The authors would like to thank Priyanka Subrahmanyam for helpful discussions. The authors have no competing financial interest. This work was supported by grants from the American Cancer Society, NIH/NCI K01 CA131487, R21 CA162273, R21 CA162277, and P30 Tumor Immunology and Immunotherapy Program to T.J. Webb. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

1. Generation of aAPC
<ol>
	<li>Before adding proteins to beads, prepare all reagents and buffers: 0.1M Borate buffer; 1X D-PBS (no Ca2+ and Mg2+); Bead Wash Buffer (1X PBS+5% Human AB serum + 0.02% sodium azide); Complete medium (RPMI medium +100 mM sodium pyruvate,10 mM non-essential vitamin solution, 100 mM MEM Vitamin solution, 1% 2-mercaptoethanol, 10 &mu;M ciprofloxacin, 5% Human AB serum); MACS buffer (1 L PBS free of Ca2+ and Mg2+, 5 g BSA, and 2 mmol EDTA).</li>
	<li>Rinse 1 ml Dynabeads M-450 Epoxy beads with 3 ml sterile 0.1 M Borate buffer (boric acid and water, pH 7.0-7.4) in a 5 ml clear borosilicate glass threaded vial.</li>
	<li>In a separate 1.5 ml microcentrifuge tube, add 100 &mu;g hCD1d-Ig dimer and 20 &mu;g costimulatory molecules (example: anti-CD28mAb) to 1 ml PBS w/o Ca2+ or Mg2+.</li>
	<li>Place bead containing glass vial on magnet and aspirate borate buffer from beads. Add protein mixture from step 1.2 to glass vial and replace cap. Mix immediately by inverting the vial, cover the cap with parafilm, and place on a rotator and incubate overnight at 4 &deg;C.</li>
	<li>The next day, place glass vial on magnet and remove protein mixture, while carefully avoiding beads. Wash the beads by adding 3 ml bead wash buffer (PBS with 5% AB serum +0.02% sodium azide), and incubating at 4 &deg;C on a rotator for 5 min. Repeat twice.</li>
	<li>Beads can be stored in this bead wash mixture. To make functional aAPC, remove a small aliquot and count the beads using a hemacytometer. Check that the proteins are stably loaded onto the beads by staining with antibodies (ex. PE- conjugated anti-mouse IgG1) and performing flow cytometric analyses.</li>
	<li>To load beads with antigen, remove 5 x 107 beads, and add to a small 1.5 ml glass vial, rinse beads with 1 ml sterile PBS. Resuspend washed beads with 1 ml sterile PBS and add antigen; example: Load with &alpha;-GalCer/KRN7000 (5mg/ml). * Note: While, we have not detected any issues with lipid solubility or micelle formation, lipids should be handled according to manufacturer's recommendations. Specifically, KRN7000 was reconstituted in DMSO (1mg/ml) for these studies. KRN7000 and other glycolipid antigens can also be dissolved in 0.2 mg/ml PBS containing 0.5% Tween-20 (sonicate 2 hr. at 37 &deg;C). IMPORTANT- The aAPC need to be loaded for at least 48-72 hr prior to use.</li>
</ol>
2. Isolation of CD161+CD3+ Cells
<ol>
	<li>Collect peripheral blood mononuclear cells (PBMC). For Ficoll density gradient centrifugation separation of lymphocytes from a buffy coat or leukopheresis pack, first dilute heparinized blood with an equal volume of 1X PBS at room temperature.</li>
	<li>Add 15 ml of Ficoll (warmed to room temperature) to 50 ml conical tubes. Slowly overlay 25 ml of the diluted blood mixture on top of the Ficoll. Centrifuge at 2,000 rpm for 30 min at room temperature with the brake off.</li>
	<li>Carefully remove the lymphocyte interface (white ring between the media and Ficoll) with a Pasteur pipette and transfer to a new 50 ml conical tube.</li>
	<li>Wash the cells by filling up the tube to 50 ml with PBS and centrifuging at 1,500 rpm for 5 min. Discard the supernatant and combine the tubes from a single individual to a single tube and wash the peripheral blood mononuclear cells (PBMC) again with 20 ml PBS. Then count the PBMC and resuspend at a concentration of 5 x 107 cells/ml in MACS buffer (1 L PBS free of Ca2+ and Mg2+, 5 g BSA, and 2 mmol EDTA).</li>
	<li>In order to isolate the T cell fraction, start with 2 ml of PBMC (108 cells) and add 100 &mu;l of Pan T cell enrichment solution from the EasySep Human T Cell Enrichment Kit. Incubate at room temperature for 10 min.</li>
	<li>Add 100 &mu;l of magnetic particles to the solution and incubate at room temperature for another 10 min. Bring the final volume of solvent to 2.5 ml and place the tube in the purple magnet for 5 min. Quickly pour off the CD3+ fraction into a 15 ml conical tube.</li>
	<li>Wash the cells by adding 5 ml cold MACS buffer, count the number of viable cells, and remove an aliquot for FACS staining.</li>
	<li>To select the CD161+ cells, first resuspend enriched T cells in 980 &mu;l ice cold MACS buffer, add 10 &mu;g anti CD161 mAb, and incubate in refrigerator for 10 min.</li>
	<li>Centrifuge the cells at 1,500 rpm at 4 &deg;C for 5 min. Then reconstitute the cell pellet in 800 &mu;l of MACS buffer. Add 200 &mu;l of anti-mouse IgG1 microbeads and incubate the solution for 10 min at 4 &deg;C.</li>
	<li>During this incubation step, equilibrate a LS column by adding 3 ml MACS buffer.</li>
	<li>Next, wash the cells by centrifuging 1,500 rpm at 4 &deg;C for 5 min. Resuspend the cells in 3 ml MACS buffer. Then pipette the cells into the LS MACS separating column. Make sure to avoid generating bubbles by pipetting slowly. Rinse the column by adding 3 ml of MACS buffer. Repeat twice.</li>
	<li>Add 3 ml fresh MACS buffer and remove column from magnet. Place column into a 15 ml conical tube. Insert plunger and push out contents to obtain purified CD161+CD3+ cells. Count NKT cell enriched fraction. You should have 2-4 million cells.</li>
</ol>
3. aAPC-mediated NKT Cell Expansion
<ol>
	<li>Set up co-culture by adding 106 enriched CD161+CD3+ T cells and 106 aAPC in 16 ml complete medium (complete medium + IL-2, 100 U/ml). Plate this mixture by adding 160 &mu;l/well final volume to a 96 well tissue-culture treated polystyrene, U-bottom plate with low-evaporation lid. Perform medium exchange every 7th day by adding 80 &mu;l of fresh medium.</li>
	<li>Harvest cells, count, and perform FACS staining on day 12-14.</li>
	<li>The expanded NKT cells can be replated as described above in step 3.1, specifically resuspend 106 expanded T cells and 106 aAPC in 16 ml complete medium. Plate this mixture by adding 160 &mu;l/well to a 96 well U-bottom plate. Continue to refresh coculture medium every 7th day by adding 80 &mu;l of fresh medium.</li>
	<li>It is best to freeze extra NKT cells following the second round of expansion (1 x106/cryovial in 1 ml-5% DMSO/ 95% FBS.)</li>
</ol>
4. Functional Test: aAPC-mediated Stimulation of NKT Cells
<ol>
	<li>Set up 5x104 NKT cells/ well with 5x105 aAPC in 200 &mu;l final volume (complete medium) in 96 well U-bottom plate.</li>
	<li>Harvest cell culture supernatant for ELISA after 24-48 hr.</li>
</ol>

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycolipid+antigen+presentation+by+CD1d+molecules.">Glycolipid antigen presentation by CD1d molecules.</a> Science. 291, 664-667 (2001).</li><li>Davodeau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+phenotypic+and+functional+similarities+between+human+and+murine+&#945;&#946;+T+cells+expressing+invariant+TCR+&#945;-chains.">Close phenotypic and functional similarities between human and murine &#945;&#946; T cells expressing invariant TCR &#945;-chains.</a> J. Immunol. 158, 5603-5611 (1997).</li><li>Exley, M., Garcia, J., Balk, S. 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Immunol. 177, 3484-3492 (2006).</li><li>Shiratsuchi, T., Schneck, J., Kawamura, A., Tsuji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD1+dimeric+proteins+as+indispensable+tools+for+research+on+CD1-binding+lipids+and+CD1-restricted+T+cells.">Human CD1 dimeric proteins as indispensable tools for research on CD1-binding lipids and CD1-restricted T cells.</a> Journal of immunological. 345, 49-59 (2009).</li><li>Webb, T. J., Bieler, J. G., Schneck, J. P., Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+natural+killer+T+cells+by+CD1d1-Ig+coated+artificial+antigen+presenting+cells.">Ex vivo induction and expansion of natural killer T cells by CD1d1-Ig coated artificial antigen presenting cells.</a> J. Immunol. Methods. 346, 38-44 (2009).</li><li>Tham, E. L., Jensen, P. L., Mescher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+antigen-specific+T+cells+by+artificial+cell+constructs+having+immobilized+multimeric+peptide-class+I+complexes+and+recombinant+B7-Fc+proteins.">Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins.</a> J. Immunol. Methods. 249, 111-119 (2001).</li><li>Goldberg, J., Shrikant, P., Mescher, M. 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No conflicts of interest declared.

aAPC can be used to study the basic requirements for NKT cell activation and it has potential clinical value for ex vivo expansion of NK T cells for adoptive immunotherapy. Mescher et al. described one of the first bead based systems, where biotinylated murine MHC class I-peptide-single chain constructs were combined with biotinylated costimulatory molecules B7.1 and B7.2 via streptavidin to the surface of latex microspheres 14, 15. This approach has successfully been used to stimulate antigen-specific T cells from transgenic mice. In addition, since this approach uses a single chain MHC-peptide complex to ensure homogenous loading of the MHC molecules, each target peptide antigen would require a new transfection for expression of the desired single chain MHC-peptide complex, thus limiting the generality of the approach. Importantly, Dr. Schneck's group pioneered the bead based-aAPC, by developing another non-cellular bead based aAPC, made by coupling HLA-Ig, signal 1, and anti-CD28, signal 2, onto magnetic beads. HLA-Ig, a unique multimeric form of HLA fused to an immunoglobulin molecular scaffold 16, 17 was developed by his group. Subsequently, they developed MHC-Ig based aAPC, which have been shown to effectively expand CMV and MART-1 specific CTL 18. Here, we have demonstrated that CD1d-Ig based aAPC can be used to expand functional NKT cells. One study has used a similar system to examine the physical interaction of NK cells with CD1d 19.
Notably, we have designed an artificial antigen presenting cell which is adaptable to any requirements we find necessary for optimal NKT cell proliferation. The aAPC expansion method provides a simple and reliable method for expanding and enriching human NKT cells. Our aAPC can be modified to systematically evaluate the role of a panel of potential costimulatory molecules and assess their role on NKT cell proliferation and function. Thus, aAPC represent a robust versatile technology useful for inducing and expanding NKT cells. The generation of aAPCs takes less than one week and is suitable for the production of large quantities of beads. However, a critical step in generating the aAPC is to confirm that CD1d-Ig is stably immobilized on the surface of the beads and to assess their functionality to ensure consistency from batch to batch. A potential limitation of the system is that there is not a mechanism in place to turn off stimulation, other than mechanical removal of the beads. Specifically, the engagement of the T cell receptor (TCR) with the antigen: CD1d/MHC complex typically generate the immunological synapse in concert with accessory/adhesion molecules, which can result in the induction of inhibitory or suppressive factors on both the T cell and antigen presenting cell. In the aAPC system, these factors may be upregulated by the T cell, but the bead will not express the cognate ligands for these receptors.
In addition, CD4+ NKT cells have been shown to suppress antitumor responses in mice and humans, therefore it is possible that nonselective activation of all NKT cells (i.e. global stimulation with &alpha;-GalCer) or activation of the wrong subset could result in unwanted immunological outcomes. Consequently, one must phenotypically and functionally characterize the aAPC-expanded NKT cell population. As shown in Figure 4, we have found that stimulation with &alpha;-GalCer-loaded aAPC expressing anti-CD28 can result NKT cells producing Th1, Th2, and Th17 type cytokines. Murine studies have reported that challenge with IL-33, a recently identified cytokine, resulted in increased levels of circulating inflammatory cytokines such as IL-5 and IL-13. Treatment of NKT cells with IL-33 enhanced their cytokine production 20. IL-33 is a specific ligand for ST2 and it has been shown that soluble ST2 can block IL-33 signaling. Thus, as an example of a future application, aAPC expressing ST2 could be generated and used to determine if one could selectively inhibit the production of Th2 cytokines while inducing Th1 cytokine secretion by NKT cells. It has also been reported that i.v. injection of Kb-expressing aAPC into C57BL/6 mice resulted in decreased lung metastasis of tumor 21. Importantly, these data demonstrate that aAPC traffic to the lung and are able to activate effector T cell subsets. Therefore, one could generate multiple types of aAPC and examine the interplay between antigen specific T cell subsets. To summarize, these studies demonstrate that CD1d-Ig based aAPC can be used to replace normal cellular APC, and have to the potential to enhance current clinical approaches for NKT cell based- adoptive immunotherapy.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Ficoll-Paque PLUS</td>
 <td>GE Life Sciences</td>
 <td>17-1440</td>
 <td></td>
 </tr>
 <tr>
 <td>EasySep Human T Cell Enrichment Kit</td>
 <td>StemCell Technologies</td>
 <td>19051</td>
 <td></td>
 </tr>
 <tr>
 <td>Allophycocyanin CD161 human mAb</td>
 <td>Pharmingen</td>
 <td>550968</td>
 <td></td>
 </tr>
 <tr>
 <td>EasySep Magnet</td>
 <td>StemCell Technologies</td>
 <td>18002 </td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse IgG1 Microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-047-101 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dimer XI Recombinant Soluble Dimeric Mouse CD1d:IG Fusion Protein</td>
 <td>BD Biosciences </td>
 <td>557599</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-CD28 mAb</td>
 <td>Biolegend</td>
 <td>302914</td>
 <td></td>
 </tr>
 <tr>
 <td>M-450 Epoxy beads</td>
 <td>Life Technologies</td>
 <td>150-11</td>
 <td></td>
 </tr>
 <tr>
 <td>Alpha-galactosylceramide (KRN7000)</td>
 <td>Axxora, LLC</td>
 <td>BML-SL232-0100</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI 1640 Medium</td>
 <td>Sigma Aldrich</td>
 <td>R 0883</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Pyruvate</td>
 <td>Gibco</td>
 <td>11360-070</td>
 <td></td>
 </tr>
 <tr>
 <td>Non-essential amino acids</td>
 <td>Gibco</td>
 <td>11140-050</td>
 <td></td>
 </tr>
 <tr>
 <td>Vitamin Solution</td>
 <td>Gibco</td>
 <td>11120-052</td>
 <td></td>
 </tr>
 <tr>
 <td>2-mercaptoethanol</td>
 <td>Gibco</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>Alexis Biochemicals</td>
 <td>380-288-G025</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-V&alpha;24J&alpha;18 </td>
 <td>Biolegend</td>
 <td>342904</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-V&alpha;24 (Clone C15) </td>
 <td>Beckman Coulter</td>
 <td>A66907</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-V&beta;11 (Clone C21) </td>
 <td>Beckman Coulter</td>
 <td>A66905</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-CD1d</td>
 <td>Biolegend</td>
 <td>123510</td>
 <td></td>
 </tr>
 <tr>
 <td>PE anti mouse IgG1</td>
 <td>BD Biosciences</td>
 <td>556650</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC anti-mouse IgG2a</td>
 <td>Biolegend </td>
 <td>407105</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS, no calcium, no magnesium</td>
 <td>Life Technologies</td>
 <td>14190250</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Azide</td>
 <td>Sigma Aldrich</td>
 <td>S8032</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>American Bioanalytical</td>
 <td>AB00440-00100</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma Aldrich</td>
 <td>431788</td>
 <td></td>
 </tr>
 <tr>
 <td>IL-2 (Proleukin)</td>
 <td>BD Biosciences</td>
 <td>354043</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB Serum</td>
 <td>Atlanta Biologicals</td>
 <td>S40110</td>
 <td></td>
 </tr>
 <tr>
 <td>Labquake Tube Rotator</td>
 <td>Fisher Scientific</td>
 <td>13-687-10Q</td>
 <td></td>
 </tr>
 <tr>
 <td>BD Falcon 5 ml polystyrene round-bottom tubes</td>
 <td>Fisher Scientific</td>
 <td>14-959-1A</td>
 <td></td>
 </tr>
 <tr>
 <td>Wheaton Glass Sample Vials with Cap</td>
 <td>Fisher Scientific</td>
 <td>Small (03-343-6A ) Large (03-343-6E) </td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Herein we describe a method for generating CD1d-Ig based aAPC, made by covalent coupling of CD1d-Ig and anti-CD28 mAb to magnetic beads to stimulate NKT cells as a standardized method for the propagation of NKT cells (Figure 1).First, one must demonstrate that the CD1d-Ig fusion proteins are stably immobilized onto the surface of the magnetic beads. As shown in Figure 2A, CD1d-Ig and anti-CD28 antibodies were both expressed on the surface of the magnetic beads. To examine the stimulatory capacity of the aAPC, we co-cultured NKT cell hybridomas with aAPC overnight, harvested the culture supernatants and measured IL-2 production by ELISA. We found that CD1d-Ig based aAPC were able to stimulate the NKT cell hybridomas at levels equal to or higher than their cellular counterparts (Figure 3, data not shown). Interestingly, we found that our mouse NKT cell hybridomas are stimulated by human CD1d-based aAPC (Figure 2B), which provides simple method for testing each batch of aAPC.
Next, we sought to demonstrate the propagation potential of aAPC, thus human T cells were isolated from the peripheral blood. First the CD161+CD3+ T cell fraction was enriched by magnetic bead separation. Then, the T cells were stimulated biweekly with &alpha;-GalCer-loaded aAPC. Importantly, we found that even with a relatively low initial NKT cell population (0.03%), we were able to expand the cells to ~67% V&alpha;24+V&beta;11+ (Figure 3). We have expanded NKT cells from the PBMC of many healthy volunteers and cancer patients and have found that &alpha;-GalCer loaded aAPC were able to expand the NKT cells population in both groups. Notably, the expansion rate was highly donor dependent. As expected the higher the initial population of V&alpha;24+ cells, the greater the percentage of expansion. In addition, when using a starting population of 2 million cells CD161+CD3+T cells, one can obtain &gt;107 cells after two rounds of expansion (Table 1). Approximately 80-90% of the expanded NKT cells are CD4+, ~5% CD8+, and the remaining are presumably CD4-CD8- double negative NKT cells. These expanded NKT cells can be used for functional studies as shown in Figure 4A-C. We have found that our ex vivo expanded NKT cells remain responsive to &alpha;-GalCer stimulation and are potent producers of IL-17A, TNF-&alpha;, and IFN-&gamma;. It should be noted that if the initial T cell enrichment population is low and one is unable to perform the second CD161 enrichment step, the aAPC-mediated expansion may not yield the expected results (see Figure 4D, Donor 1). However, if the percentage of circulating NKT cells is higher than 0.1%, one should still be able to obtain a significant expansion of iNKT cells. Collectively, these data demonstrate that CD1d based-aAPC can be used to effectively expand and stimulate primary human NKT cells.
<img alt="Figure 1" src="/files/ftp_upload/4333/4333fig1.jpg" /> 
 Figure 1. Schematic diagram of CD1d:Ig-based aAPCs Extracellular portions of the CD1d molecule are fused to the constant region of an immunoglobulin heavy chain protein separated by a short amino acid linker. These molecules can be easily loaded with lipid antigens, such as &alpha;-GalCer, simply by incubating them with an excess of the lipid of interest. aAPC were made by coupling CD1d-Ig and anti-CD Abs to magnetic beads. In this system, CD1d-Ig is used to provide the cognate antigen-specific signal through the TCR and anti-CD28 mAb provides the costimulatory signal.
<img alt="Figure 2" fo:content-width="4.25in" fo:src="/files/ftp_upload/4333/4333fig2highres.jpg" src="/files/ftp_upload/4333/4333fig2.jpg" /> 
 Figure 2. FACS staining of surface proteins on aAPCs. A) aAPCs were tested for the presence of CD1d:IgG dimer (via staining with PE-conjugated anti-mouse IgG1) as well as anti-CD28 antibody (using FITC-conjugated anti-mouse IgG2a).Open histograms indicate isotype control; filled histograms represent the indicated antibodies. CD1d-Ig Expressing aAPC can Stimulate IL-2 Production by NKT cells. B) The V&alpha;14+ mouse NKT cell hybridoma, DN32.D3, was cocultured with either medium, soluble antigen (&alpha;-GalCer), unloaded aAPC or &alpha;-GalCer-loaded aAPC. Culture supernatants were harvested and standard sandwich ELISA was used to measure IL-2 production.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/4333/4333fig3highres.jpg" src="/files/ftp_upload/4333/4333fig3.jpg" /> 
 Figure 3. Expansion of NKT cells by CD1d-Ig coated artificial antigen presenting cells. (A) Primary CD3+CD161+ double positive cells were isolated from PBMCs using magnetic separation. The sorted cells were stimulated with &alpha;-GalCer loaded,CD1d-Ig coated aAPC for 14 days. The cells were stained for V&alpha;24 and V&beta;11 following aAPC stimulation. (B) Primary human NKT cell mediated lysis of a B cell lymphoma line. C1R-CD1d cells incubated with NKT cells at the indicated ratios in the presence or absence of antigen, a-GalCer (100 ng/ml) in 96-well U- bottom plates for 20-24 hr. NKT cell mediated cell lysis was assessed by by standard 51Cr-release assay.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/4333/4333fig4highres.jpg" src="/files/ftp_upload/4333/4333fig4.jpg" /> 
 Figure 4. Cytokine profiles of aAPC-expanded NKT cells. After stimulation with &alpha; GalCer loaded aAPC for two weeks, the expanded NKT cells(1&times;105/well) were cocultured with soluble &alpha;-GalCer, PMA/Ionomycin, anti-CD3/28 microbeads, or &alpha;-GalCer loaded aAPC (2&times;105/well) for 48 hr. (A) IL-17A, (B) TNF-&alpha;, and (C) IFN-&gamma; production was measured by standard cytokine ELISA. Data shown are net cytokine production after subtracting the negative controls (media and empty beads). (D) Primary T cells were isolated from PBMC using magnetic bead separation. The sorted cells were stimulated for two weeks with the indication a-GalCer loaded-aAPC. The cells were stained using Abs specific for V&alpha;24+ and V&beta;11+ and analyzed by flow cytometry.

Watch this Scientific Journal Video about Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells at JoVE.com

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.

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 ...

artificial-antigen-presenting-cell-aapc-mediated-activation-expansion

Research

JoVE Journal

Immunology and Infection

17.9K visualizações.  University of Maryland. Aqui nós descrevemos um método para ativar e expandir células humanas NKT de granel populações de células T com células artificiais apresentando antígeno (AAPC). O uso de CD1d baseado AAPC fornece um método padronizado para a geração de números elevados de células NKT funcionais.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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 ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

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 ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...