We thank Penelope Garcia for her technical assistance and preparation of this manuscript. This work was supported by a Research Innovation Grant (RIG) from the Department of Medicine at Loma Linda University (681205-2967) and a pilot grant from National Multiple Sclerosis Society (PP1685) to XT.

All experiments were done in compliance with an Institutional Animal Care and Use Protocol approved by Animal Care and Use Committee at the University of Texas at El Paso and Loma Linda University.
1. Generation of an OLP Library Covering the Whole Length of a Protein
<ol>
	<li>Design an OLP library in which all peptides are 15-mer in length, and adjacent peptides overlap by 11 amino acids. 
	NOTE: In the MOG OLP study, sequence of the MOG precursor [Mus musculus] was retrieved from NCB...

<ol>
	<li>Aldrich, C. J., 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=Identification+of+a+Tap-dependent+leader+peptide+recognized+by+alloreactive+T+cells+specific+for+a+class+Ib+antigen.">Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen.</a> Cell. 79 (4), 649-658 (1994).</li><li>Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E., Raulet, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+CD94/NKG2A+is+a+natural+killer+cell+receptor+for+the+nonclassical+major+histocompatibility+complex+(MHC)+class+I+molecule+Qa-1(b).">Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b).</a> J Exp Med. 188 (10), 1841-1848 (1998).</li><li>Oliveira, C. 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=The+nonpolymorphic+MHC+Qa-1+mediates+CD8++T+cell+surveillance+of+antigen-processing+defects.">The nonpolymorphic MHC Qa-1 mediates CD8+ T cell surveillance of antigen-processing defects.</a> J Exp Med. 207 (1), 207-221 (2010).</li><li>Nagarajan, N. A., Gonzalez, F., Shastri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+MHC+class+Ib-restricted+cytotoxic+T+cells+monitor+antigen+processing+in+the+endoplasmic+reticulum.">Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum.</a> Nat Immunol. 13 (6), 579-586 (2012).</li><li>Hu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+regulatory+CD8+T+cells+in+Qa-1-deficient+mice.">Analysis of regulatory CD8 T cells in Qa-1-deficient mice.</a> Nat Immunol. 5 (5), 516-523 (2004).</li><li>Tang, X., 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=Regulation+of+immunity+by+a+novel+population+of+Qa-1-restricted+CD8alphaalpha+TCRalphabeta++T+cells.">Regulation of immunity by a novel population of Qa-1-restricted CD8alphaalpha+TCRalphabeta+ T cells.</a> J Immunol. 177 (11), 7645-7655 (2006).</li><li>Leavenworth, J. W., Tang, X., Kim, H. J., Wang, X., Cantor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+arthritis+through+mobilization+of+peptide-specific+CD8++regulatory+T+cells.">Amelioration of arthritis through mobilization of peptide-specific CD8+ regulatory T cells.</a> J Clin Invest. 123 (3), 1382-1389 (2013).</li><li>Wu, Y., Zheng, Z., Jiang, Y., Chess, L., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specificity+of+T+cell+regulation+that+enables+self-nonself+discrimination+in+the+periphery.">The specificity of T cell regulation that enables self-nonself discrimination in the periphery.</a> Proc Natl Acad Sci U S A. 106 (2), 534-539 (2009).</li><li>Panoutsakopoulou, V., 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=Suppression+of+autoimmune+disease+after+vaccination+with+autoreactive+T+cells+that+express+Qa-1+peptide+complexes.">Suppression of autoimmune disease after vaccination with autoreactive T cells that express Qa-1 peptide complexes.</a> J Clin Invest. 113 (8), 1218-1224 (2004).</li><li>Tang, X., Maricic, I., Kumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-TCR+antibody+treatment+activates+a+novel+population+of+nonintestinal+CD8+alpha+alpha++TCR+alpha+beta++regulatory+T+cells+and+prevents+experimental+autoimmune+encephalomyelitis.">Anti-TCR antibody treatment activates a novel population of nonintestinal CD8 alpha alpha+ TCR alpha beta+ regulatory T cells and prevents experimental autoimmune encephalomyelitis.</a> J Immunol. 178 (10), 6043-6050 (2007).</li><li>Holderried, T. A., Lang, P. A., Kim, H. J., Cantor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disruption+of+CD8++Treg+activity+enhances+the+immune+response+to+viral+infection.">Genetic disruption of CD8+ Treg activity enhances the immune response to viral infection.</a> Proc Natl Acad Sci U S A. 110 (52), 21089-21094 (2013).</li><li>Wang, X., 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=Targeting+Non-classical+Myelin+Epitopes+to+Treat+Experimental+Autoimmune+Encephalomyelitis.">Targeting Non-classical Myelin Epitopes to Treat Experimental Autoimmune Encephalomyelitis.</a> Sci Rep. 6, 36064 (2016).</li><li>Lo, W. F., Dunn, C. D., Ong, H., Metcalf, E. S., Soloski, 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=Bacterial+and+host+factors+involved+in+the+major+histocompatibility+complex+class+Ib-restricted+presentation+of+Salmonella+Hsp+60:+novel+pathway.">Bacterial and host factors involved in the major histocompatibility complex class Ib-restricted presentation of Salmonella Hsp 60: novel pathway.</a> Infect Immun. 72 (5), 2843-2849 (2004).</li><li>Xu, W., 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=The+nucleocapsid+protein+of+Rift+Valley+fever+virus+is+a+potent+human+CD8++T+cell+antigen+and+elicits+memory+responses.">The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.</a> PLoS One. 8 (3), e59210 (2013).</li><li>Schumacher, T. N., 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=Peptide+selection+by+MHC+class+I+molecules.">Peptide selection by MHC class I molecules.</a> Nature. 350 (6320), 703-706 (1991).</li><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Curr Protoc Immunol. , (2008).</li><li>Guo, H. 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=Different+length+peptides+bind+to+HLA-Aw68+similarly+at+their+ends+but+bulge+out+in+the+middle.">Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.</a> Nature. 360 (6402), 364-366 (1992).</li><li>Soloski, M. J., Metcalf, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+involvement+of+class+Ib+molecules+in+the+host+response+to+infection+with+Salmonella+and+its+relevance+to+autoimmunity.">The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity.</a> Microbes Infect. 3 (14-15), 1249-1259 (2001).</li><li>Smith, T. R., 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=Dendritic+cells+use+endocytic+pathway+for+cross-priming+class+Ib+MHC-restricted+CD8alphaalpha+TCRalphabeta++T+cells+with+regulatory+properties.">Dendritic cells use endocytic pathway for cross-priming class Ib MHC-restricted CD8alphaalpha+TCRalphabeta+ T cells with regulatory properties.</a> J Immunol. 182 (11), 6959-6968 (2009).</li><li>Kalra, A., Mukherjee, P., Chauhan, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+fine+specificity+of+the+immune+response+to+a+Plasmodium+falciparum+rhoptry+neck+protein,+PfAARP.">Characterization of fine specificity of the immune response to a Plasmodium falciparum rhoptry neck protein, PfAARP.</a> Malar J. 15, 457 (2016).</li><li>Kambayashi, T., 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=The+nonclassical+MHC+class+I+molecule+Qa-1+forms+unstable+peptide+complexes.">The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes.</a> J Immunol. 172 (3), 1661-1669 (2004).</li></ol>

Here, we have described a protocol for analyzing Qa-1 epitopes in a protein. In relation to this protocol, several other strategies were also reported previously. First, allogeneic CD8+ T cell lines and clones were used for the identification of the Qdm1. Second, a putative Qa-1-binding motif from the analysis of Qdm was used for the identification of the HSP60p216-224 and a TCRBV8.1 epitope9,18. Third, individual overlapping pe...

Qa-1 belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules in mice. Its human homolog is HLA-E. Previous evidence has demonstrated that Qa-1 molecules have important biological functions. Firstly, Qa-1 molecules play an important role in surveying cells for structural and functional integrity. In this regard, Qa-1 molecules have evolved several strategies to monitor the normal function of a cell. One such strategy enables Qa-1 molecules to form complexes with a processed leader peptide (epitope), i.e. the Qa-1 determinant modifier (Qdm) that is processed from classical MHC-Ia molecules in the endoplasmic reticulum1. These Qa-1/Qdm complexes later display on the surface of a cell and bind to inhibitory NKG2A receptors on NK cells to inhibit NK killing activity2. If the expression of MHC-Ia molecules is lost, a cell (e.g. a malignant cell) becomes sensitive to NK killing2. The other strategy enables Qa-1 molecules to form new Qa-1/epitope complexes on the surface of a cell that is deficient in TAP (transporter associated with antigen processing)3 and/or ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing)4 (both deficiencies often occur in malignant cells). The cell that expresses these new Qa-1/epitope complexes can then be recognized and eliminated by the epitope-specific Qa-1-restricted CD8+ T cells. Secondly, Qa-1 molecules induce immune regulation5. In this regard, Qa-1/epitope complexes have been shown to stimulate CD8+ regulatory T (Treg) cells that are important for the prevention of immune-mediated damage of self-tissues6,7,8,9,10. Thirdly, Qa-1-restricted CD8+ Treg cells have been shown to limit immune responses against viral infection11.
Therefore, specific augmentation of epitope-specific Qa-1-restrictred CD8+ T cells is a potentially promising strategy for the elimination of abnormal cells, for the enhancement of immune regulation, and for the control of the magnitude of virus-induced immune responses. While it has not been determined whether augmentation of epitope-specific Qa-1-restricted CD8+ T cells can enhance immune surveillance and limit virus-induced immune responses, our laboratories and others have clearly demonstrated that immunization with Qa-1 epitopes can augment the function of Qa-1-restricted CD8+ Treg cells specific for pathogenic autoimmune CD4+ T cells, leading to efficient control of CD4+ T cell-mediated autoimmune diseases in a variety of animal models such as experimental allergic encephalomyelitis (an animal model of human multiple sclerosis)6,10, collagen-induced arthritis (an animal model of human rheumatoid arthritis)7, and non-obese diabetes (an animal model of human type 1 diabetes)8. Additionally, we have discovered that immunization with a tissue-specific Qa-1 epitope leads to specific control of immune-mediated inflammation in that tissue through augmentation of CD8+ Treg cells12. The above successes of preclinical studies indicate a need for a full evaluation of Qa-1 epitope immunization for the treatment of tissue-specific immune-mediated diseases and potentially for the therapy of other diseases associated with deficiencies in TAP and ERAAP.
Accordingly, there is a demand for a technology that can reliably and quickly analyze Qa-1 epitopes in a protein. In this regard, a limited number of biologically important Qa-1 epitopes has been described. Most of these Qa-1 epitopes were identified serendipitously during the study of CD8+ T cell responses to bacteria13, cells deficient in TAP3, cells deficient in ERAAP4, and cells that cause EAE6,9. Therefore, a high throughput technique is desirable for the identification of biologically important Qa-1 epitopes in a defined protein. In the following, we describe an overlapping peptide (OLP) library strategy that maps functional Qa-1 epitopes in a protein using Qa-1-resrticted CD8+ T cell lines specific for the OLP pool (OLP_pool) of a protein.

<table>
	<tbody>
		<tr>
			<td>The protein to be analyzed</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Sequence of the protein can be obtained from NCBI</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: D2650 SIGMA</td>
			<td>DMSO should be sterile and cell culture tested.</td>
		</tr>
		<tr>
			<td>Kb-/-Db-/- mice</td>
			<td>The Lackson Laboratory</td>
			<td>Stock#: 019995</td>
			<td>We used Taconic H2-KbH2-Db doube knockout mice (Cat#: 4215-F and 4215-M) which however are not available anymore.</td>
		</tr>
		<tr>
			<td>AIM V Serum Free Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 12055091</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11140076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads CD8 Positive Isolation Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11333D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Gel P-100</td>
			<td>Bio-Rad</td>
			<td>Cat#: 150-4171</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoshate Balanced Solution (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Trasfer pipette</td>
			<td>Globe Scientific</td>
			<td>Mfg#: 137238</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine M-CSF</td>
			<td>PeproTech</td>
			<td>Cat#: 315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well tissue culture plates</td>
			<td>USA Scientific</td>
			<td>Cat#: CC7682-7548</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar TC-treated Multiple well Plates, 96-well, V-shaped bottom</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: Z372129 Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml deep 06-well PP plate, sterile</td>
			<td>USA Scientific</td>
			<td>Item#: 1896-1110</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-2</td>
			<td>PeproTech</td>
			<td>Cat#: 212-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-7</td>
			<td>PeproTech</td>
			<td>Cat#L: 217-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Capture anti-IFN-&#947; antibody</td>
			<td>BD Biosciences</td>
			<td>Cat#: 551881</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISPOT plate</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: S2EM004M99</td>
			<td></td>
		</tr>
		<tr>
			<td>C1R</td>
			<td>ATCC</td>
			<td>Cat#: ATCC CRL-1993</td>
			<td></td>
		</tr>
		<tr>
			<td>C1R.Qa-1b</td>
			<td></td>
			<td></td>
			<td>Custom made (GenBank access#: NM_010398.3)</td>
		</tr>
		<tr>
			<td>Qa-1 lentiviral vector</td>
			<td>GeneCopoeia</td>
			<td>Product#: Mm02955</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection anti-IFN-&#947; antibody</td>
			<td>BD Biosciences</td>
			<td>Cat#: 551881</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-HRP</td>
			<td>BD Biosciences</td>
			<td>Cat#: BD557630</td>
			<td></td>
		</tr>
		<tr>
			<td>AEC substrate</td>
			<td>BD Biosciences</td>
			<td>Cat#: 551951</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoSpot Analyzer</td>
			<td>ImmunoSpot</td>
			<td></td>
			<td>Any immunoSpot analyer should work for this purpose.</td>
		</tr>
	</tbody>
</table>

overlapping peptide library to map qa-1 epitopes in a protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Design of an OLP library covering the whole length of a protein
Beginning at the N-terminus of a protein, each peptide is 15 amino acids (15-mer). Hence, the first peptide spans position 1 to position 15. The N-terminus of the second peptide overlaps with the C-terminus of the first peptide by 11 amino acid. Hence, the second peptide spans the position 5 to position 19. Design the rest of the peptides to the end of C-terminus of th...

Watch this Scientific Journal Video about Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein at JoVE.com

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

The overall goal of this procedure is to identify Qa-1 epitopes in proteins. This method can help answer key questions in the field of immune regulation and immunotherapy. The main advantages of this technique are that mapped peptides are known to stimulate Qa-1 restricted CD8 T cells, and the procedure is easy to perform.The implications of this technique extend towards therapeutic designs for immune-mediated diseases, such as multiple sclerosis. Though this method can provide insight into Qa-1 mediated immune regulation in animals, it can also be applied to HLA-E mediated immune regulation in humans. To begin the procedure, design a library of 15-mer peptides, in which adjacent peptides overlap in 11 amino acids.Obtain and reconstitute the peptides under sterile conditions. For each peptide, combine five milligrams of the peptide with 100 microliters of dimethyl sulfoxide. Combine 10 microliters of each peptide stock in a clean 1-milliliter cryo tube to prepare the OLP pool stock.Then, load 20 microliters of each peptide stock into a V-bottomed 96-well plate, and dilute each stock with 80 microliters of sterile water. Store both the remaining 50 milligrams per milliliter peptide stocks and the plate of 10 milligrams per milliliter peptides stocks at 20 degrees Celsius. Next, add 10 microliters of the OLP pool stock solution to a 15 milliliter tube containing two milliliters of irradiated dendritic cells in serum-free medium, thus diluting the DMSO two hundred-fold.Store the remaining OLP pool stock at 20 degrees Celsius. Incubate the DCs with the OLP pool at room temperature for three hours. Gently shake the cells every 15 minutes by hand.During the incubation, use a kit to purify CD8+T cells from a harvested mouse spleen or lymph nodes. Prepare five milliliters of a solution with 10 million T cells per milliliter of serum-free medium with 50 units per milliliter of interleukin 2, and 100 units per milliliter of interleukin 7. The purified CD8 T cells should be free of beads, or untouched.Place 0.5 milliliters of the T-cell solution in each of 10 wells of a 48-well plate. Once the incubation of the OLP pool-pulsed DC solution is complete, add 10 milliliters of serum-free medium to the DCs. Centrifuge the cells at 300 times G for 10 minutes at room temperature.Then, reconstitute the cells in five milliliters of serum-free medium. Add 0.5 milliliters of the cell suspension to each of the 10 wells. Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide atmosphere for four days.Then, remove 400 microliters of the culture medium from each well. Add 500 microliters of fresh serum-free medium containing 100 units per milliliter each of IL-2 and IL-7 to each well. Incubate the cells for another two to three days before re-stimulating the OLP pool-primed cells.Prior to re-stimulation, obtain and irradiate mouse peritoneal macrophages. Adjust the macrophage concentration to five million cells per milliliter of serum-free medium. Combine 10 microliters of the OLP pool stock with two milliliters of the irradiated peritoneal macrophages for a final DMSO concentration of 0.5%by volume.Supplement this solution with 100 units per milliliter of murine macrophage colony-stimulating factor. Fill a 48-well plate with 200 microliters of the OLP-pool macrophage solution in each well. Incubate the plate at 37 degrees Celsius for four hours.Then, gently wash each well with 200 microliters of pre-warmed RPMI-0 medium. The macrophage solution will contain lot of beads. Before the OLP pool-primed CD8 T cells are added, the plate should be checked under a microscope to make sure that all the wells are free of beads.Next, collect and pool the OLP pool-primed CD8+T cells that are ready for re-stimulation. Adjust the T-cell concentration to one million cells per milliliter in serum-free medium containing 25 units per milliliter of IL-2, and 50 units per milliliter of IL-7. Add one milliliter of the pooled T-cell solution to each well containing OLP pool-pulsed macrophages.Culture the cells at 37 degrees Celsius in a 5%carbon dioxide atmosphere for four days. Then, remove about 400 microliters of the medium from each well. Add to each well 500 microliters of fresh serum-free medium containing 100 units per milliliter each of IL-2 and IL-7.Resume incubation under the same conditions. After three to four days, examine the re-stimulated cells for an OLP pool-specific Qa-1 restricted response with an interferon-gamma enzyme linked immuno spot assay. Refresh the CD8+T-cell medium every three to four days while waiting for the results.Perform an ELISPOT assay with the dilute peptide stocks to determine which peptides produce the OLP pool-specific Qa-1 restricted interferon-gamma secretion. Lastly, evaluate a truncated peptide series with ELISPOT to identify the optimal Qa-1 epitope. An interferon-gamma ELISPOT assay showed a significant increase in spot-forming cells when CD8+T cells stimulated by the pooled MOG overlapping peptide library were combined with C1R.Qa-1b cells. This suggested that the T cells responded to the Qa-1 binding peptides, and that the MOG_pool contained one or more Qa-1 binding epitopes. Another assay was performed to screen the individual peptides in the pool.Three peptides showed responses that were at least three times greater than the response in the presence of C1R. Qa-1b cells without peptides. Of these, the peptide with the greatest response was selected for further evaluation.A library of truncated peptides based on the 15-mer peptide of interest was evaluated to identify the optimal Qa-1 epitope. The epitope would ideally be eight to 10 mer, with nine mer preferred. Further, the optimal epitope would show a similar or stronger interferon-gamma response than the 15-mer peptide.The nine mer N-terminally truncated peptide best fit these criteria, making it the optimal Qa-1 epitope. After its development, this technique paved the way for researchers in the field of immune regulation to discover new Qa-1 epitopes for the study of immune regulation in other tissues, such as the pancreas. Don't forget, working with radiation can be extremely hazardous.Researchers should strictly follow the institutional policy and be appropriately trained.

overlapping-peptide-library-to-map-qa-1-epitopes-in-a-protein

Research

JoVE Journal

Immunology and Infection

6.8K vues.  Loma Linda University. The overall goal of this procedure is to identify Qa-1 epitopes in proteins. This method can help answer key questions in the field of immune regulation and immunotherapy. The main advantages of this technique are that mapped peptides are known to stimulate Qa-1 restricted CD8 T cells, and the procedure is easy to perform.The implications of this technique extend towards therapeutic designs for immune-mediated diseases, such as multiple sclerosis. Though this method can provide insight...