This work has been supported by the German Research Foundation (DFG) SCHU-2681/1-1 (C.S.) and KFO 146 (M.F.), the DOD Department of Defence grant PC 040972 (M.O.) and the National Institute of Health grants RO1 CA108835, RO1 Ai44129 (J.P.S.)

1. Generation of HLA-A2-Ig Based KaAPC
<ol>
	<li>Prepare sterile borate buffer. Make a 0.1 M solution of boric acid in water and adjust the pH to 7.0. Sterile filter (0.22 &#956;m) buffer and store at 4 &#176;C.</li>
	<li>Prepare sterile bead wash buffer. Use phosphate buffered saline (PBS) without magnesium and calcium. Add human AB serum for 3% final concentration. Add ethylenediaminetetraacetic acid (EDTA) for 2 mM final concentration. Add sodium azide (NaN3) for 0.01% final concentration. Sterile filter (0.22 &#956;m) buffer and store at 4 &#176;C.</li>
	<li>Transfer 250 &#956;l of epoxy beads (about 100 x 106 beads) from stock into a sterile, screw top glass vial.</li>
	<li>Add 250 &#956;l of borate buffer.</li>
	<li>Put glass vial on a magnet and let beads adhere for 2 min; aspirate buffer and remove glass vial from magnet.</li>
	<li>Wash beads once with 1 ml borate buffer.</li>
	<li>Resuspend beads into 550 &#956;l of 0.018 &#956;g/ml HLA-A2-Ig and 3.64 &#956;g/ml anti-human CD95 mAb (clone CH11) and gently mix by shaking. (Please see discussion section for additional information.)</li>
	<li>Incubate glass vial on a rotator end over end, at 4 &#176;C for 24 hr.</li>
	<li>Spin down beads at 300 x g for 2 min.</li>
	<li>Place glass vial onto a magnet for 2 min and aspirate the &#8220;KaAPC loading solution&#8221;; remove glass vial from magnet, add 1 ml of bead wash buffer and resuspend carefully by shaking.</li>
	<li>Repeat step 1.10 twice.</li>
	<li>Incubate glass vial on a rotator at 4 &#176;C for 24 hr in 1 ml bead wash buffer. At this point, all residual protein binding sites will be blocked by human AB serum contained in the bead wash buffer.</li>
	<li>Spin down beads at 300 x g for 2 min.</li>
	<li>Place glass vial onto a magnet for 2 min and aspirate bead wash buffer; remove glass vial from magnet and replace solution with 1 ml of PBS.</li>
</ol>
2. Quality Control, Peptide Loading, Washing, Storage, and Stability of KaAPC
<ol>
	<li>Transfer 1.5 x 105 KaAPC into a FACS tube and wash with 1 ml FACS wash buffer.</li>
	<li>Resuspend beads in 100 &#956;l FACS wash buffer and add 1 &#956;l of anti-mouse IgG1 PE (for detection of the Fc part of the HLA-A2-Ig) and 1 &#956;l of anti-mouse IgM FITC (clone R6-60.2, for detection of the anti-human CD95 mAb).</li>
	<li>Incubate for 15 min at 4 &#176;C, wash with 1 ml FACS wash buffer, and resuspend in 250 &#956;l FACS wash buffer and read staining by flow cytometry.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig1highres.jpg" width="500" /> 
Figure 1. QC stain of a KaAPC batch made as stated in the protocol section. Staining was performed as described in protocol section 2. HLA-A2-Ig was detected using an anti-mouse-IgG1 PE mAb, whereas anti-human-CD95 (anti-Fas) was detected with an anti-mouse-IgM FITC mAb (see protocol section 2.2) (blue line). Filled black graphs represent empty beads stained with anti-mouse-IgG1 PE mAb or anti-mouse-IgM FITC mAb, respectively. Numbers in the upper right corner indicate the mean fluorescent intensity (MFI). <a href="https://www.jove.com/files/ftp_upload/51859/51859fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>For peptide loading transfer 20 x 106 KaAPC into a new sterile glass vial, place onto a magnet for 2 min, aspirate PBS and resuspend beads into 500 &#956;l fresh PBS with 5 &#956;g peptide (Keep in mind, only HLA-A2 restricted peptides will bind onto the dimer!).</li>
	<li>Store loaded KaAPC at 4 &#176;C until use, for at least 3 days to allow sufficient peptide loading onto the HLA-A2-Ig dimer.</li>
	<li>Immediately prior to use, wash the peptide loaded KaAPC as indicated in 1.14; repeat this step at least 6x. 
	NOTE: This is a crucial step to ensure that free peptide has to be removed completely to avoid false positive killing results; residual free peptide has been shown to induce apoptosis in antigen-specific T cell cultures9. To rule out a soluble peptide effect, run supernatant control samples of peptide loaded and washed KaAPC (see 3.8).</li>
	<li>Count beads using hemocytometer and label with date, name of peptide and concentration. 
	NOTE: Loaded KaAPC remain functional at 4 &#176;C for at least one month. After one month, re-loading with the same peptide allows KaAPC to be utilized for up to one year.</li>
</ol>
3. KaAPC In Vitro Killing Assay
<ol>
	<li>Prepare co-culture media. Prepare complete RPMI medium supplemented with 3% T cell growth factor (made in the lab6) and 3% donor autologous plasma. If donor autologous plasma is unavailable, use heat-inactivated human AB serum.</li>
	<li>Prepare AnnexinV binding buffer preparation. Dissolve 8.12 g sodium chloride (NaCl) and 0.12 g calcium chloride (CaCl2) into 990 ml ddH2O.Add 10 ml 1M HEPES buffer.</li>
	<li>Generate human antigen-specific T cells. (For a detailed protocol please see a previous publication7 utilizing artificial Antigen Presenting Cells (aAPC)). Ensure that peptide specificity is at least &#62;75% as determined by tetramer staining.</li>
	<li>For each sample incubate 2 x 105 antigen-specific T cells/well (in a 96 well round bottom plate) in 200 &#956;l of co-culture medium at a 1:1 ratio with KaAPC for 48 hr in a humidified incubator at 37 &#176;C and 5% CO2.</li>
	<li>Harvest samples and transfer into a FACS tube; wash with 1 ml AnnexinV binding buffer, spin down at 300 x g for 5 min, discard supernatant and resuspend in 100 &#956;l of AnnexinV binding buffer</li>
	<li>Stain samples with 1 &#956;l AnnexinV FITC and 5 &#956;l 7-AAD for 10-15 min at RT, wash with 1 ml AnnexinV binding buffer; spin down at 300 x g for 5 min, discard supernatant and resuspend in 250 &#956;l AnnexinV binding buffer</li>
	<li>Read samples immediately on flow cytometer.</li>
	<li>Prepare the following samples to determine antigen-specific killing by KaAPC: T cells only; T cells + soluble anti-CD95; T cells + unloaded KaAPC; T cells + non-cognate peptide loaded KaAPC; T cells + cognate peptide loaded KaAPC; T cells + supernatant of cognate peptide loaded KaAPC.</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig2highres.jpg" width="500" /> 
Figure 2. KaAPC killing assay. Peptide loaded KaAPC or control beads (HLA-A2-Ig only beads) were cultured for 48 hr at a 1:1 ratio with CMVpp65 specific T cells (~80%), harvested and subsequently stained with AnnexinV and propidium iodide (PI) to determine apoptosis by flow cytometry (see protocol section 3.3-3.7). &#8220;cognate&#8221; refers to beads loaded with CMVpp65 and &#8220;non-cognate&#8221; to beads loaded with Mart-1 peptide. Numbers indicate the percentage of viable T cells in the lower left quadrant of each plot. <a href="https://www.jove.com/files/ftp_upload/51859/51859fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="9">
	<li>To perform crisscross-experiments, generate antigen-specific T cells of two different specificities.</li>
	<li>Further evaluate the antigen-specific depletion-abilities of KaAPC with a simultaneous analysis of the antigen&#8211;specific elimination of T cells in heterogeneous T cell population. (For a detailed protocol please see Sch&#252;tz&#160;et al.10).</li>
</ol>

<ol>
	<li>Bizzaro, N., Tozzoli, R., Shoenfeld, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+at+a+stage+to+predict+autoimmune+rheumatic+diseases.">Are we at a stage to predict autoimmune rheumatic diseases.</a> Arthritis and rheumatism. 56 (6), 1736-1744 (2007).</li><li>Sch&#252;tz, C., Oelke, M., Schneck, J. P., Mackensen, A., Killer Fleck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=artificial+antigen-presenting+cells:+the+synthetic+embodiment+of+a+&#8220;guided+missile.">artificial antigen-presenting cells: the synthetic embodiment of a &#8220;guided missile.</a> Immunotherapy. 2 (4), 539-550 (2010).</li><li>Hoves, S., 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=Mature+but+not+immature+Fas+ligand+(CD95L)-transduced+human+monocyte-derived+dendritic+cells+are+protected+from+Fas-mediated+apoptosis+and+can+be+used+as+killer+APC.">Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC.</a> Journal of immunology (Baltimore, Md.: 1950). 170 (11), 5406-5413 (2003).</li><li>Hoves, S., 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=Elimination+of+activated+but+not+resting+primary+human+CD4++and+CD8++T+cells+by+Fas+ligand+(FasL/CD95L)-expressing+Killer-dendritic+cells.">Elimination of activated but not resting primary human CD4+ and CD8+ T cells by Fas ligand (FasL/CD95L)-expressing Killer-dendritic cells.</a> Immunobiology. 208 (5), 463-475 (2004).</li><li>Sch&#252;tz, C., Hoves, S., Halbritter, D., Zhang, H. -. G., Mountz, J. D., Fleck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alloantigen+specific+deletion+of+primary+human+T+cells+by+Fas+ligand+(CD95L)-transduced+monocyte-derived+killer-dendritic+cells.">Alloantigen specific deletion of primary human T cells by Fas ligand (CD95L)-transduced monocyte-derived killer-dendritic cells.</a> Immunology. 133 (1), 115-122 (2011).</li><li>Oelke, M., Maus, M. V., Didiano, D., June, C. H., Mackensen, A., 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=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nature. 9 (5), 619-624 (2003).</li><li>Chiu, Y. -. L., 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=HLA-Ig+based+artificial+antigen+presenting+cells+for+efficient+ex+vivo+expansion+of+human+CTL.">HLA-Ig based artificial antigen presenting cells for efficient ex vivo expansion of human CTL.</a> Journal of Visualized Experiments : JoVE. (50), (2011).</li><li>Sch&#252;tz, 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=Killer+artificial+antigen-presenting+cells:+a+novel+strategy+to+delete+specific+T+cells.">Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells.</a> Blood. 111 (7), 3546-3552 (2008).</li><li>Snow, A. L., Pandiyan, P., Zheng, L., Krummey, S. M., Lenardo, M. 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+power+and+the+promise+of+restimulation-induced+cell+death+in+human+immune+diseases.">The power and the promise of restimulation-induced cell death in human immune diseases.</a> Immunological reviews. 236 (1), 68-82 (2010).</li><li>Sch&#252;tz, 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=A+new+flow+cytometric+assay+for+the+simultaneous+analysis+of+antigen-specific+elimination+of+T+cells+in+heterogeneous+T+cell+populations.">A new flow cytometric assay for the simultaneous analysis of antigen-specific elimination of T cells in heterogeneous T cell populations.</a> Journal of immunological. 344 (2), 98-108 (2009).</li><li>Shen, 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=artificial+antigen-presenting+cells+deplete+alloantigen-specific+T+cells+in+a+murine+model+of+alloskin+transplantation.">artificial antigen-presenting cells deplete alloantigen-specific T cells in a murine model of alloskin transplantation.</a> Immunology letters. 138 (2), 144-155 (2011).</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=Nanoscale+artificial+antigen+presenting+Cells+for+T+cell+immunotherapy.">Nanoscale artificial antigen presenting Cells for T cell immunotherapy.</a> Nanomedicine nanotechnology, biology, and medicine. 10 (1), 119-129 (2013).</li><li>Sunshine, J. C., Perica, K., Schneck, J. P., Green, 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=Particle+shape+dependence+of+CD8++T+cell+activation+by+artificial+antigen+presenting+cells.">Particle shape dependence of CD8+ T cell activation by artificial antigen presenting cells.</a> Biomaterials. 35 (1), 269-277 (2013).</li></ol>

C.S. and M.F. have nothing to disclose.
Under a licensing agreement between NexImmune and the Johns Hopkins University, Drs. Schneck and Oelke are entitled to shares of royalty received by the University on sales of aAPC products described in this article. They also own NexImmune stock, which is subject to certain restrictions under University policy. Dr. Schneck is a member of the company&#34;s Board of Directors and Scientific Advisory Board. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

The most critical step in the protocol is to ensure the appropriate ratio of signal 1 (peptide MHC) and signal 2 (anti-Fas mAb). We have performed intensive titration experiments to define the perfect ratio for signal 1 and 2; it became evident that minimal variations due to differences in the HLA-A2 Ig dimer or anti-Fas mAb concentrations can interfere with the functionality of the KaAPC. Thus, the concentration of both signals should always be verified and quality of both proteins should be frequently tested even if it was ordered from a commercial source. Furthermore, the concentrations should always be determined by the same assay as different detection assays might result in different concentrations. Functionality of the HLA-A2-Ig can also be impaired due to incomplete refolding and/or inefficient peptide loading. Furthermore dimer molecules can aggregate to different degrees, which could impact on the functional activity of the protein. Therefore, the actual amount of dimer needed for the generation of a functional and specific KaAPC may vary. In addition, we found that the ideal range for the death inducing signal anti-Fas mAb on KaAPC is extremely tight. In our hands amounts greater 3.64 &#956;g/ml lead to non-specific killing of Fas positive T cells while amounts below 3.64 &#956;g/ml resulted in a dose-depending reduced killing activity. While these conditions seem quite tight, attention to these details will result in the generation of a functional and specific KaAPC.
While the current KaAPC phenotype is functional for depletion of activated antigen-specific T cells, initial experiments targeting na&#239;ve or resting antigen-specific T cells demonstrated no significant induction of antigen-specific apoptosis as these populations have reduced Fas expression. Therefore, one might envision adding a co-stimulatory signal onto the KaAPC to induce the up-regulation of Fas on resting T cells to make turn them into a target for the KaAPC. While this is feasible all three signals will have to be carefully titrated to ensure a functional KaAPC phenotype.
Adjusting the bead platform to a biocompatible or biodegradable matrix will enhance KaAPC in vivo applicability. Recently, Shen et al. have reported the successful in vivo usage of KaAPC utilizing 5 &#956;m latex beads conjugated to anti-Fas (clone Jo2) and anti-His/H2-Kb-TRP211. We have been able to show that the general concept of artificial antigen-presenting Cells (aAPC) can be successfully transferred from &#956;m sized to nm sized particles12 and that functionality is influenced by particle geometry13. Thus, by varying the size and/or shape of future KaAPC designs one might be able to impact on the in vivo functionality and bio-distribution, which could be key for successful treatment of organ specific autoimmune diseases.
KaAPC overcome the major shortcomings of cell based depletion strategies as an easy to use &#8220;off the shelf&#8221;, time and cost efficient approach that is independent of donor condition and pre-treatment. KaAPC are not targets of self- or paracrine killing signaling and not sensitive to cytolytic effector functions of CTL. KaAPC will require identification of the relevant disease antigen and rely on their specific HLA type. While currently numerous suitable HLA-A2 restricted antigens for type 1 diabetes, GvHD and multiples sclerosis have been identified2, development of additional HLA class I dimer molecules as well as the ongoing identification of new autoimmune relevant antigens will further increase and broaden the applicability of KaAPC. Furthermore one could think of using KaAPC directed at different epitopes to target multiple antigens simultaneously.
In summary, KaAPC represent a flexible platform technology for antigen-specific T cell depletion. Their &#8220;lego-like&#8221; nature enables the inclusion of new signals such as additional co-stimulatory or inhibitory signals such as PD1 or TRAIL on various matrices, which could broaden and enhance their subsequent in vitro and in vivo applicability. We herein present the step-by-step protocol to generate KaAPC, the first bead based approach for the elimination of antigen-specific T cells from T cell mixtures with different specificities.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig3highres.jpg" width="500" /> 
Figure 3. KaAPC eliminate CTL in an antigen-specific fashion. PKH26-labelled activated Fas+ effector-memory CTL and PKH67-labelled CMVpp65-specific CTL from the same donor were mixed to a 1:1 ratio and co-cultured with CMVpp65KaAPC for 48 h. Control cultures were treated with unloadedKaAPC, or 1 &#181;g/ml of soluble anti-Fas-mAb (clone CH11). Minimal loss of viable cells was determined in untreated mixed CTL cultures (Tmix). Original FACS data for each CTL population of the Tmix are shown. Analysis of apoptosis was performed as previously published by separate gating on both T cell populations10. Numbers depicted in the upper left quadrants represents % of total cells. The illustrating figure is derived from one representative experiment out of three independent experiments. This research was originally published in Blood. Sch&#252;tz, C.&#160;et al. Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells. Blood. 2008; 111, 3546&#8211;52. Copyright the American Society of Hematology. <a href="https://www.jove.com/files/ftp_upload/51859/51859fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Over the last two decades much progress has been made in understanding the pathogenic mechanisms of autoimmune diseases. However, the most common treatments still rely on corticosteroids as well as immunosuppressive drugs such as purine analogs, alkylating agents and calcineurin inhibitors1. The use of such drugs has greatly improved the prognosis of patients, yet the treatment does not provide a cure of the underlying immunological malfunction. Moreover, patients become vulnerable to infections and to the development of cancer.
Hence the ultimate goal for treatment of T cell mediated autoimmune diseases and allograft rejection is to specifically target only disease causing T cells while, at the same time, leaving the remaining immune system untouched and competent to fight immunological disorders. Utilizing antigen-presenting cells (APC) as a treatment to suppress peripheral T cell responses was first described as early as the 1970s by several groups. Since then many cell-based approaches have been developed (reviewed in Sch&#252;tz&#160;et al.2). Strategies using FasL-expressing killer-APC as immunoregulatory cells showed promising results indicating therapeutic potential for treatment of autoimmunity, allograft rejection and chronic infections. However, there are significant limitations with strategies utilizing FasL Killer-APC that have ultimately limited their clinical applicability; (i) generation or isolation of APC is time, labor and cost intensive (ii) patients suffering from cytopenia due to immunosuppression provide limited amounts and quality of cells (iii) coating or transduction of APC with apoptosis inducing signals such as FasL results in highly variable phenotypes and (iv) Killer-APC are sensitive to cytotoxic effector functions of T cells consequently reducing their therapeutic potential. Therefore, it is difficult to guarantee a defined reproducible Killer-APC phenotype that can be utilized for antigen-specific modulation of T cell responses in vivo.
Based on our previous work with FasL expressing Killer-APC3-5 and artificial antigen-presenting cells (aAPC)6,7 we developed a bead-based approach (KaAPC) for antigen-specific inhibition or elimination of auto-reactive T cells in vitro. KaAPC consist of a HLA-A2-Ig dimer (signal 1) and an anti-Fas mAb (apoptosis inducing signal) covalently immobilized onto a surface of a 4.5 &#956;m paramagnetic latex bead. They deplete antigen-specific T cells in a Fas/FasL mediated fashion from T cell cultures of multiple specificities in an antigen-specific manner and independent of the activation induced cell death (AICD) pathway. Dose dependent apoptosis in targeted T cells is induced rapidly already after 30 min8.
KaAPC can be easily generated in a controlled fashion ensuring a defined off the shelf phenotype and overcome most of the shortcomings of cell based depletion strategies. Consequently, KaAPC display great potential for treatment of T cell mediated autoimmune diseases and allograft rejection, advancing the field of T cell specific treatment strategies.

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:185px;">Name</td>
			<td style="width:157px;">Company</td>
			<td style="width:148px;">Cat #</td>
			<td style="width:164px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human AB serum</td>
			<td>Atlanta biologicals</td>
			<td>S40110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma/Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium azide</td>
			<td>Sigma/Aldrich</td>
			<td>71289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-450 epoxybeads</td>
			<td>Invitrogen/Dynal</td>
			<td>14011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPC-1 magnet</td>
			<td>Invitrogen/Dynal</td>
			<td>12001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">screw top glass vial</td>
			<td>Fisher Scientific</td>
			<td>03-338AA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td>Miltenyi</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD95 (clone CH11)</td>
			<td>Milliore</td>
			<td>05-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HLA-A2-Ig dimer</td>
			<td>BD/Pharmingen</td>
			<td>551263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryo-tube 2ml</td>
			<td>Corning</td>
			<td>430488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse IgG1 PE</td>
			<td>BD/Pharmingen</td>
			<td>559320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse IgM FITC</td>
			<td>BD/Pharmingen</td>
			<td>553408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium chloride</td>
			<td>Merck</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride</td>
			<td>Sigma/Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td>cellgro/Mediatech</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>gibco/life technologies</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96well round bottom plate</td>
			<td>Falcon</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnnexinV FITC</td>
			<td>PromoKine</td>
			<td>PK-CA577-1001-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-AAD</td>
			<td>BD/Pharmingen</td>
			<td>51-68981E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The features of this protocol include the defined generation of a KaAPC phenotype that displays HLA-A2-Ig (signal 1) and anti-Fas mAb (apoptosis signal) at the same time, which in comparison to cell based approaches is not altered by coating or transduction efficacies and can be easily modulated during production (Figure 1). Furthermore, KaAPC, are not sensitive to cytotoxic effector functions of T cells and their functionality is not dependent on the quality of autologous antigen-presenting cells pre and post in vitro manipulation.
KaAPC are capable of depleting antigen-specific T cells in vitro when loaded with cognate peptide, while non-cognate loaded KaAPC will not deplete T cells (Figure 2). Therefore, KaAPC induce T cell apoptosis in an antigen-specific manner. Subsequently, we used KaAPC for selective depletion of antigen-specific T cells from a mixture of different T cell specificities, demonstrating that only minimal cytotoxicity was leaking into neighbor T cells (Figure 3). Thus, KaAPC represent an exquisite tool for in vitro antigen-specific depletion of human activated antigen-specific T cells with potential clinical applicability for the treatment of autoimmune diseases and allograft rejections.

Watch this Scientific Journal Video about Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells at JoVE.com

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.

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 ...

killer-artificial-antigen-presenting-cells-kaapc-for-efficient-vitro

Research

JoVE Journal

Immunology and Infection

9.7K 次观看.  Johns Hopkins University. 准则都用于杀伤人工抗原的产生呈递细胞，KaAPC，用于体外耗尽人抗原特异性T细胞的和在一个抗原的替代解决方案，以细胞免疫治疗对T细胞介导的自身免疫性疾病的治疗中的有效工具具体的方式不影响剩余的免疫系统。 

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

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 ...

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

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

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

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

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

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 ...

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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

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

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

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

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