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

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 &#8220;staple&#8221; maleimide-functionalized lipids on apposing lipid layers, and finally remaining external maleimide groups are que...

Watch this Scientific Journal Video about Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination at JoVE.com

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

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 ...

whole-animal-imaging-flow-cytometric-techniques-for-analysis-antigen

Research

JoVE Journal

Immunology and Infection

13.2K Views.  University of Michigan. 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.

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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Bioengineering

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

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

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

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Engineering

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

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

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

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.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Medicine

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

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

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

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).

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

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.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

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

Source: Simonetti, S., et al. <a href="https://app.jove.com/v/61867">A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice.</a> J. Vis. Exp. (2021)
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.

1. Preparation of medium and staining solution

	Prepare Complete Medium: Roswell Park Memorial Institute (RPMI) medium with 2 mM glutamine, 100 U/mL ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

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

To isolate antigen-specific CD8-positive T cells, macerate the spleens and lymph nodes from wild-type C57Black/6j mice through a sterile 70-micrometer cell strainer with frequent PBS rinses, and use a no-touch CD8-positive T cell isolation kit, according to the manufacturer&#39;s instructions, to eliminate the non-CD8-positive T cells.
After counting, collect the isolated CD8-positive T cells by centrifugation, and resuspend the pellet in 100 microliters of PBS, supplemented with 0.5% bovine serum albumin and 2-millimolar EDTA.
Then, incubate the cells with the nanoparticle artificial antigen-presenting cells such that there are 1 x 1011 peptide-loaded MHC-Ig, per 1 x 106 isolated CD8-positive T cells in a sterile 5-milliliter polystyrene round-bottom tube for 1 hour at 4 degrees Celsius with continual mixing.
At the end of the incubation, wash the nanoparticle cell suspension three times on the magnetic stand as demonstrated, re-suspending the artificial antigen-presenting cells and the enriched CD8-positive T cells in 500 microliters of fresh supplemented medium with 1% T cell growth factor, after the last wash.
For maximum cell recovery, it is critical to leave the particles in cells on the magnetic column for at least 2 minutes, and to aspirate the buffer carefully without disrupting the particles and cells subject to the magnetic field.
After counting, seed 2.5 x 105 enriched CD8-positive T cells and magnetic particle mixture per 160 microliters of supplemented medium plus 1% TCGF in a 96-well U-bottomed plate. On day three, feed the cells with 80 microliters of supplemented medium with 2% T cell growth factor per well, and return the plate to the cell culture incubator for four more days.
On day seven, harvest the stimulated cells into a 5-milliliter round-bottom tube for counting, and collect the cells by centrifugation. Resuspend the pellet in 0.5 milliliters of PBS supplemented with 0.05% sodium azide and 2% fetal bovine serum for counting, and aliquot 5 x 104 to 5 x 105 cells into new 5-milliliter round bottom tubes for antigen-specific staining.
Label the appropriate tubes with biotinylated MHC-Ig and anti-mouse CD8a for 1 hour at 4 degrees Celsius, followed by a centrifuge wash in fresh PBS, to remove any excess biotinylated immunoglobulin. Then, stain the samples with an appropriate streptavidin-conjugated secondary antibody, and an appropriate live/dead fixable dead cell stain for 15 minutes at 4 degrees Celsius, and read the cells on a flow cytometer to determine the specificity and number of the antigen-specific CD8-positive T cells, according to standard protocols.

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

Summary

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Chapters in this video

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

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

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

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

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

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

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

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

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

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