Name: Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination (Video) | JoVE Journal
Uploaded: 2015-04-29T15:00:00.000+00:00
Duration: 11 min 7 s
Description: Learn whole-animal imaging and flow cytometry techniques to analyze antigen-specific CD8+ T cell responses after nanoparticle vaccination in mice. This method enables non-invasive tracking of immune cell dynamics.

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5 minutes

This study was supported by the National Institute of Health grant 1K22AI097291-01 and by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000433. We also acknowledge Prof. Darrell Irvine at MIT and Prof. Matthias Stephan at Fred Hutchinson Cancer Center for their contribution on the initial work on the vaccine nanoparticles and OT-I/Luc transgenic mice.

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
<ol>
	<li>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 &#181;mol per batch (i.e., 500 &#181;g of DOPC and 630 &#181;g of MPB) in a 20 ml glass vial (diameter = 28 mm and height = 61 mm).</li>
	<li>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.</li>
	<li>Hydrate the lipid film by adding 200 &#181;l 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.</li>
	<li>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.</li>
	<li>Add 4 &#181;l of 150 mM dithiothreitol (DTT) to each batch (working concentration 2.4 mM), vortex, and briefly centrifuge using a tabletop microcentrifuge.</li>
	<li>Add 40 &#181;l of 200 mM CaCl2 and mix with the pipette (working concentration 33 mM). Incubate samples at 37 &#176;C for 1 hr to allow crosslinking of MPB-containing lipid layers with DTT.</li>
	<li>Centrifuge samples at 20,000 x g for 15 min, remove the supernatant, and resuspend in 200 &#181;l of ddiH2O.</li>
	<li>Repeat step 1.7 and centrifuge again after the second ddiH2O wash to remove CaCl2, unreacted DTT, and unencapsulated cargo materials from the supernatant.</li>
	<li>Prepare 10 mg/ml of 2 kDa polyethylene glycol-thiol (PEG-SH) in ddiH2O. Resuspend each ICMV sample in 100 &#181;l of PEG-SH solution and incubate at 37 &#176;C for 30 min.</li>
	<li>Perform two ddiH2O washes (step 1.7) and resuspend the final ICMV pellet in PBS and store at 4 &#176;C. Prior to use, mix the ICMV suspension, as particles may settle to the bottom after prolonged storage.</li>
	<li>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&#8217;s protocol).</li>
</ol>
2. Examination of Lymph Node Draining of Fluorescence-tagged ICMVs with Confocal Microscopy
<ol>
	<li>Preparation of ICMVs loaded with fluorophore-tagged antigen and lipophilic fluorescent dye
	<ol>
		<li>Prepare fluorophore-tagged protein, such as ovalbumin reacted with Alexa Fluor 555-succinimidyl ester, according to the manufacturer&#8217;s instruction.</li>
		<li>To prepare ICMVs tagged with fluorophore in the lipid shell, add lipophilic fluorescent dye, (e.g., 1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-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.</li>
	</ol>
	</li>
	<li>Subcutaneous administration of nanoparticles at tail base
	<ol>
		<li>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.</li>
		<li>Spray the base of the tail with 70% ethanol to sanitize and wet the fur&#160;Part the wet hair to expose a small patch of visible skin, which can be used to visualize the needle under the skin.</li>
		<li>Prepare particle injection suspension containing desired amount of antigen and adjuvant per 100 &#181;l of vaccination dose in PBS (e.g., 10 &#181;g OVA and 0.3 &#181;g MPLA per 100 &#181;l of injection dose has been used in the past6,9).</li>
		<li>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 &#181;l of the particle suspension22.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of lymph node cryosections and examination with confocal microscopy.
	<ol>
		<li>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 &#176;C PBS.</li>
		<li>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 &#176;C freezer.</li>
		<li>Cut tissue sections 5-10 &#181;m thick in a cryostat set at -20 &#176;C24.</li>
		<li>If necessary, perform immunofluorescence labeling, and examine the tissue with confocal microscopy as previously demonstrated24.</li>
	</ol>
	</li>
</ol>
3. Monitoring Expansion of Antigen-specific, Luciferase-expressing CD8+ T Cells after Nanoparticle Vaccination with Whole Animal Imaging
<ol>
	<li>Isolation of OVA257-264-specific, luciferase-expressing CD8+ T cells from OT-I/Luc transgenic mice
	<ol>
		<li>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 &#176;C PBS + 2% FBS for transfer to tissue culture hood.</li>
		<li>Place the spleen on a 70 &#181;m 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.</li>
		<li>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.</li>
		<li>Using a commercially available magnetic negative selection kit, isolate the CD8+ T cell population by following the manufacturer&#8217;s instructions.</li>
		<li>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 &#181;l of mouse CD16/32 antibody (0.025 mg/ml) for 10 min, then add 20 &#181;l of &#945;CD8-APC antibody (0.005 mg/ml) and incubate for 30 min. Perform all incubations at 4 &#176;C in PBS + 1% w/v BSA. Perform flow cytometric analysis25.</li>
	</ol>
	</li>
	<li>Adoptive transfer of isolated CD8+ T cells and visualization of their expansion post vaccination
	<ol>
		<li>Perform adoptive transfer of isolated OT-I/Luc CD8+ T cells into na&#239;ve C57BL/6 mice by administering 1-10 &#215; 105 cells in a 200 &#181;l 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.</li>
		<li>After one day (day 0), administer the vaccine as described previously (section 2.2).</li>
		<li>Administer 150 mg of luciferin per kg mouse body weight intraperitoneally in a 300 &#181;l 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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>At a desired time point after vaccination, collect approximately 100 &#181;l of blood (4-6 drops) from mice via submandibular bleeding technique27 into a tube coated with K2EDTA and invert several times to prevent clotting.</li>
	<li>Transfer 100 &#181;l 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 (&#60; 1 min) of lysis buffer.</li>
	<li>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.</li>
	<li>Aspirate the supernatant and resuspend the sample in 20 &#181;l of mouse CD16/32 antibody (0.025 mg/ml) to block nonspecific and FcR-mediated antibody binding. Incubate for 10 min at RT.</li>
	<li>Transfer cells from microcentrifuge tubes into 4 ml round bottom FACS tubes. Add 20 &#181;l of H-2Kb OVA Tetramer-SIINFEKL-PE solution according to the manufacturer&#8217;s specifications to each sample and incubate for 30 min on ice.</li>
	<li>Prepare the antibody cocktail (e.g., &#945;CD8-APC, &#945;CD44-FITC, and &#945;CD62L-PECy7 antibodies (0.005, 0.005, and 0.002 mg/ml concentration, respectively)). Add 20 &#181;l 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.</li>
	<li>Wash 2 times with FACS buffer and resuspend the final pellet in FACS buffer containing 2 &#181;g/ml of DAPI. The cells are now ready for flow cytometry analysis (details and examples can be found in Scheffold25).</li>
</ol>

Perkin Elmer provided the production cost incurred during the publication of this article.

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...

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 pathogens1,2. 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 activation3-5. In this report, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; 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 compartments6-9. 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 cells9,10. Finally, we describe tetramer staining of peripheral blood mononuclear cells (PBMCs) for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles6,9.
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 APCs12,13. 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 administration14,15. 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 OVA257-264 peptide (SIINFEKL) immunodominant CD8+ T cell epitope, which allows detailed immunological analyses of antigen-specific T cell responses for initial vaccine development16,17. 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-2Kb). From these OT-I/Luc mice, luciferase-expressing, OT-I CD8+ T cells can be isolated and prepared for adoptive transfer into na&#239;ve 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 system9,10. This non-invasive whole-body imaging technique has been used with other viral or tumor antigens in the past18-20, 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.

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules&lt;/strong&gt;</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-[4-(&lt;em&gt;p&lt;/em&gt;-maleimidophenyl)butyramide] (sodium salt) (MPB)</td><td>Avanti Polar Lipids, INC.</td><td>870012</td><td></td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipids, INC.</td><td>850375</td><td></td></tr><tr><td>Monophosphoryl Lipid A (Synthetic) (PHAD&amp;trade;) (MPLA)</td><td>Avanti Polar Lipids, INC.</td><td>699800</td><td></td></tr><tr><td>20 ml glass vials</td><td>Wheaton</td><td>0334125D</td><td></td></tr><tr><td>Symphny Vacuum Oven</td><td>VWR</td><td>414004-580</td><td></td></tr><tr><td>Ovalbumin (OVA)</td><td>Worthington</td><td>3054</td><td></td></tr><tr><td>Bis-Tris Propane (BTP)</td><td>Fisher</td><td>BP2943</td><td></td></tr><tr><td>Q125 Sonicator (125&amp;nbsp;W/20&amp;nbsp;kHz)</td><td>Qsonica</td><td>Q125-110</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher</td><td>BP172</td><td></td></tr><tr><td>2 kDa Thiolated Polyethylene Glycol (PEG-SH)</td><td>Laysan Bio</td><td>MPEG-SH-2000-1g</td><td></td></tr><tr><td>Malvern ZetaSizer Nano ZSP&amp;nbsp;</td><td>Malvern</td><td></td><td></td></tr><tr><td>ZetaSizer Cuvettes</td><td>Malvern</td><td>DTS1070</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy&lt;/strong&gt;</td></tr><tr><td>1,1&#039;-Dioctadecyl-3,3,3&#039;,3&#039;-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID)</td><td>Life Technologies</td><td>D-7757</td><td></td></tr><tr><td>Alexa Fluor 555-succinimidyl ester (AF555-NHS)</td><td>Life Technologies</td><td>A37571</td><td></td></tr><tr><td>Tissue-Tek OCT freezing medium&amp;nbsp;</td><td>VWR</td><td>25608-930</td><td></td></tr><tr><td>Tissue Cryomolds</td><td>VWR</td><td>25608-922</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging&lt;/strong&gt;</td></tr><tr><td>C57BL/6 mice</td><td>Jackson</td><td>000664</td><td></td></tr><tr><td>Albino C57BL/6 mice</td><td>Jackson</td><td>000058</td><td></td></tr><tr><td>OT-1 C57BL/6 mice</td><td>Jackson</td><td>003831</td><td></td></tr><tr><td>70 &amp;mu;m nylon strainer</td><td>BD</td><td>352350</td><td></td></tr><tr><td>EasySep&amp;trade; Mouse CD8+ T Cell Isolation Kit</td><td>StemCell</td><td>19853</td><td></td></tr><tr><td>IVIS&amp;reg; whole animal imaging system</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells&lt;/strong&gt;</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;EDTA tubes</td><td>BD</td><td>365974</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Life Technologies</td><td>A10492-01&amp;nbsp;</td><td></td></tr><tr><td>Anti-CD16/32 Fc Block</td><td>Ebioscience</td><td>14-0161-86</td><td></td></tr><tr><td>H-2K&lt;sup&gt;b&lt;/sup&gt; OVA Tetramer</td><td>MBL</td><td>TS-5001-1C</td><td></td></tr><tr><td>Anti-CD8-APC</td><td>BD</td><td>553031</td><td></td></tr><tr><td>Anti-CD44-FITC</td><td>BD</td><td>553133</td><td></td></tr><tr><td>Anti-CD62L-PECy7</td><td>Ebioscience</td><td>25-0621-82</td><td></td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>SIGMA</td><td>D8417-10MG</td><td></td></tr><tr><td>CyAn Flow Cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>FlowJo Software</td><td>FlowJo</td><td></td><td></td></tr></tbody></table>

whole-animal imaging and flow cytometric techniques for analysis of antigen-specific cd8+ t cell responses after nanoparticle vaccination

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

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 &#8220;pathogen-mimicking&#8221; 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.

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 quenched in a r...

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.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

The overall goal of the following experiment is to synthesize and characterize lipid-based vaccine nanoparticles and to assess their ability to elicit antigen-specific cytotoxic T lymphocyte expansion in vivo. This is achieved by engineering pathogen mimicking inter bilayer, cross-linked multilam vesicles or I CMVs co loaded with antigen and adjuvant for vaccination of naive mice. Adoptively transferred with luciferase expressing antigen specific CD eight positive T cells.In the second step, the expansion of the antigen-specific CD eight positive T cells is evaluated by whole animal in vivo imaging. Peripheral blood mononuclear cells harvested from the ICMV immunized animals are then stained with fluorescently tagged tetramers and analyzed by flow cytometry to quantify the frequency of the endogenous antigen-specific CD eight positive T cells within the systemic compartment. Ultimately, bioluminescent imaging can be used to track the expansion of the antigen-specific cytotoxic T lymphocytes in response to the nanoparticle vaccination.These methods can help answer key preliminary questions in vaccine design, such as what is the extent of cytotoxic tial lymphocyte expansion and how do these cells traffic to various tissues in response to nanoparticle vaccination? The main advantage of this technique over existing methods, like in-situ T cell analysis after tissue necropsy, is that in vivo imaging of TT cell response is non-invasive. Allowing longitudinal monitoring of the same animal Begin by mixing a one-to-one molar ratio of DOPC and MPB in chloroform, maintaining a total lipid quantity of 1.26 micromoles per batch in a 20 milliliter glass vial.Next, add lipophilic cargo to the lipid solution at the desired concentration and purge the solution with extra dry nitrogen gas to thoroughly remove the organic solvent. Place the sample under vacuum overnight the next morning at 200 microliters of 10 millimolar BTP containing the water soluble cargo of interest to hydrate the lipid film vortex the resulting solution for 10 seconds every 10 minutes for one hour at room temperature. Then transfer the contents to a 1.5 milliliter micro centrifuge tube.Next place the tube in an ice water bath with continuous sonication using the 40%intensity setting on a 125 watt 20 kilohertz probe tip soat. After five minutes, add four microliters of 150 millimolar DTT to the tube vortex to mix and spin down the sample in a micro centrifuge. Then mix 40 microliters of 200 millimolar calcium chloride into the sample, followed by cross-linking of the MPB containing lipid layers with DTT at 37 degrees Celsius for one hour.At the end of the incubation, spin down the solution three times at 200 microliters of double distilled water during the second and third centrifugation to remove the remaining calcium chloride unre reacted DTT and Unencapsulated cargo materials from the supernatant. After the last spin, we suspend the I CMVs in 100 microliters of freshly prepared pegol for 30 minutes at 37 degrees Celsius. Then wash the vesicles two more times in double distilled water.Resus suspending the final pellet in PBS and store the ICM VS at four degrees Celsius. To characterize the particles, mix the cold ICMV suspension before diluting a small aliquot of the sample in a total volume of one milliliter of double distilled water. Then place the I CMVs in a zeta sizer cell and use a dynamic light scattering and zeta potential measuring system to measure the particle size poly dispersity index and zeta potential of the samples according to the manufacturer's instructions.To isolate ova specific luciferase expressing CD eight positive T cells. First harvest the spleens from OT one luciferase transgenic mice. Place the tissues in five milliliters of ice cold PBS and 2%FBS and then in a tissue culture hood.Use the plunger from a three milliliter syringe to grind up to three spleens at once through a 70 micron strainer into a 15 milliliter conical tube. Wash the plunger and the strainer with PBS and 2%FBS. Then bring the total volume of the cyte suspension to 10 milliliters per spleen.Determine the total cell number and then spin down the cells. Next, use a commercially available magnetic negative selection kit to isolate the CD eight positive spleen cells according to the manufacturer's instructions. Then wash the T cells in PBS.Count them and incubate two to three times 10 to the fifth of the cells in 20 microliters of mouse CD 1632 blocking antibody. After 10 minutes, add 20 microliters of anti CD eight A PC antibody to the cells for 30 minutes and assess the purity of the magnetic bead isolated CD eight positive T-cell population by flow cytometry. Next adoptively transfer one to 10 times 10 to the fifth OT one luciferase CD eight positive T cells into naive shaved albino C 57 black six mice in 200 microliters of PBS via intravenous tail vein injection 24 hours later, administer the vaccine at the appropriate number of days post vaccination.Inject the animals with 150 milligrams per kilogram of Lucifer after 10 minutes, acquire the bioluminescence signal for five to 10 minutes with a whole animal imaging system to visualize the OT one Lucifer CD eight positive T cells to determine the number of antigen specific CD eight positive T cells at the appropriate time. Post vaccination, collect approximately 100 microliters of blood from the vaccinated mice via the submandibular bleeding technique into a tube coated with potassium EDTA, invert the tube several times to prevent clotting. Then transfer 100 microliters of blood to a micro centrifuge tube and add one milliliter of a CK lysis buffer.After two to three minutes, centrifuge the cells and remove the supernatant. Wash the remaining pbmc in one milliliter of fax buffer and block the nonspecific binding with CD 1632 as just demonstrated. After 10 minutes, divide the cell suspension into four milliliter round bottom fax tubes and add 20 microliters of H two KB over tetramer syn fecal PE solution to each sample according to the manufacturer's specifications.After 30 minutes on ice, add 20 microliters of the appropriate antibody cocktail to each experimental sample and the single fluoro four control antibodies for each fluoro four tag tetramer or antibody to each control sample. Finally, after another 20 minute incubation, wash all of the samples in fax buffer resuspend the pellets in fax buffer supplemented with DPI, and analyze the samples by flow cytometry I CMVs co loaded with fluoro four tact protein antigens and fluorescent lipophilic dyes allow the visualization of antigen and nanoparticle delivery in vivo. For example, confocal microscopy imaging indicates that soluble antigen delivered subcutaneously at the base of the tail, reaches the draining lymph nodes within four hours of administration and is cleared by 24 hours.In contrast, overloaded I CMVs are first detected at the periphery of the draining lymph nodes by 24 hours after administration with a continued accumulation that results in a large deposit of ova I CMVs within the draining lymph tissue. By day four, indeed bioluminescence imaging of C 57 BL six mice adoptively transferred with OT one Luciferase CD eight positive T cells on the day of vaccination demonstrates a minimal background level of OT one luciferase tcell by day four post vaccination. However, mice immunized with the ova MPI CMVs exhibit a robust bioluminescence signal within the draining inguinal lymph nodes.The soluble ova vaccinated mice, on the other hand demonstrate a much reduced expansion of the OT one Lucifer CD eight positive T-cell within the draining lymph nodes. Further mice immunized with soluble ova and MPLA without any adoptive transfer of antigen Specific T cells do not exhibit an appreciable expansion of ova specific CD eight positive T cells as indicated by weekly monitoring of the pbmc ICMV vaccination, however, elicits a significantly stronger and endogenous CD eight positive T cell response achieving a peak 28%of the syn fecal tetramer positive T cells within the CD eight positive T cell compartment among pbmc by day 41. After watching this video, you should have a good understanding of how to synthesize lipid-based nanoparticles, perform immunizations, and visualize and quantify the CTL expansion in vivo.These methods can be further modified to reveal additional information about the CTL response. For example, the teer staining essay can be supplemented with additional markers such as CD 1 27, BCL two, and K lrg one to assess the memory phenotype of the cells.

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Research

JoVE Journal

Immunology and Infection

This video describes a protocol for isolating rare-antigen-specific CD8+ T cells using magnetic nanoparticle-based artificial antigen-presenting cells (aAPCs) functionalized with antigenic peptide-loaded MHC-Igs and anti-CD28 antibodies. Interaction of the aAPCs with CD8+ T cells specifically expressing the antigenic peptide-targeting T cell receptors, followed by their expansion in vitro, results in the enrichment and expansion of this CD8+ T cell population.

Nanoparticle-Based Enrichment and Expansion of Antigen-Specific CD8+ T Cells

This video demonstrates an assay to analyze antigen-specific CD8+ T cells in different cell cycle stages. Fluorescently-labeled viable cells are selected using flow cytometry, and the cells in the different cell cycle stages are identified by their DNA content using antibodies against cell proliferation-associated nuclear protein and a nucleic acid stain.

An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells

The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

This video demonstrates an in-situ fluorescence multispectral imaging technique to analyze different subpopulations of infiltrating T cells in tumor tissue. A renal cell carcinoma tissue cryosection is treated with antibodies to label CD8+ T cells that express specific inhibitory receptors and exhibit an immunotherapy-resistant phenotype. The labeled section is visualized using a multispectral microscope to identify the distinct CD8+ T cell subpopulations.

Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

Learn whole-animal imaging and flow cytometry techniques to analyze antigen-specific CD8+ T cell responses after nanoparticle vaccination in mice. This method enables non-invasive tracking of immune cell dynamics.

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination (Video) | JoVE Journal

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

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