Protocol Tag: Immunology Assays

Prepare 1 x 10⁶ – 3 x 10⁶ PMBCs, from freshly isolated PBMCs or thawed from frozen, per stain. All staining should be performed on a single-cell suspension either in 96-well U-bottomed plates or in 5ml polypropylene tubes.

1. Centrifuge cells (300g, 4 min at 4ºC)
2. Stain cell pellet with a fixable cell viability dye diluted in PBS (Live/Dead™ stain, Invitrogen™) for 15 min at 4ºC
3. Wash cells using 1x PBS and centrifugation (300g, 4 min at 4ºC)
4. Block non-specific antibody binding using FcR blocking reagent (Miltenyi Biotec) for 15 min at 4ºC
5. Stain cells with 50ml of anti-human monoclonal antibodies of interest, in combination with Dy550 and Dy650 antigen baits at the following concentrations:

HBsAg -Dy550 and -Dy650: 10-20ug/ml

HBcAg -Dy550 and -Dy650: 300-600ng/ml

Dilute antibodies in 50%-Brilliant Stain buffer (BD Biosciences™) and 50%-PBS supplemented with 0.5%-FBS and 2mM EDTA to minimize interactions between multiple fluorescent dyes. Incubate cells with monoclonal antibodies and antigen-baits for 30 min in the dark on ice.

6. Wash cells with PBS supplemented with 0.5%-FBS and 2mM EDTA and centrifugation (300g, 4 min at 4ºC)
7. Fix cells (BD Cytofix™, BD Biosciences™) prior to acquisition

TIP: Run the cells at a low threshold rate and make sure to acquire all of the cells

Stringent gating criteria should be applied during analysis to exclude doublet, dead and CD19-negative cells. Cells stained with an identical panel minus antigen-bait staining can be used to control for non-specific binding and guide gating.

All reagents used should be stored at 4ºC; monoclonal antibodies and antigen-baits should be stored in the dark.

1. Prepare the cells you want to use for enrichment.

• Use freshly isolated peripheral blood mononuclear cells (PBMCs) or thaw frozen PBMCs in pre-warmed RPMI.
• Thawing can be performed with Bezonase nuclease (e.g. if cells are frozen for >10 years or cell clumping is expected for whatever reason).
• Use at least 10⁷ PBMCs
• Depending on the frequency of the analyzed virus-specific CD8+ T cells, enrichment can be performed also with less cells.

2. Centrifuge cells (500xg, 10 minutes) and discard the supernatant.
3. Optional: Incubation of cells with Benzonase-containing RPMI for 10-30min (50Uml ^-1 Benzonase) at 37 °C, 5% CO2 (after incubation wash cells once with RPMI (500xg, 10 minutes)).
4. Resuspend cell pellet in 100μl MACS buffer.
5. Centrifuge tetramers at full speed for 4 min at 4°C before use. Add optimized volume of tetramers (labelled with APC or PE), resuspend, incubate 30 min at room temperature in the dark.

• Avoid light exposure when working with fluorochrome-conjugated tetramers!
• Consider tetramer titration for the appropriate amount you have to add!

6. After incubation add 5ml MACS buffer.
7. Centrifuge the cells (500xg, 10 minutes), discard the supernatant.
8. Add 50μl anti-APC and/or anti-PE beads (Miltenyi Biotec) and fill up to a final volume of 250μl with MACS buffer (the optimal concentration of beads should be titrated).

• If you use more cells (e.g. 8×10⁷ PBMCs) or the frequency of your virus-specific CD8+ T cells is very high you probably have to adjust the volume of beads to catch all your tetramer+ cells (the original enrichment protocol used 100μl of beads and a final volume of 500μl).

9. Incubate 20 minutes at 4°C in the dark.
10. Add 5ml MACS buffer and centrifuge the cells (500xg, 10 minutes), discard the supernatant.
11. Resuspend cell pellet in 1ml MACS buffer.
12. Remove 5μl for counting the “pre”-fraction if you want to calculate your frequency.
    • Caution! Your total volume is about 1.2ml (because the cells also have a volume), consider this in your calculation of the total “pre” cell number.

13. Remove 5μl for staining of the “pre”-fraction and plate them in your staining plate.

• before removing the cells you can add 5µl into the “pre” wells of the staining plate to prevent evaporation of the low volume of 5μl.
• in general, staining of the pre-fraction is required for the calculation of the frequency of enriched virus-specific CD8+ T cells (number of tetramer+ cells/ number of CD8+ cells).

14. Perform a magnetic separation with according to the manufacturer’s instructions (use a LS column even if your initial cell count was very low – the enrichment does not really work well with MS columns).

• Place the column into the MACS magnet.
• Place a 15ml falcon tube under the column.
• Equilibrate LS column with 3ml of MACS buffer, let the whole buffer run through the column.
• Discard the falcon tube and place a new tube under the column.
• Add your cell suspension onto the column, let run through.
• Add 3ml of MACS buffer onto the column, let run through -> the cells collected in the falcon represent the “depleted” fraction.
• Place the column onto a fresh 15ml falcon tube (outside of the MACS magnet) and add 5ml MACS buffer.
• Elute the labeled cells with the plunger -> this is your “enriched” fraction.

All further steps are for staining the enriched population (for flow cytometric analysis).

15. Centrifuge eluted cells (500xg, 10 minutes).
16. During centrifugation remove cells from the depleted fraction for the single stains you may need for compensation (about 100μl each).
17. After centrifugation discard supernatant (but leave about 150μl).
18. resuspend cells in the leftover MACS buffer and transfer them to your staining plate (you can also rinse the falcon once again with another 100-150μl MACS buffer).

Handling & storage

All reagents used for this protocol should be stored at 4°C. In the case of tetramers and antibodies: avoid light exposure. The benzonase is stored at -20 °C.

1. Splenocyte preparation

a. Collect the mouse spleen in a 15 mL conical tube containing 3-5 mL HBSS. As an alternative to HBSS, either serum free RPMI or RPMI containing 1% FBS can also be used.

b. For each spleen, place a 70 μm pore strainer on the top of a 50 mL conical tube.

c. Transfer HBSS and spleen into the strainer, allowing the HBSS to pass through into the tube.

d. Disrupt the spleen through the strainer using a plunger from a 1 mL syringe. As the connective tissue of the spleen is disrupted, the splenocytes will pass through the strainer into the tube. Continually wash the strainer by addition of 3 – 5 mL HBSS while disrupting the spleen.

e. Centrifuge at 300 × g for 5 min at 4^o C to pellet the cells.

f. Remove liquid from the tube and dissociate the cell pellet from the bottom by gently flicking andshaking the tube. Using too much mechanical force during suspension steps can damage the cells and reduce yields. Suspend the pellet in 2 mL of ACK lysis buffer and incubate at room temperature for 5 min. Do not leave cells in ACK lysis buffer for extended periods of time, as doing so can result in lysis of lymphocytes.

g. Add 8 mL of HBSS to the tube and immediately centrifuge at 300 × g for 5 min at 4^o C.

h. Decant the supernatant and wash cells in 5 mL HBSS, followed by centrifugation at 300 × g for 5 min at 4^o C. Suspend pellet in 5 mL of complete medium and keep on ice.

i. Count cells using a hemocytometer or automated cell counter.

2. IFN-gamma ELISPOT assay

a. This procedure is adapted from the manufacturer’s protocol for the BD Biosciences mouse IFN-gamma ELISPOT set (catalog #551083). Procedures for other manufacturers may vary.

b. On the day before the assay will be performed, dilute anti-mouse IFN-gamma capture antibody according to the manufacturer’s lot-specific recommendation in PBS, and add 100 μL of diluted antibody per well to an ELISPOT plate.

c. Incubate the plate overnight at 4° C. Although the ideal incubation period is overnight, a minimum time of 4 hours can be done.

d. Remove the capture antibody solution, and wash wells once with complete media. Block wells by adding 200 μL/well complete media and incubating at room temperature for 2 hrs.

e. Prepare peptides for stimulation by diluting in complete media at a concentration of 20 µg/mL (final concentration will be 10 µg/mL after 1:1 dilution with cell suspension). Although mouse CD8⁺ T cell responses to many HBV antigen epitopes have been described, the responses to some epitopes are stronger than others. The HBV S protein 191-202 (IPQSLDSWWTSL; L^d), 353-360 (VWLSVIWM; K^b), and 364-372 (WGPSLYSIL; D^d) epitopes, the HBcAg 87-95 (SYVNTNMGL; K^d)and 93-100 (MGLKFRQL; K^b) epitopes, and the Pol 140-148 (HYFQTRHYL; K^d) epitope are particularly immunogenic [3-10]. Alternatively, pools of overlapping peptides that cover the entire antigen coding sequence can be used.

f. Dilute cells in complete media to a concentration of 2 × 10⁶ cells/mL. Cells may need to be diluted more or less depending on the responses expected in a given experiment. It is recommended that a pilot experiment be performed to determine the optimal cell concentration. Keep in mind that the responses obtained using different cell numbers may not be linear, as both the interactions between T cells and antigen presenting cells as well as the actual number of T cells will be affected by dilution.

g. Discard blocking solution and add 100 µL each of diluted cells (2 × 10⁵ cells/well) and diluted peptides to the appropriate wells. It is recommended that duplicate wells be used for each experimental condition.

h. As an unstimulated negative control, a well for each cell sample should also be included that contains cells but lacks peptide (add 100 µL of complete media without peptide). This control will be important to subtract the nonspecific responses. As a positive control, a well can be included for each sample in which the cells are stimulated with a polyclonal T cell activator such as PMA (20 ng/mL) plus ionomycin (1 µg/mL).

i. To exclude that false spots might be generated by the reagents alone, a background control well without the cells can also be included.

j. Depending on the parameters of the specific experiment, other controls are also appropriate and recommended. For example: cells from a control group of mice subjected to a mock/sham experimental manipulation that are stimulated with peptide; cells from an experimental group of mice stimulated with an irrelevant peptide (rather than no peptide).

k. Incubate the ELISPOT plate overnight at 37^o C / 5% CO₂ in a cell culture incubator. To prevent formation of irregular spots, avoid disturbing the plate after addition of the cells.

l. Discard cells and media from the plate, and wash wells twice with deionized or Milli-Q water, allowing wells to soak for 3-5 min during each wash.

m. Wash wells 3× with PBS-Tween. Avoid touching the surface of the membrane in the wells during washing of the plate. Generally, to quickly remove cells or wash buffer, the plate can be inverted and flicked over a sink (or if appropriate, removed by vacuum suction in a biological safety cabinet) and tapped against absorbent paper to remove residual wash buffer.

n. Dilute biotinylated anti-mouse IFN-gamma detection antibody according to the manufacturer’s lot-specific recommendation in PBS-FBS. After removal of the final wash, add 100 µL of diluted antibody to each well.

o. Incubate the plate at room temperature for 2 hrs. Dispose of detection antibody solution, and wash wells 3× with PBS-Tween.

p. Dilute streptavidin-HRP reagent 1:100 in PBS-FBS, and add 100 µL of the diluted enzyme conjugate to each well. Incubate at room temperature for 1 hr.

q. After incubation, discard the streptavidin-HRP solution and wash wells 4× with PBS-Tween, soaking 1-2 min during each wash. Wash wells an additional 2× with PBS.

r. Prepare substrate solution by adding 20 µL of AEC chromogen (1 drop) to 1 mL of AEC substrate. Add 100 µL of final solution to each well.

s. While monitoring spot development, incubate the plate for 5-60 min at room temperature. When spots have developed sufficiently so that they are readily visible, stop the reaction by washing wells with water. Do not allow the plate to overdevelop.

t. Allow the plate to dry at room temperature, removing the rubber backing to facilitate drying. Spots will continue to develop as the plate dries, and contrast between spots and background will improve.

3. Quantification of T cell response

a. Using a dissecting microscope to magnify the wells, count the number of spots in each well. Ideally, the number of spots will fall in the range between a minimum of 10-20 and maximum of ~100-200. Numbers fewer than 10 may be difficult to distinguish from background, and greater than 200 are difficult to accurately quantify. Alternatively, an automated spot counter can be used to enumerate the spots (Cellular Technology Ltd.).

b. Spots will vary in size and intensity, and it is important to maintain well-to-well consistency in how the spots are counted. In wells with very few spots, there will be a tendency to count very small or light spots, but to undercount these in wells that have many or larger/darker spots, which should be avoided.

c. To quantify the specific response, subtract the corresponding “no peptide” control sample from each animal from the peptide-stimulated sample. The number of non-specific background spots in the unstimulated and background control wells should ideally be relatively low in comparison to the number of specific spots in the peptide-stimulated wells.

d. Data are often enumerated as specific IFN-gamma spot forming cells (SFC), and normalized to 10⁶ total cells (i.e. “Specific IFN-gamma SFC per 10⁶ splenocytes”).

e. One limitation of the ELISPOT assay using total splenocytes is the inability to rigorously ascribe a response to a specific cell type, such as CD8⁺ T cells. However, if using well-defined peptide epitopes for stimulation that are known to not be cross-reactive for CD4⁺ T cells, there is a reasonable degree of likelihood that the response is CD8⁺T cell-specific. However, specificity can be further confirmed by using magnetic bead separation (Miltenyi Biotech) to enrich or deplete specific cell populations from the splenocytes prior to use in the assay.