Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Summary

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Abstract

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8⁺ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Introduction

Cytotoxic T lymphocytes (CTL) inducing vaccines are key therapeutic interventions that have been developed to fight certain types of cancer¹. CTL are also important for prophylactic vaccines against intracellular pathogens². Moreover, CTL are one of the few immune defense mechanisms functionally active in risk populations such as neonates^3,4 whom also depend on CTL to combat early life infections⁵. In this regard, vaccines against Respiratory Syncytial Virus (RSV) that were developed with an adjuvant that does not elicit CTL responses (alum) resulted in a complete failure of the vaccine leading to serious complications upon infections in infants⁶. These negative effects of vaccination can be reversed by a CD8⁺ T cell response⁷. We have previously demonstrated that the main cytokines (type I interferons) elicited by some stimulator of interferon genes (STING) agonists are essential for the CTL responses generated by these adjuvants⁸, in part by measuring the proliferation of OT-I T cells after vaccination and using these results as a measure of CTL inducing capabilities observed in extended vaccination schedules⁹. The measurement of the proliferation of OT-I CD8⁺ T cells in a wild type (WT) C57BL/6 recipient mouse by carboxyfluorescein succinimidyl ester (CFSE) dye dilution is a robust estimation of the capability of the adjuvant of a vaccine to generate cross-priming of SIINFEKL, (the immuno-dominant peptide of ovalbumin, OVA). Variations of this technique are widely used for the assessment of the proliferation of OT-I CD8⁺ and OT-II CD4⁺ T cells. For example, it has been used in the absence of selected cytokines (KO mice) or to measure vaccine efficacy after antigen recall in WT animals. We devised a short protocol (4 days experiment) in which after passive transfer of CFSE-stained OT-I CD8⁺ T cells, a subcutaneous (s.c.) immunization consisting of one dose of 50 µg of endotoxin-free OVA supplemented with test adjuvants is administered (Figure 1). The follow up of the results 48 h after vaccination provides a reliable proof of the capacity of the adjuvant to generate CTL responses. By this strategy, it is possible to assess the potency of the local immune response in the draining lymph node after immunization as well as the extent of the response by measuring the CTL activity in the spleen (or distant lymph nodes).

Protocol

All mice used in this study were from the C57BL/6 background. All the animals were kept under pathogen-free conditions. All experiments were performed according to the normative of the German animal protection law (TierSchG BGBl. I S 1105; 25.05.1998) and were approved by the Lower Saxony Committee on the Ethics of Animal Experiments and the state office (Lower Saxony State Office of Consumer Protection and Food Safety), under permit number 33.4-42502-04-13/1281 and 162280.

1. CFSE Staining of OT-I T Cells and Adoptive Transfer

NOTE: OT-I mice are transgenically generated animals that express a T cell receptor (TCR) with fixed α and β chains that together recognize the immuno-dominant peptide of OVA, SIINFEKL^10,11. As a result, these mice have a considerably high number of SIINFEKL-specific CD8⁺ T cells (97%)¹² when compared to normal or OVA-vaccinated mice (≤ 1%)¹³.

1. Isolation of traceable CD8⁺ T cells from OT-I mice expressing T lymphocyte-specific Thy1^a (Thy1.1) allele:
   1. Euthanize 6-9 weeks old OT-I mice by CO₂ inhalation followed by cervical dislocation¹⁴. Dissect the spleen and major lymph nodes (inguinal, axillary and cervical pairs only) by cutting the skin with surgical scissors, detaching the skin from the body with the help of surgical pliers and forceps and place them in Petri dishes (60 x 15 mm) each containing a 100 µm pore mesh cup for tissue grinding and cell release.
   2. Maintain petri dishes (each with one mesh cup) in 3-5 mL of complete RPMI medium (RPMI 1640, 10% v/v FCS, 100 U/mL penicillin, 50 µg/mL streptomycin) kept on ice.
   3. Mash the organs with the help of a syringe plunger or a similar sterile instrument (prior cutting is not needed) and collect the resulting single cell suspension in 15 mL centrifuge tubes.
   4. Centrifuge the cell suspension at 300 x g for 10 min at 4 °C. Discard the supernatant. Wash the cells in 10 mL of cold PBS by centrifugation and lyse the erythrocytes from the spleen by re-suspending the pellet in 1 mL/spleen of ammonium chloride buffer (ACK buffer, commercially available). Incubate the cells for 1.5 min on ice and subsequently wash the cells with 10 mL of cold PBS by centrifugation (as done before).
   5. Re-suspend the pellet in the same tube in 1 mL of PBS (pH 7.2) containing 5% fetal bovine serum for magnetic isolation.
2. To perform magnetic isolation, proceed to the negative selection of CD8⁺ T cells by using a magnetic isolation kit according to the manufacturer instructions or published protocols¹⁵.
3. To perform CFSE staining, first count the number of CD8⁺ T cells obtained from OT-I mice by using an automated cell counter (particle counter). Stain 1 - 5 x 10⁷ cells/mL in a volume of 1-5 mL with 5 µM CFSE in PBS for 7 min at 37 °C, protected from light in a 15-ml falcon tube.
4. Quench the CFSE staining by adding the same volume (1:1) of fetal bovine serum to the cells, and incubate them for additional 7 min at 37 °C protected from light. Wash the cells with 10 mL of PBS twice.
5. Count the cells using an automated cell counter. Set the cell number to 3-5 x 10⁷ cells/mL of PBS in order to inject 3-5 x 10⁶ cells/mouse in 100 µL intravenously (i.v.) via the tail vein.
6. To perform tail vein injections, immobilize the mice in appropriate restrainers. Warm the back area and tail of the mice to be injected by using a red-light lamp, placed between 20 to 25 cm away from the mice for 1-3 min to allow tail vein vasodilation.
   NOTE: This helps to ease vein detection and administration of the cell suspension.
7. Ensure (with the hand placed in between the mice and the lamp) that it is not too hot for the mice and when the tail veins are clearly visible, proceed to injection. Inject the cells into the lateral or dorsal tail vein using an 1mL syringe with a 25-gauge needle¹⁶.

2. Immunization (Endo-free OVA +/- Adjuvant)

1. Place the mice transplanted with OT-I cells (step 1.7, Figure 1) in an anesthesia chamber and administer isoflurane in oxygen by an anesthesia machine¹⁴.
2. When the mouse is completely asleep under anesthesia, take it out from the chamber and shave the fur of the mouse on the area over the gluteus superficialis (lateral lower back) by using an electric hair trimming machine, in order to perform a clean injection with a good view of the application area.
3. Inject 50 µL of the vaccine, s.c. in the shaved area using a 25-gauge needle.

3. Isolation of Lymphocytes and Staining for Flow Cytometry Analysis

1. Isolation of lymphocytes and splenocytes
   1. Euthanize the vaccinated mice by CO₂ inhalation followed by cervical dislocation.
   2. Extract the draining and distant lymph nodes and spleen and place them in separate Petri dishes containing 100 µm pore mesh cups for tissue grinding and cell release. Follow steps 1.1.2 through 1.1.3.
   3. Decant the supernatant after centrifugation and re-suspend the cell pellets in the same volume of remnant PBS (approx. 100 µL).
2. Staining for flow cytometry analysis
   1. In a 15-mL centrifuge tube prepare a master mix containing the staining antibodies in the concentrations depicted in Table 1, thus yielding a 2x concentrated staining mix. Prepare enough volume of master mix to have 100 µL per sample. Mix the cells and the master mix 1:1 and incubate it at 4°C for 30 min.
      NOTE: The final staining volume per sample should be 200 µL (100 µL of cell pellet +100 µL of antibody master mix).
   2. Wash the cells twice by centrifugation as described in 1.1.3, adding 10 mL of PBS, twice.
   3. Re-suspend the stained cells in 0.5-1 mL of PBS for acquisition and transfer the suspension to flow cytometer tubes. Always keep the samples on ice and protected from light.

4. Flow Cytometry

1. Always pre-filter the cell samples using 70-100 µm filters.
2. Prepare single staining compensation controls for the fluorophores detailed in Table 1 (beads or cells).
   NOTE: Compensation should be performed in cytometer or analysis software^17,18,19 and applied to all samples.
3. Follow the gating strategy depicted in Figure 2. Briefly, gate populations in forward scatter height vs. forward scatter area (Figure 2A), and again side scatter wide vs. side scatter area (SSA, Figure 2B) in order to exclude doublets.
4. Gate the population from Figure 2B by plotting BV 650 (auto-fluorescence) vs. FITC (CFSE) in order to discriminate true CFSE stained cells from high auto-fluorescent cells. Include beads or cells stained with antibody conjugated to BV 650 into compensation controls. This plot (Figure 2C) of BV 650 vs. FITC is used to gate the OT-I CFSE stained cells.
5. Gate the population from Figure 2C for Pe-Cy7 (Thy1.1 -CD90.1-) vs. APC (CD8) in order to distinguish cells derived from donor vs. recipient mice.
   NOTE: Cells derived from OT-I donor mice are Thy1.1⁺ (CD90.1), whereas WT (C57BL/6, recipient) mice-derived cells are Thy1.2⁺ (CD90.2) (Figure 2D). Some laboratories have OT-I mice in a Thy1.2 background and WT (C57BL/6) in a Thy1.1. In this case you have to use an antibody against Thy1.2 for OT-I cell gating.
6. Re-gate CD8⁺ cells from Thy1.1⁺ population by plotting it on a gate comprising BV 450 (CD4) vs. APC (CD8) (Figure 2E).
7. Display the population gated in Figure 2E by using a histogram display of the CD8⁺ cells showing CFSE (FITC, 530/15 channel -blue laser-), gate the proliferated population by including intensities from 10² up to the level where the undivided control populations are (intensities ~10⁵, depending on the efficacy of the CFSE staining and the voltage setting on the flow cytometer) (Figure 2F).
8. Make an additional gate (not displayed in figure 2) plotting FITC vs. L/D marker (450/20 channel, UV laser) in order to assess dead cells in parallel to the proliferation evaluation.
9. Acquire at least 5000 events for the compensation controls and 10.000 events (gate E, Figure 2) from the samples at a flow cytometer. Run the cytometer at low or medium flow (do not exceed flow rates of 20.000 events/s).

Results

In order to test the treatments using a different combination of adjuvants (ADJ1 and ADJ2), we have assessed the CTL generation capacity by measuring the proliferation of adoptively transferred OT-I CD8⁺ T cells by flow cytometry (Figure 2). For this, we previously stained isolated cells from the draining lymph nodes and spleen (Table 1). By measuring the proliferation of CD8⁺ T cells in lymph nodes and spleen, we were a...

Discussion

Modern vaccines are ideally composedof purified antigen and adjuvants, with the possible addition of a delivery system like liposomes, virus-like particles, nanoparticles or live vectors. A key aspect when designing a vaccine is to choose the right adjuvant according to the clinical needs. Part of the scope could involve favoring a humoral vs. cellular immune response (or both), the election of a local vs. a systemic immune response (or both), and the kind of memory that the vaccine must generate in the target population...

Materials

Name	Company	Catalog Number	Comments
BD LSR Fortessa Cell Analyzer	BD	Special Order	Flow Cytometer
CFSE	Molecular Probes	C34554	Proliferation Dye
MojoSort Mouse CD8 T Cell Isolation Kit	Biolegend	480007	Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation	Molecular Probes	L23105	Dead Cell Marker
CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7	eBioscience	25-0900-82	antibody
APC anti-mouse CD8a Antibody	BioLegend	100712	antibody
BV421 Rat Anti-Mouse CD4	BD	740007	antibody
Z2 coulter Particle count and Size Analyzer	Beckman Coulter	9914591DA	Cell counter. Z2 Automated particle/cell counter
EndoGrade Ovalbumin (10 mg)	Hyglos(Germany)	321000	Ovalbumin endotoxin free tested.
Cell Strainer 100µm nylon	Corning	352360	Cell strainer (100 µm pore mesh cups).
Sample Vials	Beckman Coulter	899366014	Sample vials for Z2 automated counter
C57BL/6 mice (CD90.2)	Harlan (Rossdorf, Germany)		Company is now Envigo
OT-I (C57BL/6 background, CD90.1)	Harlan (Rossdorf, Germany)		Inbreed at our animal facility. Company from where adquired is now Envigo
FACS tubes	Fischer (Corning)	14-959-5	Corning Falcon Round-Bottom Polystyrene Tubes
Falcon 15 mL tubes	Fischer (Corning)	05-527-90	Falcon 15mL Conical Centrifuge Tubes
PBS (500 mL)	Fischer (Gibco)	20-012-027	Gibco PBS (Phosphate Buffered Saline), pH 7.2
Red lamp (heating lamp)	Dirk Rossmann GmbH (Germany)	405096	Heating infrred lamp (100 wats)
IsoFlo (Isoflurane)	Abbott Laboratories (USA)	5260.04-05.	Isoflurane anesthesic (250 mL flask).
Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber	Parkland Scientific	V3000PK	Isoflurane anesthesia machine.
RPMI 1640 medium	Gibco (distributed by ThermoFischer)	11-875-093	Base medium with Glutamine (500 mL)
Pen-Strept antibiotic solution (Gibco)	Gibco (distributed by ThermoFischer)	15-140-148	Gibco Penicillin-Streptomycin (10,000 U/mL)
Fetal Bobine Serum (Gibco)	Gibco (distributed by ThermoFischer)	10082147	Fetal Bovine Serum, certified, heat inactivated, US origin
ACK Lysing Buffer (100 ml)	Gibco (distributed by ThermoFischer)	A1049201	Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also
Plastic Petri Dishes	Nunc (distributed by ThermoFischer)	150340	60 x 15mm Plastic Petri Dish, Non-treated
Cell Clump Filter	CellTrics (Sysmex)	04-004-2317	CellTrics® 50 μm, sterile