EBA (DGE-1746891) and KRR (DGE-1232825) thank the NSF Graduate Research Fellowship program for support. RAM thanks the National Research Service Award NIH NCI F31 (F31CA214147) and the Achievement Rewards for College Scientists Fellowship for support. The authors thank the NIH (R01EB016721 and R01CA195503), the Research to Prevent Blindness James and Carole Free Catalyst Award, and the JHU Bloomberg-Kimmel Institute for Cancer Immunotherapy for support.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University.
1. Fabrication of Spherical PLGA Particles of Tunable Size
<ol>
	<li>Preparation of materials for particle synthesis
	<ol>
		<li>Prepare 5% w/w polyvinyl alcohol (PVA) solution.
		<ol>
			<li>Add 500 mL of deionized (DI) water to an Erlenmeyer flask with a magnetic stir bar and place on hot plate stirrer at 500 rpm and monitor temperature with thermometer. Cover flask with tinfoil to prevent evaporation.</li>
			<li>When water temperature reaches approximately 70 &#176;C, add 25 g total of PVA in small batches over time, waiting for PVA to dissolve before adding more.</li>
			<li>Once all PVA is dissolved (typically 30-60 min), let solution cool and sterile filter. Store at 4 &#176;C for future use.</li>
		</ol>
		</li>
		<li>Prepare film casting solution of 10% w/w PVA and 2% w/w glycerol.
		<ol>
			<li>Add 500 mL of DI water to an Erlenmeyer flask with a magnetic stir bar. Add 8 mL of glycerol at room temperature and mix by trituration.</li>
			<li>Place flask on hot plate stirrer at 500 rpm and monitor temperature with thermometer. Cover flask with tinfoil to prevent evaporation.</li>
			<li>When solution temperature reaches approximately 70 &#176;C, add 50 g total of PVA in small batches over time, waiting for PVA to dissolve before adding more.</li>
			<li>Once all PVA is dissolved (typically 60 min), let solution cool and sterile filter using a bottle-top vacuum filter system with a pore size of 0.22 &#956;m. Store at room temperature for future use.</li>
		</ol>
		</li>
		<li>Prepare a 50 mL 1% w/w PVA solution. Add 40 mL of DI water and 10 mL of 5% PVA solution (made in 1.1.1) to a 100-150 mL beaker.</li>
		<li>Prepare a 100 mL 0.5% w/w PVA solution. Add 90 mL of DI water and 10 mL of 5% PVA solution to a 150-250 mL beaker.</li>
		<li>Add a magnetic stir bar to the 0.5% PVA solution and place in a chemical hood on a stir plate at room temperature at 500 rpm.</li>
	</ol>
	</li>
	<li>Microparticle synthesis
	<ol>
		<li>Weigh out 100 mg of poly(lactic-co-glycolic acid) (PLGA) into a scintillation vial and dissolve in 5 mL of dichloromethane (DCM). Vortex to dissolve the PLGA.</li>
		<li>Place homogenizer in the 50 mL 1% PVA solution so that the homogenizer is as close to the bottom of the beaker as possible without touching it. Turn on homogenizer and adjust to desired speed&#8212;3,200 rpm for 5 &#956;m diameter particles, 5,000 rpm for 3 &#956;m, 15,000 rpm for 1 &#956;m (increasing homogenization speed decreases particle size). Once at the desired speed, add the PLGA solution to the beaker and homogenize for 1 minute.</li>
		<li>After homogenization, pour the 1% PVA, PLGA microparticle solution into the 100 mL 0.5% PVA solution on a stir plate and stir for at least 4 hours for solvent evaporation in a chemical hood.</li>
		<li>Wash the particles 3 times in DI water.
		<ol>
			<li>Pour the particle solution into 50 mL conical tubes and centrifuge at 3000 x g for 5 minutes. Pour out supernatant and add approximately 20 mL of DI water.</li>
			<li>Resuspend particles by vortexing. Once resuspended, fill up conical tubes to 50 mL with DI water.</li>
			<li>Wash again the same way two more times.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Nanoparticle synthesis
	<ol>
		<li>Weigh out 200 mg of PLGA into a scintillation vial and dissolve in 5 mL of DCM. Vortex to dissolve the PLGA.</li>
		<li>Place a beaker with 50 mL 1% PVA solution into container filled with ice. Place the sonicator probe in the beaker as close to the bottom as possible without touching. Begin sonication and immediately add PLGA solution into the beaker. Sonicate with a power of 12 W for 2 minutes to generate nanoparticles with an approximate diameter of 200 nm.</li>
		<li>After sonication, pour the 1% PVA, PLGA nanoparticle solution into a 100 mL 0.5% PVA solution on a stir plate and stir for at least 4 hours for solvent evaporation in a chemical hood.</li>
		<li>Pour the particle solution into 50 mL conical tubes and centrifuge at 3,000 x g for 5 minutes to remove microparticles. Remove supernatant and pour into high speed centrifuge tubes.</li>
		<li>Wash the particles 3 times with DI water. Centrifuge at 40,000 x g for 15 minutes. Pour out supernatant and resuspend in DI water by vortexing. Wash again the same way two more times.</li>
	</ol>
	</li>
</ol>
2. Fabrication of Polymeric Particles of Tunable Shape
<ol>
	<li>After washing PLGA particles three times, resuspend the particles in approximately 1 mL of DI water. Add the film casting solution to particles for final particle concentration of 2.5 mg/mL particles.</li>
	<li>Pipette the particle suspension in 10 mL aliquots into 75 x 50 mm rectangular Petri dishes for one-dimensional stretching or in 15 mL aliquots into 100 x 100 mm square Petri dishes for two-dimensional stretching. Remove bubbles by either pipette or pushing bubbles to the side and let films dry overnight in a chemical hood.</li>
	<li>Once films have dried, remove films from plastic dishes with tweezers and cut off edges of films with scissors. Save edges in a 50 mL conical tube to be used as spherical particles.
	<ol>
		<li>Load a film onto the automated thin film stretching device by mounting a film onto aluminum blocks. For 1D stretching, mount film onto aluminum blocks of one axis of the stretcher (Figure 2) by placing two short edges of film in between two pieces of neoprene rubber. Using an Allen wrench, screw the metal grips on top of rubber to hold the film in place. For 2D stretching, mount all four edges onto four aluminum blocks to stretch on both axes (Figure 2).</li>
		<li>Measure and record the length of film in between aluminum blocks on one axis for 1D stretching or both axes for 2D stretching. Calculate the distance required to stretch the film in one or two directions based on desired fold-stretch.</li>
		<li>Place the film-loaded stretching device in an oven at 90 &#176;C and bring the film to temperature over 10 minutes. Place a large beaker in the oven with a small amount of water.</li>
		<li>Stretch film.</li>
		<li>When stretching is completed, remove the stretching device from oven and let the film cool to room temperature for 20 minutes.</li>
		<li>For 1D stretching, cut the film out of the stretching device at the edges. For 2D stretching, cut out and save the center square of film that is uniformly stretched on both axes. Place films in conical tubes with 2 films per tube and discard the rest of the film.</li>
		<li>Add approximately 25 mL of DI water to each conical tube and vortex until the films are dissolved.</li>
		<li>Once the films are dissolved, wash particles 3 times with DI water.
		<ol>
			<li>For microparticles, fill up conical tubes to 50 mL and centrifuge at 3000 x g for 5 minutes, pour out supernatant, add approximately 20 mL of DI water, and vortex to resuspend particles.</li>
			<li>For nanoparticles, transfer particles to high speed centrifuge tubes and centrifuge at 40,000 x g for 15 minutes, pour out supernatant, add approximately 20 mL of DI water, and vortex to resuspend particles.</li>
		</ol>
		</li>
		<li>After the third wash, resuspend particles in approximately 1 mL of DI water. Record the weight of the microcentrifuge tube and add particles to the tube. Freeze particles in a -80 &#176;C freezer for 1 hour or flash freeze in liquid nitrogen. Once frozen, lyophilize particles overnight.</li>
		<li>Once lyophilized, weigh particles in the microcentrifuge tube and subtract recorded weight of the empty tube to determine the weight of lyophilized particles.</li>
	</ol>
	</li>
</ol>
3. Surface Protein Conjugation to Create Artificial Antigen Presenting Cells
<ol>
	<li>Prepare 2-(N-Morpholino)ethanesulfonic acid (MES) buffer. Make a 0.1 M solution of MES in water and adjust the pH to 6.0 by titration with 1M sodium hydroxide (NaOH).</li>
	<li>Resuspend lyophilized PLGA/PBAE micro/nanoparticles at 20 &#956;g/mL in MES buffer by vortexing. Fill a polypropylene microcentrifuge tube with 900 &#956;L of MES buffer and add 100 &#956;L of the particle solution to the tube.</li>
	<li>Prepare EDC/NHS solution. Dissolve 40 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 48 mg N-hydroxysulfoxuccinimide (NHS) in 1 mL MES buffer.</li>
	<li>Add 100 &#956;L EDC/NHS solution to the particles and vortex to mix.</li>
	<li>Incubate the tube on an inverter at room temperature for 30 minutes.</li>
	<li>Spin down microparticles at 5,000 x g for 5 minutes, or nanoparticles at 17,000 x g for 5 minutes. Discard the supernatant and resuspend in 1 mL PBS by vortexing (microparticles) or sonicating at 2-3 W for 5 seconds (nanoparticles).</li>
	<li>For microparticles, add 8 &#956;g of the desired signal 1 protein, and 10 &#956;g anti-mouse CD28 (clone 37.51) to the particles. For nanoparticles, add 16 &#956;g signal 1 and 20 &#956;g anti-mouse CD28. Add PBS to bring the volume in the tube to 1.1 mL.</li>
	<li>Incubate the tube on an inverter at 4 &#176;C overnight.</li>
	<li>The next day, wash the particles. Spin down microparticles at 5,000 x g for 5 minutes, or nanoparticles at 17,000 x g for 5 minutes. Discard the supernatant, and resuspend particles in 1 mL of sterile PBS by vortexing (microparticles) or sonication at 2-3 W for 5 seconds (nanoparticles). Repeat twice.</li>
	<li>Resuspend the particles at the desired concentration in culture medium for immediate use. For long-term storage, resuspend particles at 10 mg/mL in a 100 mM sucrose solution. Freeze, lyophilize, and store the particles at -80 &#176;C.</li>
</ol>
4. Characterization and Evaluation of aAPC
<ol>
	<li>Characterization of aAPC size and shape (Figure 3)
	<ol>
		<li>Characterize microparticle size and shape by imaging particles using scanning electron microscopy (SEM). For SEM imaging, spread lyophilized particles onto carbon tape adhered to an aluminum tack and sputter coat with gold-palladium.</li>
		<li>Analyze size and aspect ratio of particles using image analysis software.</li>
		<li>To determine particle size, set scale using scale bar and measure particle diameter. Repeat for approximately 100 particles to determine the average particle diameter and generate a histogram of particle sizes.</li>
		<li>To determine aspect ratio, measure the distance across the long axis and the short axis of particles and divide long axis by short axis. Repeat for approximately 50 particles of each shape to determine the average particle aspect ratio for each shape and generate histograms.</li>
		<li>Characterize nanoparticle size and shape by imaging particles using transmission electron microscopy (TEM). Analyze size and aspect ratio of nanoparticles using image analysis software as described in 5.1.2. Alternatively, measure spherical nanoparticle size using dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA).</li>
	</ol>
	</li>
	<li>Protein Conjugation Efficiency
	<ol>
		<li>Prepare aAPC according to Methods 1-3, but in Step 3.7 use fluorescently labeled signal 1 protein and anti-mouse CD28. After conjugation and washing, resuspend the aAPC in 1 mL PBS for a final concentration of 2 mg/mL aAPC.</li>
		<li>Prepare protein standards in a black polystyrene 96-well microplate. Add 5 &#956;g of fluorescently labeled signal 1 protein to PBS in the first well of the plate and make 10 1:2 dilutions in PBS across the row of the plate. Leave the last well blank. Repeat this step to generate another set of standards with fluorescently labeled anti-CD28.</li>
		<li>Pipette 100 &#956;L of the aAPC solution into replicate wells of the black polystyrene microplate in triplicate.</li>
		<li>Read the fluorescence on a fluorescence plate reader at the appropriate wavelengths. Use the fluorescence readings from the protein standards to generate standard curves for each fluorescent antibody. Using the standard curve, calculate the concentrations of signal 1 and anti-CD28 in each sample well, and then calculate the amount of surface protein and conjugation efficiency (Figure 4A, Figure 5A).</li>
	</ol>
	</li>
	<li>Evaluation of aAPC for in vitro stimulation of CD8+ T cells
	<ol>
		<li>Prepare B&#8217; media (RPMI medium supplemented with L glutamine, 10% FBS, 1% Non-essential amino acid solution, 1% Sodium pyruvate, 1% MEM Vitamin solution, 92 &#956;M &#946;-mercaptoethanol, 10 ng/mL ciprofloxacin, and 30 U/mL IL-2.</li>
		<li>Sacrifice a Black 6 mouse via carbon dioxide exposure.</li>
		<li>Harvest the spleen from the mouse according to a previously established protocol16. Collect the spleen in a 50 mL conical tube with 10- 15 mL PBS. Using a pestle, mash the spleen through a 70 &#181;m cell strainer into a 50 mL conical tube. During mashing, wash the strainer with 40 mL PBS.</li>
		<li>Spin the splenocytes at 300 x g for 5 minutes. Pour off the supernatant and resuspend the splenocytes in 4 mL of Ack Lysing Buffer to lyse the red blood cells. Allow the tube to sit undisturbed for 1 minute, then add PBS to bring the volume in the tube to 20 mL.</li>
		<li>Spin the cells at 300 x g for 5 minutes. Pour off the supernatant and resuspend the cells in 1 mL of PBS. Count the cells using a hemocytometer.</li>
		<li>Spin the cells at 300 x g for 5 minutes and resuspend in the desired volume of cell separation buffer. Isolate CD8+ T cells from the single cell suspension using a CD8+ negative selection T cell isolation kit, following the manufacturer&#8217;s protocol.</li>
		<li>Following magnetic separation, spin CD8+ T cells at 300 x g for five minutes and remove the cell separation buffer. Resuspend cells in 1 mL of PBS and count the cells. Label cells with carboxyfluorescein succinyl ester (CFSE) according to the manufacturer&#8217;s protocol.</li>
		<li>Incubate 8,333 CD8+ T cells and 0.0833 mg (or desired dose) of aAPC in 150 &#956;L B&#8217; media in each well of a 96 well U-bottom tissue culture-treated plate.</li>
		<li>Incubate at 37 &#176;C for 7 days. After 3-4 days, refresh the culture medium by adding 75 &#956;L fresh medium to each well.</li>
		<li>After 3 days of incubation, analyze CFSE labelled cells on a flow cytometer to assess proliferation. Each peak on the flow cytometry CFSE histogram represents a generation of cells due to the CFSE dilution with each successive cell division.</li>
		<li>After 7 days, use a hemocytometer to count the number of cells in each well. Prior to counting, stain dead cells with a Trypan Blue solution. Exclude dead cells from final cell counts. Normalize the final cell concentration to the initial concentration to calculate the fold-expansion (Figure 4 and Figure 5).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol details a versatile method for the precise generation of anisotropic polymeric particles. The thin film stretching technique described here is scalable, highly reproducible and inexpensive. Alternative techniques for generating anisotropic particles suffer from many limitations, including high cost, low throughput, and limited particle size. The thin film stretching approach is also advantageous because the particles are modified to be anisotropic after synthesis, and, as a result, is compatible with a wide...

Artificial antigen presenting cells (aAPC) have shown promise as immunomodulatory agents because they can generate a robust antigen-specific T cell response. Essential to these platforms are their ability to efficiently present crucial signals for T cell activation. Acellular aAPC are an attractive alternative to cell-based aAPC because they are easier and less costly to fabricate, face fewer challenges during scale-up and translation, and alleviate risks associated with cell-based therapies. Acellular aAPC also allow for a high degree of control over signal presentation parameters and physical properties of the surface that will interface with T cells1.
aAPC must recapitulate a minimum of two signals essential for T cell activation. Signal 1 provides antigen recognition and occurs when the T cell receptor (TCR) recognizes and engages with an MHC class I or II bearing its cognate antigen, culminating in signaling through the TCR complex. To bypass the antigen specificity requirement, aAPC systems often bear an agonistic monoclonal antibody against the CD3 receptor, which nonspecifically stimulates the TCR complex. Recombinant forms of MHC, particularly MHC multimers, have also been used on the surface of aAPC to provide antigen specificity2,3. Signal 2 is a costimulatory signal that directs T cell activity. To provide the costimulation necessary for T cell activation, the CD28 receptor is generally stimulated with an agonistic antibody presented on the aAPC surface, although other costimulatory receptors such as 4-1BB have been successfully targeted4. Signal 1 and 2 proteins are typically immobilized on the surface of rigid particles to synthesize aAPC. Historically, aAPC have been fabricated from a variety of materials, including polystyrene4,5 and iron dextran6. Newer systems utilize biodegradable polymers like poly(lactic-co-glycolic acid) (PLGA) to generate aAPC that can be easily coupled to signal proteins, are suitable for direct administration in vivo, and can facilitate the sustained release of encapsulated cytokines or soluble factors to augment T cell activation7,8.
In addition to the presence of necessary signal proteins, receptor engagement over a sufficiently large surface area during the aAPC/T cell interaction is essential for T cell activation. Thus, physical parameters of the aAPC such as size and shape drastically alter their available contact area and affect their ability to stimulate T cells. Micron-sized aAPC have been shown to be more effective at stimulating T cells than their nanoscale counterparts9,10. However, nano-aAPC can have superior biodistribution and better drainage to the lymph nodes that may enhance their performance in vivo over micro-aAPC11. Shape is another variable of interest in particle-based aAPC systems. Anisotropic aAPC have recently been shown to be more effective than isotropic particles at stimulating T cells, mainly due to enhanced interaction with target cells coupled with reduced non-specific cell uptake. Cells preferentially bind to the long axis of ellipsoidal particles, and the larger radius of curvature and flatter surface allow for more contact between the aAPC and T cell12. The long axis of ellipsoidal particles also discourages phagocytosis, resulting in increased circulation time compared to spherical particles following in vivo administration12,13. Because of these advantages, ellipsoidal particles mediate greater expansion of antigen-specific T cells in vitro and in vivo compared to spherical particles, an effect observed at both the micro and nanoscales12,13. There are various strategies to fabricate anisotropic particles, but thin-film stretching is a simple, widely accepted method used to generate a range of diverse particle shapes14. Following synthesis, particles are cast into films and stretched in one or two dimensions at a temperature above the glass transition temperature of the particle material. The film is then dissolved to retrieve the particles. Despite growing interest in anisotropic particles, current approaches for fabricating particle-based aAPC are mostly limited to isotropic systems, and methods of altering particle shape can be difficult to implement, incompatible with certain aAPC synthesis strategies, and lack precision and reproducibility15. Our thin-film stretching technique can be performed manually or in an automated fashion to rapidly generate anisotropic particles synthesized from a variety of biodegradable polymers, stretched to a desired aspect ratio in one or two dimensions15.
Based on our previous work, we developed a biodegradable particle-based approach combined with scalable thin-film stretching technology to rapidly generate aAPC with tunable size and shape in a standardized fashion for T cell expansion ex vivo or in vivo. Our protein conjugation strategy can be used to couple any protein(s) of interest to carboxyl groups on the particle surface at a desired density, giving this aAPC system a high degree of flexibility. We also describe methods to characterize the size, morphology, and surface protein content of aAPC, and to evaluate their functionality in vitro. This protocol can be easily adapted to expand immune cells ex vivo or in vivo for a variety of immunotherapeutic applications.

<table><tbody><tr><td>Poly(vinyl alcohol), MW 25000, 88% hydrolyzed</td><td>Polysciences, Inc.</td><td>02975-500</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>Digital Thermometer</td><td>Fluke</td><td>N/A</td><td>Model name: Fluke 52 II</td></tr><tr><td>Immersion Temperature Probe</td><td>Fluke</td><td>N/A</td><td>Model name: Fluke 80PK 22</td></tr><tr><td>Digital Hotplate &amp;amp; Stirrer</td><td>Benchmark Scientific</td><td>H3760-HS</td><td></td></tr><tr><td>Multipoint stirrer</td><td>Thermo Fisher Scientific</td><td>50093538</td><td></td></tr><tr><td>Resomer RG 504 H, Poly(D,L-lactide-co-glycolide)</td><td>Sigma-Aldrich</td><td>719900</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich</td><td>D65100</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>&amp;nbsp;0003725001</td><td></td></tr><tr><td>Sonicator</td><td>Sonics &amp;amp; Materials, Inc.</td><td>N/A</td><td>Model number: VC 505</td></tr><tr><td>Sonicator sound abating enclosure</td><td>Sonics &amp;amp; Materials, Inc.</td><td>N/A</td><td>Part number: 630-0427</td></tr><tr><td>Sonicator probe</td><td>Sonics &amp;amp; Materials, Inc.</td><td>N/A</td><td>Part number: 630-0220</td></tr><tr><td>Sonicator microtip</td><td>Sonics &amp;amp; Materials, Inc.</td><td>N/A</td><td>Part number: 630-0423</td></tr><tr><td>High speed centrifuge</td><td>Beckman Coulter</td><td>N/A</td><td>Model number: J-20XP (discontinued), alternative model: J-26XP</td></tr><tr><td>High speed centrifuge rotor</td><td>Beckman Coulter</td><td>369691</td><td>Model number: JA-17</td></tr><tr><td>High speed polycarbonate centrifuge tubes</td><td>Thermo Fisher Scientific</td><td>3118-0050</td><td>50 mL, screw cap</td></tr><tr><td>Rectangular disposable petri dish</td><td>VWR International</td><td>25384-322</td><td>75 x 50 x 10 mm</td></tr><tr><td>Square disposable petri dish</td><td>VWR International</td><td>10799-140</td><td>100 mm x 100 mm</td></tr><tr><td>LEAF&amp;nbsp;Purified anti-mouse CD3&amp;epsilon; Antibody</td><td>Biolegend</td><td>100314</td><td></td></tr><tr><td>InVivoMab anti-mouse CD28, clone 37.51</td><td>Bio X Cell</td><td>BE0015-1</td><td></td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&amp;prime;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>E6383</td><td></td></tr><tr><td>N-Hydroxysulfosuccinimide sodium salt</td><td>Sigma-Aldrich</td><td>56485</td><td></td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M3671</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse CD3 Antibody</td><td>Biolegend</td><td>100212</td><td></td></tr><tr><td>APC anti-mouse CD28 Antibody</td><td>Biolegend</td><td>102109</td><td></td></tr><tr><td>Corning&amp;nbsp;96 Well Solid Polystyrene Microplate</td><td>Sigma-Aldrich</td><td>CLS3915</td><td>flat bottom, black polystyrene</td></tr><tr><td>Protein LoBind Tubes, 1.5 mL</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>RPMI 1640 Medium (+ L-Glutamine)</td><td>ThermoFisher Scientific</td><td>11875093</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F4135</td><td>Heat Inactivated, sterile-filtered</td></tr><tr><td>Ciprofloxacin</td><td>Sigma-Aldrich</td><td>17850</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Recombinant Human IL-2 (carrier-free)</td><td>Biolegend</td><td>589102</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>ThermoFisher Scientific</td><td>11360070</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>ThermoFisher Scientific</td><td>11140050</td><td></td></tr><tr><td>MEM Vitamin Solution (100X)</td><td>ThermoFisher Scientific</td><td>11120052</td><td></td></tr><tr><td>CD8a+&amp;nbsp;T Cell Isolation Kit, mouse</td><td>Miltenyi Biotech</td><td>130-104-075</td><td></td></tr><tr><td>CellTrace CFSE Cell Proliferation Kit</td><td>ThermoFisher Scientific</td><td>C34554</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>MidiMACS Separator</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>MACS Multistand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Flow Cytometer</td><td>Accuri C6</td><td></td><td></td></tr><tr><td>Synergy 2 Multi-Detection Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>autoMACS Running Buffer</td><td>Miltenyi BIotech</td><td>130-091-221</td><td></td></tr><tr><td>Cell Strainer</td><td>ThermoFisher Scientific</td><td>22363548</td><td>Sterile, 70 &amp;micro;m nylon mesh</td></tr><tr><td>ACK Lysing Buffer</td><td>ThermoFisher Scientific</td><td>A1049201</td><td></td></tr><tr><td>C57BL/6J (Black 6) Mouse</td><td>The Jackson Laboratory</td><td>000664</td><td>Male, at least 7 weeks old</td></tr><tr><td>U-Bottom Tissue Culture Plates</td><td>VWR</td><td>353227</td><td>Sterile, 96-well tissue culture treated polystyrene plates</td></tr><tr><td>40 V DC Power Supply</td><td>Probotix</td><td>LPSK-4010</td><td></td></tr><tr><td>PTFE Coated Wire</td><td>Mouser</td><td>602-5858-100-01</td><td>This is for a 100 ft. spool but an equivalent wire will work</td></tr><tr><td>Stepper Motor Driver</td><td>Probotix</td><td>MondoStep5.6</td><td></td></tr><tr><td>IDC Connector Kit</td><td>Probotix</td><td>IDCM-10-12</td><td></td></tr><tr><td>Microcontroller</td><td>Probotix</td><td>PBX-RF</td><td></td></tr><tr><td>4A Fuses</td><td>Radio Shack</td><td>2701026</td><td>Equivalent fuses will work as well</td></tr><tr><td>DB25 Male to Male Cable</td><td>Probotix</td><td>DB25-6</td><td></td></tr><tr><td>USB-A to USB-B Cable</td><td>Staples</td><td>2094915</td><td>Equivalent cable will work as well</td></tr><tr><td>8-Pin Amphenol Connectors Male and Female</td><td>Mouser</td><td>654-97-3100A-20-7P and 654-97-3106A20-7S</td><td></td></tr><tr><td>Stepper Motor</td><td>Probotix</td><td>HT23-420-8</td><td></td></tr><tr><td>Right Hand Lead Screw</td><td>Roton</td><td>60722</td><td></td></tr><tr><td>Left Hand Lead Screw</td><td>Roton</td><td>60723</td><td></td></tr><tr><td>Screws</td><td>McMaster Carr</td><td>92196A151</td><td></td></tr><tr><td>Neoprene Rubber</td><td>McMaster Carr</td><td>8698K51</td><td></td></tr><tr><td>Right Handed Flanged Lead Nut</td><td>Roton</td><td>91962</td><td></td></tr><tr><td>Left Handed Flanged Lead Nut</td><td>Roton</td><td>91963</td><td></td></tr><tr><td>Linux Control Computer</td><td>Probotix</td><td>LCNC-PC</td><td>Any computer with matching specification and Linux operating system will work</td></tr><tr><td>Corning bottle-top vacuum filter system</td><td>Sigma-Aldrich</td><td>CLS431097</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4 %</td><td>ThermoFisher Scientific</td><td>15250061</td><td></td></tr></tbody></table>

fabrication of anisotropic polymeric artificial antigen presenting cells for cd8+ t cell activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

A schematic for the automated 2D thin film stretching device is given in Figure 1. A schematic and description for a 1D thin film stretching device is given in Ho et al.17 The stretcher is constructed from aluminum parts using standard milling and machining techniques. Similar to the 1D stretcher, the 2D stretcher consists of metallic grips and guide rails. Bidirectional lead screws are used to translate linear to rotational motion. Th...

Watch this Scientific Journal Video about Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation at JoVE.com

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

fabrication-anisotropic-polymeric-artificial-antigen-presenting-cells

Research

JoVE Journal

Environment

7.9K Views.  Johns Hopkins University School of Medicine. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het gebied van amino-engineering. Zoals, wat is de rol van deeltjes fysische eigenschappen op biomaterieel immuuncel interacties en activering. Het belangrijkste voordeel van deze techniek is dat het een onafhankelijke controle van de grootte en de vorm van biomimetische deeltjes mogelijk maakt.De implicaties van deze techniek uit te breiden tot de therapie van kanker als het is een off the shelf deeltjestherapie di...

Video: Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Immunology and Infection

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Cancer Research

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Bioengineering

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

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

Source: Hickey, J. W., et al. <a href="https://app.jove.com/v/58640">Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles.</a> J. Vis. Exp. (2018).
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.

Source: Hickey, J. W., et al. Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles. J. Vis. Exp. (2018).
This video describes a ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

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