. Remember to resuspend the cells prior to washing (see Note 4).
6.
After the last spin, resuspend the cells in 1 mL RPMI medium with 10% FCS. Count cells and adjust to the desired concentration.

3.2 Isolation of Cells from Spleen and Lymph Nodes

1.
Work aseptically. Grind the spleen/lymph node through a cell strainer or metal grid to obtain a single cell suspension. The cells are washed twice in RPMI medium as above (see Note 4) and resuspended in RPMI medium with 10% FCS. Count cells and adjust to appropriate cell concentration.

3.3 Cell Culture and Antigen Recall

1.
Use sterile reagents and work aseptically. Dilute the antigens for stimulation in RPMI medium + 10% FCS at the double concentration relative to the desired final concentration (final concentration in the range of 0.2–5 μg/mL; see Note 5). Add antigens at 100 μL/well, though avoid using the outermost wells. Remember to include negative (RPMI medium + 10% FCS) and positive (Con A 5 μg/mL) control wells. Fill outermost wells with 200 μL PBS to avoid evaporation during incubation (see Note 6).
2.
Cell suspensions, e.g. PBMCs, spleen or lymph node cells, are adjusted to 2 × 10⁶ cells/mL in RPMI medium + 10% FCS and added at 100 μL/well, leaving a total of 200 μL/well (2 × 10⁵ cells/well).
3.
Incubate the plates (covered with lid) in an incubator at 37°C; 5% CO₂ for 72 h (see Note 7) and subsequently collect supernatants (150–175 μL/well), for cytokine-specific ELISAs and/or CBA. If not used immediately, store at –20°C.

3.4 Conventional Capture-ELISAs—A: IFN-γ; B: IL-17A

1.
Coat plates with 100 μL/well of capture antibody diluted in carbonate buffer (final concentration 1 μg/mL). Incubate plates overnight at 4°C.
1. (a)
  For IFN-γ ELISAs, use anti-mouse IFN-γ (clone R4-6A2) as capture antibody.
2. (b)
  For IL-17A ELISAs, use anti-mouse IL-17A (clone: TC11-18H10.1) as capture antibody.
2.
The following day, discard the coating buffer and add 200 μL/well of 2% milk powder in PBS. Incubate for 1.5 h at room temperature. Subsequently wash plates 3x in wash buffer.
3.
Standards and samples: If necessary dilute samples in 2% assay buffer (see Note 8) and add 100 μL/well. Samples should be run in duplicate or triplicate.
1. (a)
  For IFN-γ ELISA: Dilute the IFN-γ standard to a top-standard of 5,000 pg/mL. Perform six 2-fold serial dilutions in PBS + 10% FCS (5,000, 2,500, 1,250, 625, 312, 156, 78 pg/mL + blank). Each standard is added at the volume of 50 μL to each well. Subsequently add 50 μL of 2% assay buffer to the standard wells, leaving a total volume of 100 μL/well. Each plate should contain a column of standard concentrations.
2. (b)
  For IL-17A ELISA: Dilute the IL-17A standard to a top-standard of 4,000 pg/mL. Perform six 2-fold serial dilutions in PBS + 10% FCS (4,000, 2,000, 1,000, 500, 250, 125, 62.5 pg/mL + blank). Each standard is added at the volume of 50 μL to each well. Subsequently add 50 μL of 2% assay buffer to the standard wells, leaving a total volume of 100 μL/well. Each plate should contain a column of standard concentrations.
4.
Incubate plates for 2 h at rt. Wash plates 3x in wash buffer.
5.
Detection step: Incubate with biotinylated detection antibody for 1 h at rt. Subsequently wash plates 3x in wash buffer
1. (a)
  For IFN-γ ELISA: Add 100 μL/well of biotin-conjugated anti-mouse IFN-γ (0.1 μg/mL).
2. (b)
  For IL-17A ELISA: Add 100 μL/well of biotin-conjugated anti-mouse IL-17A (0.25 μg/mL).
6.
Following the washing steps, add 100 μL/well HRP-Streptavidin (0.25 μg/mL) and incubate for 30 min at rt. Wash plates 5x in wash buffer.
7.
Develop plates by adding 100 μL/well of TMB substrate. Incubate in the dark for 5–15 min at rt.
8.
Stop reaction with 100 μL/well 0.2 M H₂SO₄. Read absorbance at 450 nm (see Note 9).
9.
Make a standard curve (log OD(450 nm) against log cytokine concentration (pg/mL)) and determine the unknown cytokine concentrations. Remember dilution factors if samples were diluted and take into account the 1:2 dilutions of standards (3A and 3B).

3.5 Cytometric Bead Analysis—Th1/Th2

1.
Reconstitute a vial of the lyophilised recombinant cytokine standards in 0.2 mL of Assay Diluent (bulk standard (10x); 50 ng/mL).
2.
Dilute the cytokine standards 1:10 by mixing 25 μL bulk standard (10x) with 225 μL Assay Diluent. Perform eight 2-fold serial dilutions in Assay Diluent (75 + 75 μL Assay Diluent; hence 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256).
3.
Determine the total number of samples, including standards (nine dilutions) and blank (Assay Diluent). Make the Capture Bead mix: Vortex the Capture Bead suspensions and aliquot 2.4 μL (see Note 10) of each Capture Bead for each assay tube to be analysed into a single tube (“Mixed Beads”) and vortex vigorously.
4.
If necessary, dilute samples in Assay Diluent (standard range 20–5,000 pg/mL) (see Note 8). Add 10 μL standard/blank/unknown samples to each well.
5.
Vortex the “Mixed Beads” and add 10 μL to each of the wells. Finally, add 10 μL PE detection reagent to each well.
6.
Briefly spin plate to collect reagents and samples at the well bottom and then carefully resuspend using a multipipette to ensure mixing.
7.
Incubate plate in the dark for 2 h at rt.
8.
After incubation, add 140 μL wash buffer to each well and centrifuge the plate 5 min at 300 × g. Carefully aspirate and discard the supernatant, avoid touching the bead pellet.
9.
Resuspend in 150 μL wash buffer and transfer to cluster tubes.
10.
Before acquiring samples, prepare the Cytometer Setup Beads: Vortex Setup Beads and add 50 μL to three separate tubes (A, B and C). Add 50 μL of FITC Positive Control Detector to tube B and 50 μL of PE Positive Control Detector to tube C. Mix well.
11.
Incubate tubes (A–C) in the dark for 30 min. Add 450 μL wash buffer to tube A and 400 μL to tube B and C.
12.
Analyse samples on a flow cytometer the same day as performing the staining procedure. Set up instrument using the Setup Beads for adjusting the PMT levels and carry out compensation. Collect at least 1,800 events/beads per sample.
13.
Make standard curves for each cytokine and determine the cytokine concentrations for the unknown samples based on the fluorescent intensity value of each circulating analyte relative to their standards. Remember any dilutions factors.

3.6 ELISPOT—A: IFN-γ; B: IL-5

1.
Use sterile reagents and work aseptically. Coat ELISPOT plates with 50 μL/well of capture antibody diluted in PBS. Cover with tinfoil and incubate overnight at rt
1. (a)
  For IFN-γ ELISPOTs, use anti-mouse IFN-γ (clone R4-6A2) as capture antibody (4 μg/mL).
2. (b)
  For IL-5 ELISPOTs, use anti-mouse IL-5 (clone: TRFK5) as capture antibody (2 μg/mL).
2.
The following day, discard the capture antibody and wash plates 5x with sterile PBS (100 μL/well). Subsequently block plates in blocking buffer (RPMI medium + 10% FCS) 100 μL/well for 1.5 h at 37°C in a CO₂ incubator.
3.
Dilute the antigens for stimulation in RPMI medium + 10% FCS at the double concentration relative to the desired final concentration (final concentration in the range of 0.2–5 μg/mL; see Note 5). Remove the blocking solution by flicking the plates and add the diluted antigens at a volume of 100 μL/well. Include negative controls (RPMI medium + 10% FCS).
4.
Cell suspensions, e.g. PBMCs, spleen or lymph node cells, are adjusted to 2 × 10⁶ cells/mL (see Note 11) and added 100 μL/well, leaving a total volume of 200 μL/well (2 × 10⁵ cells/well).
5.
Incubate the plates (covered with lid) in an incubator at 37°C; 5% CO₂ for 48 h (see Note 12).
6.
From this point, aseptic working conditions are no longer needed. Wash the plates thoroughly 5x with Wash Buffer (0.05% Tween20 in PBS).
7.
Incubate with biotinylated detection antibody for 2 h at rt. Subsequently flick plates and wash 5x with wash buffer.
1. (a)
  For IFN-γ ELISPOT: Add 100 μL/well of biotin-conjugated anti-mouse IFN-γ (final concentration 1.25 μg/mL).
2. (b)
  For IL-5 ELISPOT: Add 100 μL/well of biotin-conjugated anti-mouse IL-5 (final concentration 1 μg/mL).
8.
Following the washing steps, add 100 μL/well AP-Streptavidin
1. (a)
  For IFN-γ ELISPOT: SA-AP 0.5 μg/mL; incubate for 45 min at rt.
2. (b)
  For IL-5 ELISPOT: SA-AP 2 μg/mL; incubate for 1 h 15 min at rt.
9.
Flick plates and wash 5x in wash buffer. Carefully disassemble the bottom plate and rinse under running water. Reassemble the plate, taking care not to destroy the cellulose-ester-bottomed wells.
10.
Dissolve BCIP/NBT substrate tablets in double-distilled dH₂O, filter through a 0.2 μm filter and develop plates in the dark for 10–20 min.
11.
Stop the reaction by washing plates under running water.
12.
Allow plates to dry at rt and keep in the dark until reading the plates.
13.
Enumerate spots using an ELISPOT reader or manually using a stereomicroscope. Calculate the frequency of spot forming units (SFU) by expressing the number of SFUs per 10⁶ cells.

3.7 Proliferation: CFSE Dilution Assay

1.
Use sterile reagents and work aseptically. Resuspend cells, e.g. PBMCs, lymph node or splenic cells in 1 mL RPMI medium without FCS (max. cell concentration: 2×10⁷ cells/mL).
2.
Predilute the CFSE stock (20 mM) 1:100 in RPMI medium without FCS. Add 25 μL/mL cells to a final concentration of 5 μM CFSE (see Note 13). Mix well to ensure uniform staining and incubate in the dark for 10 min at 37°C.
3.
Stop staining and bind surplus CFSE by adding 5 volumes of ice-cold RPMI medium + 10% FCS and put on ice for 5 min.
4.
Spin cells at 800 × g for 3 min at 4°C and repeat washing three times in ice-cold RPMI medium + 10% FCS. Finally resuspend in RPMI medium + 10% FCS at a cell concentration of 2 × 10⁶ cells/mL and stimulate with appropriate antigen(s), including positive and negative controls, as described in Section 3.3. Incubate at 37°C; 5% CO₂ (2 × 10⁵ cells/well) for 3–5 days (see Note 13).
5.
Harvest cells and transfer to cluster tubes. Set up instrument and acquire samples—if cell are stained for surface markers, remember to compensate (see Note 14). Refer to Fig . 15.2 for data analysis and calculate the percentage of original cells dividing, the precursor frequency and the proliferative index.

3.8 Intracellular FACS Staining for Multifunctional T Cells

1.
Adjust cell suspensions, e.g. PBMCs, spleen or lymph node cells, to 1–2 × 10⁷ cells/mL and add 100 μL/well. Add cells to 7–8 extra wells for compensation purposes (6 wells) and for unstained controls (1–2 wells)—otherwise use compensation beads.
2.
Dilute the antigens for stimulation in RPMI medium + 10% FCS at the double concentration relative to the desired final concentration (final concentration in the range of 1–5 μg/mL; see Note 5) and add at a volume of 100 μL/well leaving a total volume of 200 μL/well. Likewise add 100 μL/well of RPMI medium + 10% FCS to unstimulated controls as well as unstained/compensation controls.
3.
Add/well anti-CD28 and anti-CD49d antibodies (final concentration each 1 μg/mL).
4.
Incubate plate 1 h at 37°C in a CO₂-incubator (see Note 15).
5.
Dilute BFA (5 mg/mL) 25x in RPMI medium + 10% FCS to a concentration of 200 μg/mL. Add 10 μL/well media containing Brefeldin A (final concentration 10 μg/mL) and 0.7 μL/well of Monensin/GolgiStop. Mix well using a multichannel pipette.
6.
Incubate for 5–6 h in the ThermostatPlus heatblock (see Note 16). Programme it to cool down to 4°C after the 5–6 h incubation at 37°C. Alternatively, incubate at 37°C in a CO₂-incubator for 5–6 h and subsequently transfer the plate to 4°C for the next day.
7.
Spin plate at 1,000 × g for 3 min at 4°C. Discard the supernatant.
8.
Wash plate 1x in 200 μL FACS buffer. Spin plate at 1,000 × g for 3 min at 4°C. Discard the supernatant.
9.
Stain cells for surface markers in 50 μL/well. Calculate the volume needed (add extra for pipetting errors) and make a surface stain mix by diluting antibodies for surface markers (CD4:APC-Cy7; CD8:PerCp-Cy5.5 and CD44:FITC) 1:200 in FACS buffer (final concentration of each antibody: 1 μg/mL). Vortex surface stain mix and add 50 μL/well. Do not add surface stain mix to compensation wells. Add 50 μL/well single colour antibodies (anti-CD4 or anti-CD8) at 1 μg/mL for each fluorescence channel (FITC, PE, PerCp-Cy5.5, PE-Cy7, APC and APC-Cy7: 6 wells) and FACS buffer for the negative unstained control. Incubate in the cold and dark for 30 min.
10.
Add 150 μL FACS buffer to each well. Spin at 1,000 × g for 3 min at 4°C. Discard the supernatant. Repeat the wash in FACS buffer 200 μL/well. Spin plate at 1,000 × g for 3 min at 4°C. Discard the supernatant.
11.
Fix cells by adding 100 μL Cytofix/Cytoperm/well. Mix by pipetting. Incubate in the cold and dark for 30 min.
12.
Spin plate at 1,000 × g for 3 min at 4°C. Discard the supernatant. Wash plates with Perm Wash Buffer, 200 μL/well. Spin at 1,000 × g for 3 min at 4°C. Discard the supernatant.
13.
Intracellular stain; 50 μL/well: Make an intracellular stain mix by diluting antibodies for cytokines (IFN-γ-PE:Cy7; TNF-α:PE; IL-2:APC) 1:200 in Perm wash buffer (final concentration of each antibody: 1 μg/mL). Vortex and add 50 μL/well, except to compensation wells. Add Perm wash buffer 50 μL to each of these. Incubate the plate for 30 min in the cold and dark.
14.
Add 150 μL Perm wash buffer to each well. Spin plate at 1,000 × g for 3 min at 4°C. Discard the supernatant. Wash plates with Perm wash buffer, 200 μL/well. Let the plate stand for 2–3 min, then spin plate at 1,000 × g for 3 min at 4°C. Discard the supernatant.
15.
Finally resuspend cells in 200 μL FACS buffer and transfer to cluster tubes.
16.
Set up instrument by adjusting the PMT levels and perform compensation using either unstained and single stain tubes or compensation beads.
17.
When running samples: Acquire as many cells as possible—preferentially no less than 200,000 events within the lymphocyte gate. Gate as in Fig . 15.3 by making IFN-γ, TNF-α and IL-2 +ve gates within the CD4 and CD8 T cells followed by creating Boolean combination gates to asses the frequency of each cytokine co-expressing subset and their share of the total responding T-cell population.

4 Notes

1.
We have found these capture–detection antibody pairs to work excellently for ELISAs and ELISPOT assays. Numerous competitive reagents are available from other commercial sources.
2.
Cytometric Bead Analysis is a multiplexing assay in which a series of soluble analytes are detected and quantified simultaneously in a single tube by virtue of catching particles with discrete fluorescent intensities. Numerous competitive reagents are available from commercial sources with slightly different setups—e.g. Bio-Plex (23-plex, Bio-Rad), Flowcytomix (10-plex; Bender MedSystems) and CBA (BD Biosciences). Here we describe a fixed Th1/Th2-cytokine CBA—but in most cases, the “Flex Set” can be customised for specific needs.
3.
The employment of anti-CD28 and anti-CD49d co-stimulation is an optional step. It was originally introduced for optimising intracellular staining techniques (14, 15). It increases Ag-specific responses thereby revealing the full functional Ag-specific response and thus increases the sensitivity resulting in better signal–noise ratios. In our experiences it results in a 2- to 4-fold enhancement in the frequency of cytokine-secreting CD4⁺ T cells following Ag-specific stimulation without affecting background levels. However, controls are essential to ensure that background levels/non-specific staining do not increase.
4.
Resuspend cells thoroughly by flicking tubes over a rack.
5.
Antigen concentrations for stimulation need to be established according to the specific experimental settings. In most cases, a final concentration in the range of 0.2–5 μg/mL can be employed.
6.
The usage of outermost wells is avoided, and these are filled with 200 μL PBS to prevent evaporation of supernatant from sample wells. Otherwise, this could impact the concentration of cytokines to be subsequently measured.
7.
Incubation times can vary according to the specific experimental settings. In most cases, 48–72 h incubation will be adequate.
8.
Experimental samples need to be diluted to be within the linear range of O.D. values obtained by the cytokine standards.
9.
If microplate reader can perform wavelength correction, set to subtract readings at 540 or 570 nm from the readings at 450 nm. This will correct for optical imperfections in the plate(s).
10.
The supplier recommends the use of 10 μL of each Capture Bead for each assay tube. In our experience this can be down-scaled 1:5 without compromising read-outs. The use of 2.4 μL is suggested to ensure adequate volume for pipetting errors.
11.
Cell concentrations for ELISPOT assays should be adjusted according to the specific experimental settings to ensure clear resolution of spots. Initially, a titration experiment should be performed.
12.
Incubation times should be adjusted according to the specific experimental settings; in most cases 24–48 h would be ideal. During incubation, it is imperative to avoid any disturbances of the sedimented cells to obtain distinct and well-demarcated spots.
13.
The concentrations of CFSE to be used depend on the duration of analysis/stimulation. For experiments examining division up to a week (<7 days), use 5–10 μM for staining 2×10⁷ cells/mL or less. Duration of stimulation varies, but in most cases, 3–7 days would be a good starting point.
14.
CFSE is detected in the Fl-1 channel (FITC). There is a substantial overlap of fluorescein emission into the PE-channel, especially when CFSE staining is bright. This can make compensation of CFSE challenging and indeed very tricky. Although it can be done, avoid using the PE-channel, if possible. Make sure to have an unstimulated control for determining the undivided peak and to compensate for CFSE-bleeding into adjacent channels. The unstimulated control must be incubated for the same duration as the stimulated cells. Also include non-CFSE-labelled cells to determine the degree of autofluorescence of divided cells, as this will aid in determining the number of divisions resolvable.
15.
Incubating 1 h before addition of BFA works well when stimulating with both protein and peptide antigens. However, stimulation time needs to be established according to the specific experimental settings.
16.
The use of a thermostat heatblock is dispensable, but eases the workflow greatly.

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Acknowledgments

Linda Christensen and Maria Nørtoft Sørensen are acknowledged for their excellent technical assistance. We are indebted to all colleagues, present and former, at the Department of Infectious Disease Immunology for input to the described protocols.

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Authors and Affiliations

Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark
Thomas Lindenstrøm, Peter Andersen & Else Marie Agger

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Thomas Lindenstrøm
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Peter Andersen
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Else Marie Agger
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Correspondence to Thomas Lindenstrøm .

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Dept. Cardio & Vascular Sciences, St. George's University of London, Cranmer Terrace, London, SW17 0RE, United Kingdom
Gwyn Davies

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Lindenstrøm, T., Andersen, P., Agger, E.M. (2010). Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells. In: Davies, G. (eds) Vaccine Adjuvants. Methods in Molecular Biology, vol 626. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-585-9_15

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DOI: https://doi.org/10.1007/978-1-60761-585-9_15
Published: 28 December 2009
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-584-2
Online ISBN: 978-1-60761-585-9
eBook Packages: Springer Protocols

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