Name: enrich and expand rare antigen-specific t cells with magnetic nanoparticles
Uploaded: 2018-11-17T14:30:00
Description: Watch this Scientific Journal Video about Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles at JoVE.com

J.W.H. thanks the NIH Cancer Nanotechnology Training Center at the Johns Hopkins Institute for NanoBioTechnology, the National Science Foundation Graduate Research Fellowship (DGE-1232825), and the ARCS foundation for fellowship support. This work was funded by support from the National Institutes of Health (P01-AI072677, R01-CA108835, R21-CA185819), TEDCO/Maryland Innovation Initiative, and the Coulter Foundation (JPS).

All mice were maintained per guidelines approved by the Johns Hopkins University&#39;s Institutional Review Board.
1. Load Dimeric Major Histocompatibility Complex Immunoglobulin Fusion Protein (MHC-Ig) with Desired Antigen Peptide Sequence.
NOTE: If using H-2Kb:Ig, then follow the protocol detailed in Step 1.1; if using H-2Db:Ig, then follow the protocol detailed in Step 1.2.
<ol>
	<li>Active loading of peptide sequence into H-2Kb:Ig.
	<ol>
		<li>Prepare necessar...

<ol>
	<li>Prasad, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=immunotherapy:+Tisagenlecleucel-the+first+approved+Car-t-cell+therapy:+implications+for+payers+and+policy+makers.">immunotherapy: Tisagenlecleucel-the first approved Car-t-cell therapy: implications for payers and policy makers.</a> Nature Reviews Clinical Oncology. 15 (1), 11 (2018).</li><li>Jenkins, M. K., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+naive+T+cell+precursor+frequency+and+recruitment+in+dictating+immune+response+magnitude.">The role of naive T cell precursor frequency and recruitment in dictating immune response magnitude.</a> The Journal of Immunology. 188 (9), 4135-4140 (2012).</li><li>Rizzuto, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-antigen-specific+CD8++T+cell+precursor+frequency+determines+the+quality+of+the+antitumor+immune+response.">Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response.</a> Journal of Experimental Medicine. 206 (4), 849-866 (2009).</li><li>Newell, E. W., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+model+antigens:+high-dimensional+methods+for+the+analysis+of+antigen-specific+T+cells.">Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells.</a> Nature biotechnology. 32 (2), 149 (2014).</li><li>Perica, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+and+expansion+with+nanoscale+artificial+antigen+presenting+cells+for+adoptive+immunotherapy.">Enrichment and expansion with nanoscale artificial antigen presenting cells for adoptive immunotherapy.</a> ACS nano. 9 (7), 6861-6871 (2015).</li><li>Kosmides, A. K., Necochea, K., Hickey, J. W., Schneck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+T+Cell+Targeting+Components+onto+Magnetically+Clustered+Nanoparticles+Boosts+Activation.">Separating T Cell Targeting Components onto Magnetically Clustered Nanoparticles Boosts Activation.</a> Nano Letters. , (2018).</li><li>Hickey, J. W., Vicente, F. P., Howard, G. P., Mao, H. Q., Schneck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+Inspired+Design+of+Nanoparticle+Artificial+Antigen-Presenting+Cells+for+Immunomodulation.">Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation.</a> Nano Letters. 17 (11), (2017).</li><li>, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+enrichment+of+antigen-specific+T+cells+by+engineering+particle+properties.">Efficient magnetic enrichment of antigen-specific T cells by engineering particle properties.</a> Biomaterials. , (2018).</li><li>Oelke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+purification+of+CD8++melan-A-specific+cytotoxic+T+lymphocytes+for+adoptive+transfer+in+tumor+immunotherapy.">Generation and purification of CD8+ melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy.</a> Clinical Cancer Research. 6 (5), 1997-2005 (2000).</li><li>Riccione, K., Suryadevara, C. M., Snyder, D., Cui, X., Sampson, J. H., Sanchez-Perez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+CAR+T+cells+for+adoptive+therapy+in+the+context+of+glioblastoma+standard+of+care.">Generation of CAR T cells for adoptive therapy in the context of glioblastoma standard of care.</a> Journal of visualized experiments: JoVE. (96), (2015).</li><li>Ho, W. Y., Nguyen, H. N., Wolfl, M., Kuball, J., Greenberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+generating+CD8++T-cell+clones+for+immunotherapy+from+the+naive+repertoire.">In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire.</a> Journal of immunological methods. 310 (1-2), 40-52 (2006).</li><li>Rudolf, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+costimulation+of+human+CD8+T+cells+by+anti-4-1BB+and+anti-CD28+on+synthetic+artificial+antigen+presenting+cells.">Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells.</a> Cancer immunology, immunotherapy : CII. 57 (2), 175-183 (2008).</li><li>Gulukota, K., Sidney, J., Sette, A., DeLisi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+complementary+methods+for+predicting+peptides+binding+major+histocompatibility+complex+molecules1.">Two complementary methods for predicting peptides binding major histocompatibility complex molecules1.</a> Journal of molecular biology. 267 (5), 1258-1267 (1997).</li><li>Castle, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+mutanome+for+tumor+vaccination.">Exploiting the mutanome for tumor vaccination.</a> Cancer research. 72 (5), 1081-1091 (2012).</li><li>Duan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+bioinformatic+profiling+of+mutational+neoepitopes+reveals+new+rules+to+predict+anticancer+immunogenicity.">Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity.</a> Journal of Experimental Medicine. 211 (11), 2231-2248 (2014).</li><li>Srivastava, P. K., Duan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+antigenic+fingerprint+of+each+individual+cancer+for+immunotherapy+of+human+cancer:+genomics+shows+a+new+way+and+its+challenges.">Harnessing the antigenic fingerprint of each individual cancer for immunotherapy of human cancer: genomics shows a new way and its challenges.</a> Cancer Immunology, Immunotherapy. 62 (5), 967-974 (2013).</li><li>Yadav, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+immunogenic+tumour+mutations+by+combining+mass+spectrometry+and+exome+sequencing.">Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing.</a> Nature. 515 (7528), 572 (2014).</li><li>Gros, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+neoantigen-specific+lymphocytes+in+the+peripheral+blood+of+melanoma+patients.">Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.</a> Nature medicine. 22 (4), 433 (2016).</li></ol>

We have created a novel antigen-specific T cell isolation technology based on nanoparticle artificial antigen presenting cells (aAPCs). Nanoparticle aAPCs have peptide-loaded MHC on the surface that allows antigen-specific T cell binding and activation alongside co-stimulatory activation. aAPCs are also paramagnetic, and thus can be used to enrich rare antigen-specific T cells using a magnetic field. We have optimized and studied key nanoparticle properties of size, ligand density, and ligand choice and their influence o...

With the rise of many immunotherapies, there is a need to be able to characterize and control immune responses. In particular, the adaptive immune response is of interest because of the specificity and durability of the cells. Recently, chimeric-antigen-receptor T cell therapies have been approved for cancer therapy; however, the antigen-receptors are based off the common cell surface antigen CD19, instead of the antigens specific to the cancer1. Beyond the specificity, immunotherapies can also suffer from the lack of control, and limited understanding the dynamic immune response within cancer or autoimmunity.
One of the challenges of studying antigen-specific responses is their extremely low frequency, e.g., antigen-specific T cells are 1 of every 104 to 106 T cells2,3. Thus, to investigate which T cells are present or responding, the cells need to either be enriched and expanded, or their signal need to be amplified. It is expensive and difficult to maintain the feeder cells using current techniques that focus on the expansion of antigen-specific cells. Current techniques that focus on amplifying the signal of antigen-specific T cells, like the enzyme-linked immunospot (ELISPOT) assay, limit the re-use of those T cells4. Finally, because of low sensitivity, often these two techniques need to be combined for antigen-specific enumeration.
To address these issues, we have developed the magnetic nanoparticle-based artificial antigen presenting cell (aAPC)5,6,7,8. The aAPC can be functionalized with an antigen-specific signal-peptide loaded major histocompatibility complex (pMHC)-and co-stimulatory molecules-e.g., an anti-CD28 antibody-to both enrich antigen-specific T cells and then subsequently stimulate their expansion (Figure 1). The particles can thus be a cost-effective off-the-shelf product that can be both customized to meet antigen-specific stimulations yet standardized across experiments and patients. Performing the enrichment and expansion process results in hundreds to thousands-fold expansion of antigen-specific CD8+ T cells and can result in frequencies up to 60 percent after just one week, enabling the characterization or therapeutic use of the large number of cells. Herein, we describe how to make nanoparticle aAPCs, some critical design considerations in choosing the nanoparticle properties, and demonstrate some typical results from utilizing these particles in isolating and expanding rare antigen-specific CD8+ T cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1136px;" width="1136">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">DimerX I: Recombinant Soluble Dimeric Human HLA-A2:Ig Fusion Protein</td>
			<td style="width:207px;">BD Biosciences</td>
			<td style="width:168px;">551263</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">DimerX I: Recombinant Soluble Dimeric Mouse H-2D[b]:Ig</td>
			<td style="width:207px;">BD Biosciences</td>
			<td style="width:168px;">551323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">DimerX I: Recombinant Soluble Dimeric Mouse H-2K[b]:Ig Fusion Protein</td>
			<td style="width:207px;">BD Biosciences</td>
			<td style="width:168px;">550750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Vivaspin 20 MWCO 50 000</td>
			<td style="width:207px;">GE Life Sciences</td>
			<td style="width:168px;">28932362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Vivaspin 2 MWCO 50 000</td>
			<td style="width:207px;">GE Life Sciences</td>
			<td style="width:168px;">28932257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Purified Human Beta 2 Microglobulin</td>
			<td style="width:207px;">Bio-Rad</td>
			<td style="width:168px;">PHP135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">nanomag-D-spio, NH2, 100 nm nanoparticles</td>
			<td style="width:207px;">Micromod</td>
			<td style="width:168px;">79-01-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Super Mag NHS Activated Beads, 0.2 &#181;m</td>
			<td style="width:207px;">Ocean Nanotech</td>
			<td style="width:168px;">SN0200&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Anti-Biotin MicroBeads UltraPure</td>
			<td style="width:207px;">Miltenyi</td>
			<td style="width:168px;">130-105-637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">EZ-Link NHS-Biotin</td>
			<td style="width:207px;">ThermoFisher</td>
			<td style="width:168px;">20217</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Sulfo-SMCC Crosslinker&#160;</td>
			<td style="width:207px;">ProteoChem</td>
			<td style="width:168px;">c1109-100mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">2-Iminothiolane hydrochloride</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:168px;">I6256 Sigma&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">96 Well Half-Area Microplate, black polystyrene</td>
			<td style="width:207px;">Corning</td>
			<td style="width:168px;">3875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">FITC Rat Anti-Mouse Ig, &#955;1, &#955;2, &#38; &#955;3 Light Chain&#160; Clone &#160;R26-46&#160;&#160;</td>
			<td style="width:207px;">BD Biosciences</td>
			<td style="width:168px;">553434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">FITC Mouse Anti-Armenian and Syrian Hamster IgG&#160; Clone &#160;G192-1</td>
			<td style="width:207px;">BD Biosciences</td>
			<td style="width:168px;">554026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J (transgenic PMEL) mice</td>
			<td style="width:207px;">Jackson Laboratory</td>
			<td style="width:168px;">005023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">C57BL/6J (B6 wildtype) mice</td>
			<td style="width:207px;">Jackson Laboratory</td>
			<td style="width:168px;">000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">CD8a+ T Cell Isolation Kit, Mouse</td>
			<td style="width:207px;">Miltenyi</td>
			<td style="width:168px;">130-104-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">MS Columns</td>
			<td style="width:207px;">Miltenyi</td>
			<td style="width:168px;">130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">LS Columns</td>
			<td style="width:207px;">Miltenyi</td>
			<td style="width:168px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">Streptavidin-Phycoerythrin, SAv-PE</td>
			<td style="width:207px;">Biolegend</td>
			<td style="width:168px;">405203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">N52 disk magnets of 0.75 inches&#160;</td>
			<td style="width:207px;">K&#38;J Magnetics</td>
			<td style="width:168px;">DX8C-N52</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">APC anti-mouse CD8a Antibody, clone 53-6.7</td>
			<td style="width:207px;">Biolegend</td>
			<td style="width:168px;">100711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:517px;">LIVE/DEAD Fixable Green Dead Cell Stain Kit, for 488 nm excitation&#160;</td>
			<td style="width:207px;">ThermoFisher</td>
			<td style="width:168px;">L-34969</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To complete a successful enrichment and expansion of antigen-specific T cells, the peptide-loaded MHC-Ig and co-stimulatory molecules should be successfully attached to the aAPC particle. Based on the 3 methods of particle attachment, we provide some representative data for a successful conjugation procedure outcome (Figure 5a). Indeed, if the ligand density is too low, then there will not be effective stimulation of antigen-specific CD8+ T cells where this o...

Watch this Scientific Journal Video about Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles at JoVE.com

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

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 ...

So the main advantage of our novel technique, we termed it E plus E, which stands for enrichment plus expansion, is that we can enrich and expand these cells, rare antigen-specific cells, simultaneously. This allows us to develop sufficient number of cells that we're gonna be able to use to investigate their role in different settings and in different clinical settings or even allows us to develop now large number of cells that we can utilize for immunotherapy in a fashion that we couldn't before. The method that we have developed, this E plus E, can help answer key questions in basic immunology about the presence of neoepitopes.What are neoepitopes? Those are the personalized epitopes that we know are now very important in terms of cancer immunotherapy and also antigen-specific dynamics and how these factors affect disease states and for effective cellular therapy. The implications of this technique extend towards immunotherapy, as the nanoparticle artificial antigen-presenting cell could be an off-the-shelf product that is easily tailored for individual patient immune responses.To generate nanoparticle artificial antigen-presenting cells, resuspend the lyophilized particles in one milliliter of resuspension buffer and vigorously vortex the suspension for at least 15 minutes until no aggregates are visible. Particles not sufficiently vortexed and resuspended prior to the reaction with protein will result in larger aggregated particles that are not able to be dissociated. Place the magnetic particles on a magnetic stand to remove the supernatant and resuspend them with 0.5 milliliters of fresh resuspension buffer.Transfer the particle suspension to a glass scintillation vial for vortexing until no aggregates are visible, and add 0.1 milligrams of total stimulatory signals per one milligram of resuspended particles. After vortexing, allow the suspension to react for 2.5 hours at room temperature with mixing in a rotator, followed by the addition of 0.1 milliliters of quenching buffer for an additional 30-minute incubation at room temperature with mixing. At the end of the second reaction, place the scintillation vial onto the magnetic stand and wash the particles in one milliliter of resuspension buffer four times, removing the buffer when it turns clear after each wash and replacing it with fresh resuspension buffer for the subsequent wash on the magnet.After the last wash, aspirate the solution and remove the particles from the magnetic stand, then store the particles in one milliliter of fresh resuspension buffer at four degrees Celsius for up to six months. To isolate antigen-specific CD8+T cells, macerate the spleens and lymph nodes from wild type C57 black 6J mice through a sterile, 70-micrometer cell strainer with frequent PBS rinses and use a no-touch CD8+T cell isolation kit according to the manufacturer's instructions to eliminate the non-CD8+T cells. After counting, collect the isolated CD8+T cells by centrifugation and resuspend the pellet in 100 microliters of PBS supplemented with 0.5%Bovine Serum Albumin and two-millimolar EDTA, then incubate the cells with the nanoparticle artificial antigen-presenting cells such that there are one times 10 to the 11th peptide-loaded MHC-ig per one times 10 to the sixth isolated CD8+T cells in a sterile, five-milliliter polystyrene round-bottomed tube for one hour at four degrees Celsius with continual mixing.At the end of the incubation, wash the nanoparticle cell suspension three times on the magnetic stand as demonstrated, resuspending the artificial antigen-presenting cells and the enriched CD8+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 and cells on the magnetic column for at least two minutes and to aspirate the buffer carefully without disrupting the particles and cells subject to the magnetic field. After counting, seed 2.5 times 10 to the fifth enriched CD8+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 five-milliliter round-bottomed 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 five times 10 to the fourth to five times 10 to the fifth cells into new five-milliliter round-bottomed tubes for antigen-specific staining.Label the appropriate tubes with biotinylated MHC-ig and anti-mouse CD8a for one hour at four 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 strain for 15 minutes at four degrees Celsius and read the cells on a flow cytometer to determine the specificity and number of the antigen-specific CD8+T cells according to standard protocols. Here a successful protein conjugation was achieved using three different methods of protein attachment to particles. If the aAPC ligand density is too low, there will not be an effective stimulation of the antigen-specific CD8+T cells.Quality control of the biotinylated dimer can be completed on transgenic CD8+T cells to verify staining. For example, these representative results demonstrate a positive staining with gp100-specific CD8+T cells with non-antigen-specific b6 CD8+T cells as a background control. Further, knowledge of the background staining of the biotinylated dimer is critical, as any percentage lower than this staining in the cognate-stained tubes should be considered a negative result.After seven days of enrichment and expansion, between two times 10 to the fourth and two times 10 to the fifth antigen-specific and five to 50%CD8+T cells can be expected from an original five times 10 to the sixth endogenous CD8+T cell starting population. The same methodology can be used to isolate and stimulate human antigen-specific CD8+T cells with similar increases in percentages and numbers of antigen-specific CD8+T cells observed after only one week of expansion following enrichment. While attempting this procedure, it's important to remember to perform the appropriate reagent quality checks as described.Following this procedure, other methods like flow cytometry for intracellular cytokine staining or surface protein expression can be performed to answer additional questions about the functionality or phenotype of the cells. This technique paves the way for researchers in the basic immunology field to explore the breadth, depth, and dynamics of antigen-specific responses in other biomedical fields such as cancer, infectious disease, or autoimmunity research.

enrich-expand-rare-antigen-specific-t-cells-with-magnetic

Research

JoVE Journal

Engineering

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen 1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion2. Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition8, 10, 13, 14 ("coking") and sulfur poisoning11, 15 and the manner in which surface modifications stave off this degradation16. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.

We present a unique platform for characterizing electrode surfaces in solid oxide fuel cells (SOFCs) that allows simultaneous performance of multiple characterization techniques (e.g. in situ Raman spectroscopy and scanning probe microscopy alongside electrochemical measurements). Complementary information from these analyses may help to advance toward a more profound understanding of electrode reaction and degradation mechanisms, providing insights into rational design of better materials for SOFCs.

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ...

Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers

Nitrogen-doped carbon nanotubes consist of many cup-shaped graphitic compartments termed as nitrogen-doped carbon nanotube cups (NCNCs). These as-synthesized graphitic nanocups from chemical vapor deposition (CVD) method were stacked in a head-to-tail fashion held only through noncovalent interactions. Individual NCNCs can be isolated out of their stacking structure through a series of chemical and physical separation processes. First, as-synthesized NCNCs were oxidized in a mixture of strong acids to introduce oxygen-containing defects on the graphitic walls. The oxidized NCNCs were then processed using high-intensity probe-tip sonication which effectively separated the stacked NCNCs into individual graphitic nanocups. Owing to their abundant oxygen and nitrogen surface functionalities, the resulted individual NCNCs are highly hydrophilic and can be effectively functionalized with gold nanoparticles (GNPs), which preferentially fit in the opening of the cups as cork stoppers. These graphitic nanocups corked with GNPs may find promising applications as nanoscale containers and drug carriers.

We discussed the synthesis of individual graphitic nanocups using a series of techniques including chemical vapor deposition, acid oxidation and probe-tip sonication. By citrate reduction of HAuCl4, the graphitic nanocups were effectively corked with gold nanoparticles due to the chemically reactive edges of the cups.

Nitrogen-doped carbon  nanotubes consist of many cup-shaped graphitic compartments termed as  nitrogen-doped carbon nanotube cups (NCNCs).  These ...

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step process that finishes with Negishi coupling. Negishi coupling is a cross-coupling process in which a chemical precursor is first lithiated, followed by transmetallation with ZnCl2. The resultant organozinc compound can be coupled to a dibrominated aromatic precursor to give the conjugated monomer. Polymer films can be prepared via electropolymerization of the monomer and characterized using cyclic voltammetry and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. Nanoparticles (NPs) are prepared via emulsion polymerization of the monomer using a two-surfactant system to yield an aqueous dispersion of the polymer NPs. The NPs are characterized using dynamic light scattering, electron microscopy, and UV-Vis-NIR-spectroscopy. Cytocompatibility of NPs is investigated using the cell viability assay. Finally, the NP suspensions are irradiated with a NIR laser to determine their effectiveness as potential materials for photothermal therapy (PTT).

A protocol is presented for the synthesis and preparation of nanoparticles consisting of electroactive polymers.

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step ...

Frequency Mixing Magnetic Detection Scanner for Imaging Magnetic Particles in Planar Samples

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. It consists of two magnetic measurement heads on both sides of the sample mounted on the legs of a u-shaped support. The sample is locally exposed to a magnetic excitation field consisting of two distinct frequencies, a stronger component at about 77 kHz and a weaker field at 61 Hz. The nonlinear magnetization characteristics of superparamagnetic particles give rise to the generation of intermodulation products. A selected sum-frequency component of the high and low frequency magnetic field incident on the magnetically nonlinear particles is recorded by a demodulation electronics. In contrast to a conventional MPI scanner, p-FMMD does not require the application of a strong magnetic field to the whole sample because mixing of the two frequencies occurs locally. Thus, the lateral dimensions of the sample are just limited by the scanning range and the supports. However, the sample height determines the spatial resolution. In the current setup it is limited to 2 mm. As examples, we present two 20 mm &#215; 25 mm p-FMMD images acquired from samples with 1 &#181;m diameter maghemite particles in silanol matrix and with 50 nm magnetite particles in aminosilane matrix. The results show that the novel MPI scanner can be applied for analysis of thin biological samples and for medical diagnostic purposes.

A scanner for imaging magnetic particles in planar samples was developed using the planar frequency mixing magnetic detection technique. The magnetic intermodulation product response from the nonlinear nonhysteretic magnetization of the particles is recorded upon a two-frequency excitation. It can be used to take 2D images of thin biological samples.

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation displays and solid-state lighting technologies. The brightness and efficiency of blue QDs-based light-emitting diodes (LEDs) remain inferior to their red and green counterparts, due to the inherently unfavorable energy levels of different colors of light. To solve these problems, a device structure should be designed to balance the injection holes and electrons into the emissive QD layer. Herein, through a simple autoxidation strategy, pure blue QD-LEDs which are highly bright and efficient are demonstrated, with a structure of ITO/PEDOT:PSS/Poly-TPD/QDs/Al:Al2O3. The autoxidized Al:Al2O3 cathode can effectively balance the injected charges and enhance radiative recombination without introducing an additional electron transport layer (ETL). As a result, high color-saturated blue QD-LEDs are achieved with a maximum luminance over 13,000 cd m-2, and a maximum current efficiency of 1.15 cd A-1. The easily controlled autoxidation procedure paves the way for achieving high-performance blue QD-LEDs.

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum cathode.

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle&#8217;s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...