An Optimized Ex Vivo Flurospot assay to identify multi-functional HBV-specific T cells in Chronic Hepatitis B patients

POSTED ON: 07 Mar, 2022

Preparation of OLP peptide pools:

  • 313 peptides in total
  • Spans 15 amino acids (ie. 15-mers)
  • Peptide offset: 5 amino acids apart
  • Peptide pools:
    • PreCore/Core = 41 peptides
    • X = 29 peptides
    • Env – 1 =   peptides   1 – 181  (37 peptides)
    • Env – 2 =   peptides   186 – 376 (36 peptides – 3 peptides not synthesized: 241, 246, 251)
    • Pol-1 = peptides  1 – 206  (42 peptides)
    • Pol-2 = peptides  211 – 416  (42 peptides)
    • Pol-3 =  peptides 421 – 626  (42 peptides)
    • Pol-4 =  peptides  631 – 831 (41 peptides)

 

Preparation of 50X OLP peptide pool

  • PreCore/Core 50x
    • 41 peptides x 10 μl /peptide = 410 μl of peptides + 590 ml Aim-V + Prim

 

  • X 50x stimulation pool
    • 29 peptides x 10 μl /peptide =  290 μl of peptides + 710 ml Aim-V + Prim

 

  • Envelope 50x stimulation pool
    • Env-1     37 peptides x 10 μl /peptide =  370 μl of peptides + 630 ml Aim-V + Prim
    • Env-2     36 peptides x 10 μl /peptide =  360 μl of peptides + 640 ml Aim-V + Prim
  • Polymerase 50x stimulation pool
    • Pol-1 42 peptides x 10 μl /peptide =  420 μl of peptides + 580 ml  Aim-V + Prim
    • Pol-2 42 peptides x 10 μl /peptide =  420 μl of peptides + 580 ml  Aim-V + Prim
    • Pol-3 42 peptides x 10 μl /peptide =  420 μl of peptides + 580 ml  Aim-V + Prim
    • Pol-4 41 peptides x 10 μl /peptide =  410 μl of peptides + 590 ml  Aim-V + Prim

 

Ex Vivo 3-color FluoroSpot – Cell prepping (ImmunoSpot)

Materials:

  • 107 frozen PBMCs (per donor; maximum 6 donors per plate)
  • 30ml polypropylene tube (one per donor sample)
  • Eppendorf tubes
  • 3-color FluoroSpot kit (ImmunoSpot; 6 donors per plate)
  • HBSS medium (Gibco, Ref# 24020-117)
  • AIM V medium (Gibco, Ref# 12055-091)
  • Knock out serum Replacement (KSR) (Lifetech: 10828028)
  • Primocin ( 1ml of 50mg/ml: InvivoGen)
  • Human Blood type-AB serum (VWR, CA45001-062)
  • HBV overlapping peptides, genotype C (GenScript)
  • CEF purified peptide library (GenScript): (USED to monitor treatment effect on unrelated virus-specific T cells)
  • DMSO solution (Sigma, Ref# D8418)
  • DynabeadsTM Human T-activator CD3/28 (Gibco, Ref# 11131D) (USED as positive/Assay Control)

Day 1: Resting cells (pre-warm media)

  • Thaw 107 cells with HBSS medium (as per protocol) in a 30mL polypropylene tube
    • Minimum count required for experiment: 6 x 106cells/donor
  • Centrifuge at 300xg for 5mins., aspirate
  • Resuspend at approximately 4×106 cells/ml with AIM V (+2% human serum)
  • Take counting aliquot for pre-rest counts
    • fix cell concentration if necessary (3.5-4.5×106 cells/ml are suitable as well)
  • Incubate O/N in 37oC incubator @ 5% CO2
  • Coat IPFL plates, fridge O/N in 4oC (as per manufacturer’s, see next pages)

 

Day 2a: Pulsing cells (pre-warm media)

  • 16-18h later, resuspend samples and take counting aliquot for post-rest counts
  • Aliquot two Eppendorf tubes with 4.5 x 105 cells each (per donor)
    • 1 tube for HBV OLP stimulation, another for DMSO vehicle control
  • Centrifuge at 300xg for 5mins., aspirate
  • Resuspend both tubes with 84µl AIM V (+2% human serum) each
  • Pulsing scheme: final volume of 100µl
    • HBV OLP sample: add 2µl of 250µg/ml/peptide per OLP pool (8 pools: 16µl)
      • HBV OLP final concentration: 5µg/ml/peptide
    • DMSO control sample: add 16µl of 19.38% DMSO (in AIM V)
      • DMSO final concentration: 3.1% DMSO (equivalent to OLP sample)
  • Incubate for 1hr in 37oC incubator @ 5% CO2
  • Block IPFL plate simultaneously (as per protocol, see next page)
  • Centrifuge both tubes of pulsed cells at 300xg for 5mins., aspirate
    • Resuspend cells with 225µl AIM V (no serum) (ie. 2 x 106 cells/ml)
  • Aliquot two Eppendorf tubes with 8 x 106 cells each (per donor); centrifuge, aspirate
    • Resuspend cells with 450µl AIM V (no serum) (ie. 4 x 106cells/ml)
    • Pool respectively with pulsed cells
    • Wash tube with another 450µl AIM V (no serum) and pool respectively
    • Final volume:25×106 PBMCs in 1.125ml AIM V (ie. 2 x 106cells/ml)
  • Aliquot 1.2 x 106cells for CEF controls; centrifuge, aspirate
    • Resuspend cells with 588µl AIM V and 12µl 50x CEF (ie. 2 x 106 cells/ml)
  • Aliquot 1 x 105cells for CD3/28 controls; centrifuge, aspirate
    • Resuspend cells with 400µl AIM V and 0.4µl CD3/28 (ie. 2.5 x 105 cells/ml)

 

Day 2b: Plating cells

 

 

 

  • Color scheme for plating:
    • Blue wells: 100µl CD3/28 controls (2.5×104cells per well)
    • Purple wells: 200/200/100µl CEF controls (4×105cells/2×105cells per well)
    • Red wells: 200µl DMSO controls (4×105cells per well)
    • Green wells: 200µl OLP pulsed cells (4×105cells per well)
  • Plating totals:
    • CD3/28: 5×104 cells in 3 wells
    • CEF: 1×106 cells in 3 wells
    • DMSO:               2×106 cells in 5 wells
    • OLP: 2×106 cells in 5 wells
  • Incubate for 20hr in 37oC incubator @ 5% CO2

 

Day 3: Plate development (20 hours after plating cells)

  • Develop IPFL plates the next day (as per manufacturer’s protocol, see next pages)

 

Ex Vivo 3-color FluoroSpot –Plate prepping (ImmunoSpot)

Materials:

  • ImmunoSpot S6 Universal Analyzer
  • 3-color FluoroSpot kit (ImmunoSpot)
  • IPFL plate (Merck Millipore, Ref# S53J104I07-MB) (comes with the kit)
  • 50ml multi-pipette troughs (Optional)
  • Multi-channel pipette (Optional)
  • Repeater pipette (Optional)
  • Syringes
  • 22µm filter (Merck Millipore, Ref# SLGP033RS)
  • Vacuum manifold or plate washer
  • 70% Ethanol (in PBS)
  • AIM V medium (Gibco, Ref# 12055-091)
  • Blocking solution (AIM V media with 10% KSR)

 

Day 1: Plate coating

  • Prepare coating Abs as per manufacturer’s protocol
  • Activate wells by adding 15µl of 70% ethanol for 1 minute at room temperature
    • NOTE: Activate all the well even if not be used. Need to activate for vacuum manifold to work. Wells, that’s not bee used for experiments do not let them dry, just add PBS during incubation steps.
  • Wash IPFL plate 3x with 200µl sterile PBS (optional: use multi-channel + troughs)
  • Plate 80µl of coating Ab solution into each well (optional: use repeater)
  • Parafilm (optional) and incubate plate at 4oC O/N

 

Day 2: Setting up plate

  • Flick coating solution, wash 3x with 200µl sterile PBS
  • Make blocking solution, AIM V – 10% KSR
    • Add 45ml of AIM V to 5ml of KSR
  • Add 100µl blocking solution into each well
  • Incubate plate at room temperature for at least 30 minutes
  • Remove via flicking
  • Add PBMCs to the plate after pulsing (as per protocol, see previous page)
  • Incubate the plate @ 37oC for 20hr

 

 

Day 3: Plate development (20 hours after plating cells)

  • Prepare secondary Abs as per manufacturer’s protocol:
    • Filtration subtracts 0.5ml from total volume, prepare excess solution as necessary
    • Attach 0.22µm filter to syringe; apply and filter solution as per manufacturer’s protocol
  • Wash IPFL plate 3x with 200µl PBS
  • Add secondary Ab solution, plate 80µl solution into each well
  • Incubate plate at room temperature for 2hr in the dark
  • Prepare detection Abs as per manufacturer’s protocol:
    • Filtration subtracts 0.5ml from total volume, prepare excess solution as necessary
    • Attach 0.22µm filter to syringe; apply and filter solution as per manufacturer’s protocol
  • Wash IPFL plate 3x with 200µl PBS
  • Add detection Ab solution, plate 80µl solution into each well
  • Incubate plate at room temperature for 1hr in the dark
  • Wash and dry with vacuum manifold using plate washer (necessary step)
    • With Microplate washer:
      • wash with 200µ PBS and dry via vacuum filtration at -187mmHg for 20s, repeat 4x
    • Remove plate underdrain and wash underside with running water, flick and repeat ~2-3x; flick dry as much as possible
    • Note: avoid flicking on paper towels to minimize dust/particulates in wells
    • Dry ~20-30mins in the BSC (with underside upwards) in the dark
  • Scan and count plates, store long-term at 4oC

 

Fluorescent-bait labelling for the ex vivo detection of HBV antigen-specific B cells

POSTED ON: 30 Apr, 2021

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.

  1. Wash cells with PBS supplemented with 0.5%-FBS and 2mM EDTA and centrifugation (300g, 4 min at 4ºC)
  2. 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.

Tetramer enrichment for HBV-specific CD8+ T cells

POSTED ON: 03 Jul, 2020
  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.
  1. Centrifuge cells (500xg, 10 minutes) and discard the supernatant.
  2. 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)).
  3. Resuspend cell pellet in 100μl MACS buffer.
  4. 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!
  1. After incubation add 5ml MACS buffer.
  2. Centrifuge the cells (500xg, 10 minutes), discard the supernatant.
  3. 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).
  1. Incubate 20 minutes at 4°C in the dark.
  2. Add 5ml MACS buffer and centrifuge the cells (500xg, 10 minutes), discard the supernatant.
  3. Resuspend cell pellet in 1ml MACS buffer.
  4. 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.
  1. 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).
  1. 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).

  1. Centrifuge eluted cells (500xg, 10 minutes).
  2. During centrifugation remove cells from the depleted fraction for the single stains you may need for compensation (about 100μl each).
  3. After centrifugation discard supernatant (but leave about 150μl).
  4. 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.

Evaluating mouse HBV-specific CD8+ T cell responses by ELISPOT assay

POSTED ON: 21 May, 2019

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 4C 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 4C.

h. Decant the supernatant and wash cells in 5 mL HBSS, followed by centrifugation at 300 × g for 5 min at 4C. 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 CD8T 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; Ld), 353-360 (VWLSVIWM; Kb), and 364-372 (WGPSLYSIL; Dd) epitopes, the HBcAg 87-95 (SYVNTNMGL; Kd)and 93-100 (MGLKFRQL; Kb) epitopes, and the Pol 140-148 (HYFQTRHYL; Kd) 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 × 10cells/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 × 10cells/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 37C / 5% COin 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 10total cells (i.e. “Specific IFN-gamma SFC per 10splenocytes”).

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 CD8T cells. However, if using well-defined peptide epitopes for stimulation that are known to not be cross-reactive for CD4T 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.