Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Summary

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Abstract

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

Introduction

Traditional vaccine development has mainly employed the empirical approach of trial-and-error. However, with the recent development of a wide array of biomaterials and discovery of molecular determinants of immune activation, it is now possible to rationally design vaccine formulations with biophysical and biochemical cues derived from pathogens^1,2. In particular, various particulate drug delivery platforms have been examined as vaccine carriers as they can be co-loaded with subunit antigens and immunostimulatory agents, protect vaccine components from degradation, and enhance their co-delivery to antigen presenting cells (APCs) residing in lymph nodes (LNs), thus maximizing immune stimulation and activation^3-5. In this report, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs), which have been previously demonstrated as a potent vaccine platform for elicitation of robust cytotoxic T lymphocyte (CTL) and humoral immune responses in both systemic and mucosal tissue compartments^6-9. In particular, vaccination with ICMVs achieved substantially enhanced serum IgG levels against a malaria antigen, compared with vaccination with conventional adjuvants (e.g., alum and Montanide)⁷ and also elicited potent CTL responses against tumor cells and viral challenge models in mice⁹. Here, using ICMVs as a model vaccine nanoparticle system, we describe methods for characterization of vaccine nano-formulations, including particle size and zeta potential measurements and tracking of particle trafficking to draining LNs (dLNs) utilizing confocal imaging of cryosectioned tissues⁷. In addition, we present a whole-animal imaging-based method of analyzing expansion of CTL responses in mice after adoptive transfer of luciferase-expressing antigen-specific CD8+ T cells^9,10. Finally, we describe tetramer staining of peripheral blood mononuclear cells (PBMCs) for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles^6,9.

ICMVs are a lipid-based nanoparticle formulation synthesized by controlled fusion of simple liposomes into multilamellar structures, which are then chemically stabilized by cross-linking maleimide-functionalized phospholipid head groups within lipid layers with dithiol crosslinkers⁶. Once ICMVs are synthesized, a small fraction of nanoparticles can be used to determine particle size and zeta potential (i.e., surface charge of particles) with a dynamic light scattering (DLS) system and a zeta potential analyzer. DLS measures changes in light scattering in particle suspensions, allowing determination of the diffusion coefficient and the hydrodynamic size of particles¹¹. Achieving consistent particle size from batch to batch synthesis is critical since particle size is one of the major factors influencing lymphatic draining of vaccine particles to dLNs and subsequent cellular uptake by APCs^12,13. In addition, zeta potential can be obtained by measuring the particle velocity when an electric current is applied, which allows determination of the electrophoretic mobility of particles and particle surface charge¹¹. Ensuring consistent zeta potential values of particles is important since surface charge of particles determines colloidal stability, which has direct impact on particle dispersion during storage and after in vivo administration^14,15. In order to track the particle localization to dLNs, ICMVs can be labeled with desired fluorophores including lipophilic dyes and covalently-tagged antigens. Following immunization, mice can be euthanized at various time points, dLNs resected, cryosectioned, and analyzed with confocal microscopy. This technique allows visualization of lymphatic draining of both the nanoparticle vaccine carriers and antigen to dLNs. The tissue sections can additionally be stained with fluorescently labeled antibodies and utilized to obtain more information, such as types of cells associated with the antigen and formation of germinal centers as we have shown previously⁷.

Once the particle synthesis is optimized and trafficking to the dLNs is confirmed, it is important to validate elicitation of in vivo CTL expansion. In order to analyze elicitation of antigen-specific CD8+ T cells in response to nanoparticle vaccination, we have utilized a model antigen, ovalbumin (OVA), with OVA_257-264 peptide (SIINFEKL) immunodominant CD8+ T cell epitope, which allows detailed immunological analyses of antigen-specific T cell responses for initial vaccine development^16,17. In particular, to interrogate the dynamics of expansion and migration of antigen-specific CD8+ T cells, we have generated a double-transgenic mouse model by crossing firefly luciferase-expressing transgenic mice (Luc) with OT-I transgenic mice that possess CD8+ T cells with T-cell receptor (TCR) specific for SIINFEKL (in association with H-2K^b). From these OT-I/Luc mice, luciferase-expressing, OT-I CD8+ T cells can be isolated and prepared for adoptive transfer into naïve C57BL/6 mice. Once seeded, successful immunization with OVA-containing nanoparticles will result in expansion of the transferred T cells, which can be tracked by monitoring the bioluminescence signal with a whole animal imaging system^9,10. This non-invasive whole-body imaging technique has been used with other viral or tumor antigens in the past^18-20, revealing processes involved in T cell expansion in lymphoid tissues and dissemination to peripheral tissues in a longitudinal manner.

Complementary to analysis of adoptively transferred antigen-specific CD8+ T cells, endogenous T cell responses post vaccination can be examined with the peptide-major histocompatibility complex (MHC) tetramer assay²¹, in which a peptide-MHC tetramer complex, consisting of four fluorophore-tagged MHC-class I molecules loaded with peptide epitopes, is employed to bind TCR and label CD8+ T cells in an antigen-specific manner. The peptide-MHC tetramer assay can be performed either in terminal necropsy studies to identify antigen-specific CD8+ T cells in lymphoid and peripheral tissues or in longitudinal studies with peripheral blood mononuclear cells (PBMCs) obtained from serial blood draws. After staining lymphocytes with peptide-MHC tetramer, flow cytometry analysis is performed for detailed analyses on the phenotype of CTLs or quantification of their frequency among CD8+ T cells.

Protocol

All experiments described in this protocol were approved by the University Committee on Use and Care of Animals (UCUCA) at University of Michigan and performed according to the established policies and guidelines.

1. Synthesis and Characterization of ICMVs Co-loaded with Protein Antigen and Adjuvant Molecules

1. Mix 1:1 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] (MPB) in chloroform, keeping the total lipid amount at 1.26 µmol per batch (i.e., 500 µg of DOPC and 630 µg of MPB) in a 20 ml glass vial (diameter = 28 mm and height = 61 mm).
2. Add lipophilic drugs, such as monophosphoryl lipid A (MPLA) or lipophilic dyes (e.g., DiD), to the lipid solution at desired concentration. Thoroughly remove the organic solvent by purging with extra dry nitrogen gas and placing the samples under vacuum O/N.
3. Hydrate the lipid film by adding 200 µl of 10 mM bis-tris propane (BTP, pH 7.0) containing water-soluble drugs (e.g., protein antigens). Vortex for 10 sec every 10 min for 1 hr at RT.
4. Transfer the contents from the glass vial into a 1.5 ml microcentrifuge tube, place samples in an ice-water bath, and sonicate continuously for 5 min using the 40% intensity setting on a 125 W/20 kHz probe-tip sonicator.
5. Add 4 µl of 150 mM dithiothreitol (DTT) to each batch (working concentration 2.4 mM), vortex, and briefly centrifuge using a tabletop microcentrifuge.
6. Add 40 µl of 200 mM CaCl₂ and mix with the pipette (working concentration 33 mM). Incubate samples at 37 °C for 1 hr to allow crosslinking of MPB-containing lipid layers with DTT.
7. Centrifuge samples at 20,000 x g for 15 min, remove the supernatant, and resuspend in 200 µl of ddiH₂O.
8. Repeat step 1.7 and centrifuge again after the second ddiH₂O wash to remove CaCl₂, unreacted DTT, and unencapsulated cargo materials from the supernatant.
9. Prepare 10 mg/ml of 2 kDa polyethylene glycol-thiol (PEG-SH) in ddiH₂O. Resuspend each ICMV sample in 100 µl of PEG-SH solution and incubate at 37 °C for 30 min.
10. Perform two ddiH₂O washes (step 1.7) and resuspend the final ICMV pellet in PBS and store at 4 °C. Prior to use, mix the ICMV suspension, as particles may settle to the bottom after prolonged storage.
11. For characterization of particles, remove a small aliquot (~10%) of ICMVs from each batch and dilute individually in a total volume of 1 ml of ddiH₂O. Place a single sample in a Zetasizer cell and measure particle size, polydispersity index, and zeta potential of the samples using a DLS and zeta potential measuring system (according to the manufacturer’s protocol).

2. Examination of Lymph Node Draining of Fluorescence-tagged ICMVs with Confocal Microscopy

1. Preparation of ICMVs loaded with fluorophore-tagged antigen and lipophilic fluorescent dye
   1. Prepare fluorophore-tagged protein, such as ovalbumin reacted with Alexa Fluor 555-succinimidyl ester, according to the manufacturer’s instruction.
   2. To prepare ICMVs tagged with fluorophore in the lipid shell, add lipophilic fluorescent dye, (e.g., 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine, (DiD)) during preparation of the lipid film (Step 1.2) at 0.05% molar lipid amount. For lipid film hydration (Step 1.3), use buffer containing fluorophore-tagged antigen, and complete ICMV synthesis as outlined in steps 1.4-1.11.
2. Subcutaneous administration of nanoparticles at tail base
   1. Anesthetize mouse using a controlled flow vaporizer equipped with an induction chamber utilizing 3% isoflurane and 1.5 L/min of oxygen flow according to an IACUC approved animal protocol. Once the mouse is unconscious, perform the following steps quickly prior to the anesthesia wearing off to allow optimal access to the site of the injection and minimize discomfort to the animal. Alternatively, use a proper fitting nose cone to maintain anesthesia. If mice are anesthetized for longer than 5 min, apply eye lube necessary to minimize irritation after the procedure.
   2. Spray the base of the tail with 70% ethanol to sanitize and wet the fur Part the wet hair to expose a small patch of visible skin, which can be used to visualize the needle under the skin.
   3. Prepare particle injection suspension containing desired amount of antigen and adjuvant per 100 µl of vaccination dose in PBS (e.g., 10 µg OVA and 0.3 µg MPLA per 100 µl of injection dose has been used in the past^6,9).
   4. Draw the particle suspension into a syringe with a 27-29 G needle and insert the needle at the base of the tail (~5 mm from hairline) with the bevel facing up and inject 50 µl of the particle suspension²².
   5. Wait a few seconds for pressure to equalize to prevent excessive back-flow and pull the needle out. Repeat the injection on the other side of the tail base to target both draining inguinal LNs.
3. Preparation of lymph node cryosections and examination with confocal microscopy.
   1. Euthanize the mouse with CO₂ asphyxiation, followed by induced pneumothorax according to an IACUC approved animal protocol. Extract inguinal LNs according to protocol demonstrated in Bedoya²³ and wash out the blood by placing the tissues in 1 ml of 4 °C PBS.
   2. Absorb the PBS from the tissues with tissues and place tissue in tissue cryomolds (10 x 10 x 5 mm³) pre-filled to the top with OCT freezing medium²⁴. Snap freeze the tissue block in liquid nitrogen for 30 sec. Alternatively, place tissue block on dry ice for 30 min. Store frozen tissue in -80 °C freezer.
   3. Cut tissue sections 5-10 µm thick in a cryostat set at -20 °C²⁴.
   4. If necessary, perform immunofluorescence labeling, and examine the tissue with confocal microscopy as previously demonstrated²⁴.

3. Monitoring Expansion of Antigen-specific, Luciferase-expressing CD8+ T Cells after Nanoparticle Vaccination with Whole Animal Imaging

1. Isolation of OVA_257-264-specific, luciferase-expressing CD8+ T cells from OT-I/Luc transgenic mice
   1. Euthanize an OT-I/Luc transgenic mouse with CO₂ asphyxiation and induce a pneumothorax according to an IACUC approved animal protocol. Harvest the spleen in a sterile manner by accessing the peritoneal cavity and carefully detaching the tissue from the pancreas²³, and place in 5 ml of 4 °C PBS + 2% FBS for transfer to tissue culture hood.
   2. Place the spleen on a 70 µm nylon strainer over a 50 ml conical centrifuge tube (up to 3 spleens at a time). Using a plunger from a 3 ml syringe, grind the cells through the strainer.
   3. Wash the plunger and the strainer with PBS + 2% FBS and discard. Bring the total volume to 10 ml/spleen in the 50 ml tube, take a small sample of the cell suspension to count with a hemocytometer, and centrifuge for 10 min at 300 x g.
   4. Using a commercially available magnetic negative selection kit, isolate the CD8+ T cell population by following the manufacturer’s instructions.
   5. After washing cells with PBS, count the number of isolated CD8+ T cells. To assess purity of the isolated CD8+ T cells, incubate ~20,000-30,000 cells in 20 µl of mouse CD16/32 antibody (0.025 mg/ml) for 10 min, then add 20 µl of αCD8-APC antibody (0.005 mg/ml) and incubate for 30 min. Perform all incubations at 4 °C in PBS + 1% w/v BSA. Perform flow cytometric analysis²⁵.
2. Adoptive transfer of isolated CD8+ T cells and visualization of their expansion post vaccination
   1. Perform adoptive transfer of isolated OT-I/Luc CD8+ T cells into naïve C57BL/6 mice by administering 1-10 × 10⁵ cells in a 200 µl volume of PBS via intravenous tail vein injection²² (day -1). Considering that fur and black skin patches in C57BL/6 mice may interfere with the bioluminescent signal, shaved albino C57BL/6 mice are ideal for these studies.
   2. After one day (day 0), administer the vaccine as described previously (section 2.2).
   3. Administer 150 mg of luciferin per kg mouse body weight intraperitoneally in a 300 µl volume of PBS. After 10 min, anesthetize the mice with isoflurane (as in step 2.2.1) and visualize OT-I/Luc CD8+ T cells by acquiring the bioluminescence signal for 5-10 min with a whole animal imaging system (IVIS; refer to Wilson²⁶ for detailed instruction). Repeat as necessary for longitudinal studies.

4. Peptide-MHC Tetramer Staining of PBMCs for Analysis of Antigen-specific CD8+ T Cells

Note: The following protocol procedure can be performed using either C57BL/6 mice adoptively transferred with OT-I/Luc CD8+ T cells or C57BL/6 mice without the adoptive transfer.

1. At a desired time point after vaccination, collect approximately 100 µl of blood (4-6 drops) from mice via submandibular bleeding technique²⁷ into a tube coated with K₂EDTA and invert several times to prevent clotting.
2. Transfer 100 µl of blood to a microcentrifuge tube, add 1 ml of lysis buffer, and incubate for 2 to 3 min in order to remove red blood cells (RBCs). Centrifuge samples for 5 min at 1,500 x g and remove the supernatant. If the pellet still appears red (indicating incomplete removal of RBCs), repeat the lysis step with a brief incubation (< 1 min) of lysis buffer.
3. Wash the remaining PBMCs with 1 ml of FACS buffer (PBS + 1% w/v BSA) and centrifuge at 1,500 x g for 5 min.
4. Aspirate the supernatant and resuspend the sample in 20 µl of mouse CD16/32 antibody (0.025 mg/ml) to block nonspecific and FcR-mediated antibody binding. Incubate for 10 min at RT.
5. Transfer cells from microcentrifuge tubes into 4 ml round bottom FACS tubes. Add 20 µl of H-2K^b OVA Tetramer-SIINFEKL-PE solution according to the manufacturer’s specifications to each sample and incubate for 30 min on ice.
6. Prepare the antibody cocktail (e.g., αCD8-APC, αCD44-FITC, and αCD62L-PECy7 antibodies (0.005, 0.005, and 0.002 mg/ml concentration, respectively)). Add 20 µl to each experimental sample, and incubate for 20 min on ice. Prepare single fluorophore controls by labeling cells with each fluorophore-tagged tetramer or antibody at the concentration indicated above.
7. Wash 2 times with FACS buffer and resuspend the final pellet in FACS buffer containing 2 µg/ml of DAPI. The cells are now ready for flow cytometry analysis (details and examples can be found in Scheffold²⁵).

Results

The steps involved in the synthesis of ICMVs are illustrated in Figure 1⁶. Briefly, a lipid film containing any lipophilic drugs or fluorescent dyes is hydrated in the presence of hydrophilic drugs. Divalent cations, such as Ca²⁺, are added to drive fusion of anionic liposomes into multilamellar vesicles. Dithiol crosslinker, such as DTT, is added to “staple” maleimide-functionalized lipids on apposing lipid layers, and finally remaining external maleimide groups are que...

Discussion

The protocol provided in this article describes the synthesis and characterization of a new lipid-based nanoparticle system, termed ICMVs, and provides the process of validating effectiveness of nanoparticle-based vaccine formulations to induce antigen-specific CD8+ T cell responses. ICMV synthesis is completed in all aqueous condition, which is a major advantage compared with other commonly used polymeric nanoparticle systems (e.g., poly(lactide-co-glycolide) acid particles), which typically require organic sol...

Materials

Name	Company	Catalog Number	Comments
1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (MPB)	Avanti Polar Lipids, INC.	870012
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)	Avanti Polar Lipids, INC.	850375
Monophosphoryl Lipid A (Synthetic) (PHAD™) (MPLA)	Avanti Polar Lipids, INC.	699800
20 mL glass vials	Wheaton	0334125D
Symphny Vacuum Oven	VWR	414004-580
Ovalbumin (OVA)	Worthington	3054
Bis-Tris Propane (BTP)	Fisher	BP2943
Q125 Sonicator (125W/20kHz)	Qsonica	Q125-110
Dithiothreitol (DTT)	Fisher	BP172
2 kDa Thiolated Polyethylene Glycol (PEG-SH)	Laysan Bio	MPEG-SH-2000-1g
Malvern ZetaSizer Nano ZSP	Malvern
ZetaSizer Cuvettes	Malvern	DTS1070
2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID)	Life Technologies	D-7757
Alexa Fluor 555-succinimidyl ester (AF555-NHS)	Life Technologies	A37571
Tissue-Tek OCT freezing medium	VWR	25608-930
Tissue Cryomolds	VWR	25608-922
3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging
C57BL/6 mice	Jackson	000664
Albino C57BL/6 mice	Jackson	000058
OT-1 C57BL/6 mice	Jackson	003831
70 μm nylon strainer	BD	352350
EasySep™ Mouse CD8+ T Cell Isolation Kit	StemCell	19853
IVIS® whole animal imaging system	Perkin Elmer
4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells
K2EDTA tubes	BD	365974
ACK lysis buffer	Life Technologies	A10492-01
Anti-CD16/32 Fc Block	Ebioscience	14-0161-86
H-2Kb OVA Tetramer	MBL	TS-5001-1C
Anti-CD8-APC	BD	553031
Anti-CD44-FITC	BD	553133
Anti-CD62L-PECy7	Ebioscience	25-0621-82
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)	SIGMA	D8417-10MG
CyAn Flow Cytometer	Beckman Coulter
FlowJo Software	FlowJo