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 NCBI protein database by following this link: https://www.ncbi.nlm.nih.gov/protein/NP_034944. The MOG precursor (247 amino acids), which contained both signal and mature peptides, was used because most of reported Qa-1 (HLA-E) epitopes were located in signal peptides (e.g. Qdm1). Beginning at its N-terminus, the sequences of 15-mer peptides were identified such that two adjacent peptides overlapped by 11 amino acids (Figure 1). Hence, exactly 59 OLPs were identified in the 247 amino acid MOG precursor. However, the last OLP can range from 12 to 15-mer depending on the length of the protein. Additionally, we choose 15-mer library because we and others have shown that 15-mer peptides, when added into dendritic cells (DCs) or macrophages, can be efficiently processed into epitopes for recognition by CD8+ T cells14,15.</li>
	<li>Purchase each individual peptide commercially. 5 mg per peptide should be enough for the screening. OLPs and truncated peptides (Step 6.1) can be desalted peptides (purity is approximately 50 - 70%). The purity of optimal peptide (Step 6.2) that will be used for the generation of tetramer and for future biological analyses should be &#62;90%. Reconstitute the peptides under sterile condition.</li>
	<li>Make 50 mg/mL individual peptide stocks in 100% DMSO. For 5 mg of each peptide, add 100 &#956;L of DMSO into each tube, mix, and store the peptides at -20 &#176;C. These stocks will be used to make an OLP_pool stock for the generation of CD8+ T cell lines reactive to the OLP_pool. In addition, these stocks will also be used to make 10 mg/mL individual peptide stocks for determining the ability of each individual peptide to stimulate a Qa-1-restricted response in an OLP_pool-reactive CD8+ T cell line.</li>
	<li>Make OLP_pool stock by adding an equal volume of each peptide into a fresh tube. This OLP_pool stock contains 100% DMSO. In the MOG OLP study, there were 59 OLPs12. Hence, concentration of each peptide in the OLP_pool was 847.46 &#956;g/mL (50 mg/mL &#247; 59).</li>
	<li>Make 10 mg/mL individual peptide stocks by diluting (5x) the 50 mg/mL stock in sterile H2O in a V-bottom 96-well plate (this stock contains 20% DMSO). Make these stocks in a 96-well plate because the determination of the Qa-1-restricted response of each individual peptide will be performed in a 96-well plate using an 8- or 12-channel multichannel pipette. 
	NOTE: All peptides, including OLPs and truncated peptides, should be diluted in either a V-bottom 96-well plate or a 96-well sample rack filled with 1 mL tubes since the peptide library screening is performed in 96-well plates using a multichannel pipette. If it is difficult to dilute a peptide, add 1 N NaOH drop wise to help dissolve the peptide.</li>
</ol>
2. Priming of Kb-/- Db-/- CD8+ T Cells with the OLP_pool-pulsed Kb-/- Db-/- Dendritic cells (DCs).
NOTE: There are two major Qa-1 alleles: one is Qa-1a, and the other is Qa-1b. Since the animals commonly used for academic research, e.g. C57BL/6 and Balb/c mice, carry Qa-1b, this protocol describes the procedure for mapping Qa-1b epitopes in a protein. CD8+ T cells used in this protocol are purified from Kb-/-Db-/- mice (C57BL/6 background) in which CD8+ T cells are restricted mostly by non-classical MHC-Ib molecules including Qa-1.
<ol>
	<li>Produce bone marrow-derived DCs as previously described12.
	<ol>
		<li>Briefly, culture bone marrow single cell suspensions (1 x 106 cells/mL) in RPMI-10 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 5.5 x 10-5 M 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acid) containing 10 U/mL IL-4 and 100 U/mL GM-CSF in a 6-well plate (4 mL/well) at 37 &#176;C, 5% CO2.</li>
		<li>Two days later, remove non-adherent cells carefully and add fresh media and cytokines.</li>
		<li>After culturing the cells for another two days, transfer non-adherent cells containing fresh media and cytokines into a new 6-well plate.</li>
		<li>Culture the cells for another two days and replenished with fresh media and cytokines containing LPS (0.1 &#181;g/mL) to activate the DCs.</li>
		<li>24 h later, collect the DCs for experiments.</li>
	</ol>
	</li>
	<li>Irradiate the DCs with 3000 Rads.
	<ol>
		<li>Alternatively, treat the DCs (5 x 107 cell/mL) with mitomycin C (50 &#956;g/mL) in PBS at 37 &#176;C for 20 min. Add RPMI-0 (RPMI 1640 without serum) to fill the tube (~12 mL) and spin the cells for 10 min in a table-top centrifuge at 300 x g. Discard supernatants and repeat the washing procedure two more times. These three washes are critical because any trace amount of mitomycin C may inhibit the response of CD8+ T cells during the DC-T cell co-culture.</li>
	</ol>
	</li>
	<li>Adjust the DC concentration to 5 x 106 cells/mL in a serum-free medium (AIM-V serum-free medium supplemented with 5.5 x 10-5 M 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acid).</li>
	<li>Add the OLP_pool stock solution, which contains 100% DMSO, to the DCs such that final DMSO concentration will be 0.5% (200x). In the MOG OLP study, the concentration of each OLP in the OLP_pool was 847.46 &#956;g/mL; hence the final concentration of each OLP in the DC culture was 4.2 &#956;g/mL (847.46 &#956;g/mL &#247; 200). However, this concentration can be scaled up to 100 &#956;g/mL as long as the DMSO concentration in the cell culture remains less than 1%.</li>
	<li>Incubate the DCs at room temperature for 3 h and shake the cells gently every 15 min.</li>
	<li>During this incubation period, purify CD8+ T cells from the harvested spleen or lymph nodes of Kb-/-Db-/- mice using a commercial CD8+ T cell purification kit, adjust the cell concentration to 10 x 106 cells/mL in the serum-free medium containing 50 U/mL interleukin 2 (IL-2) and 100 U/mL of IL-7.</li>
	<li>Now, add the CD8+ T cells into a 48-well plate as 0.5 mL/well (after the addition of 0.5 mL/well of the OLP_pool-pulsed DCs in Step 2.8, the final concentration of the CD8+ T cells will be 5 x 106 cells/mL). 
	NOTE: CD8+ T cells from mouse spleen and lymph nodes can be purified using CD8 Positive Isolation Kit by following the instruction provided by the manufacturer. CD8+ T cells obtained are free of beads.</li>
	<li>Spin down the OLP_pool-pulsed DCs (300 x g, 10 min) at RT. Reconstitute the OLP_pool-pulsed DCs to 2 x 106 cells/mL in the serum-free medium and add 0.5 mL/well into the 48-well plate that contains the CD8+ T cells (final concentration of the OLP_pool-pulsed DCs is 1 x 106 cells/mL, final concentration of CD8+ T cells is 5 x 106 cells/mL, final concentration of IL-2 is 25 U/mL, and final concentration of IL-7 is 50 U/mL). Culture the cells at 37 &#176;C and 5% CO2.</li>
	<li>On day 4, remove and discard about 400 &#956;L of the culture medium and add 500 &#956;L of the fresh serum-free medium containing 100 U/mL IL-2 and 100U/mL of IL-7 to each well. Incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>On day 7 or 8, re-stimulate the OLP_pool-primed CD8+ T cells with the OLP_pool.</li>
</ol>
3. Restimulation of the Primed CD8+ T Cells with Macrophages Pulsed with the OLP_pool
<ol>
	<li>Four days before re-stimulation of the OLP_pool-primed CD8+ T cells, prepare 2% (v/v) polyacrylamide bead solution: wash 2 g of polyacrylamide beads twice in 20 mL endotoxin-free H2O or PBS. Pellet the polyacrylamide beads by centrifugation (400 x g) for 5 min and resuspend in 100 mL of PBS. Autoclave at 15 lb/m for 20 min. Store at room temperature.</li>
	<li>Inject mice intraperitoneally with 1 mL/mouse of the sterile 2% polyacrylamide bead solution to attract the migration of monocytes/macrophages into the peritoneal cavity16.</li>
	<li>Four days later, sacrifice the animals by CO2 overdose.
	<ol>
		<li>Under sterile conditions, cut a small opening at the center of the abdomen such that the opening is just enough for passing a 5 mL transfer pipette. Fill the transfer pipette with RPMI-0 and insert the pipette into the abdomen cavity through the opening.</li>
		<li>Rinse the abdomen cavity by pipetting. Pipette out as much liquid as possible from the abdomen cavity into a sterile tube (these are peritoneal macrophages). Repeat the rinse step 4 - 5 times.</li>
	</ol>
	</li>
	<li>Irradiate the peritoneal macrophages with 3000 Rads.
	<ol>
		<li>Alternatively, treat the peritoneal macrophages with mitomycin C (follow the procedure described in Step 2.2).</li>
	</ol>
	</li>
	<li>Adjust the concentration of the peritoneal macrophages to 5 x 106 cells/mL in the serum-free medium. Add OLP_pool stock (the final DMSO concentration is less than 1%) and M-CSF (final concentration=100 U/mL). 
	NOTE: In this MOG OLP study, we used 0.5% DMSO. Thus, MOG OLP_pool stock was diluted 200x (e.g. 1 &#956;L of MOG OLP_Pool stock was added into 199 &#956;L of peritoneal macrophages). Hence, the final concentration of each OLP was 4.2 &#956;g/mL).</li>
	<li>Add 200 &#956;L/well (1 x 106 cells/well) in a 48-well tissue culture plate and incubate the plate at 37 &#176;C for 4 h.</li>
	<li>Remove the non-adherent cells and the polyacrylamide beads by gentle washing with 200 &#956;L/well of the pre-warmed RPMI-0.</li>
	<li>Collect and pool the OLP_pool primed CD8+ T cells from Step 2.10. Adjust the OLP_pool primed CD8+ T cell concentration to 1 x 106 cells/mL in the serum-free medium containing 25 U/mL IL-2 and 50 U/mL of IL-7. Add 1 mL/well to the 48-well plate that contains the OLP_pool-pulsed peritoneal macrophages.</li>
	<li>Four days later, replenish the medium in the 48-well plate. Remove and discard about 400 &#956;L of the culture medium in the 48-well culture plates. Add 500 &#956;L of fresh serum-free medium containing 100 U/mL IL-2 and 100 U/mL of IL-7 into each well. Culture the cells at 37 &#176;C and 5% CO2.</li>
	<li>Three or four days later, examine the OLP_pool-restimulated CD8+ T cells for OLP_pool-specific, Qa-1-restricted response by enzyme-linked immunospot assay (ELISPOT). After this point, re-stimulate the CD8+ T cells every 7 - 10 days. 
	NOTE: Macrophages are preferred for the re-stimulation of the OLP_pool-primed CD8+ T cells. We noticed that CD8+ T cells re-stimulated by macrophage grew better than DCs in vitro.</li>
</ol>
4. Determination of OLP_pool-specific, Qa-1-restricted Response in an OLP_pool-restimulated CD8+ T Cell Line
NOTE: OLP_pool-specific, Qa-1-restricted response in an OLP_pool-restimulated CD8+ T cell line is determined by IFN&#947; secretion following stimulation by the OLP_pool in the presence of C1R or C1R.Qa-1b cells using an IFN&#947; ELISPOT assay. C1R cells can be obtained commercially. C1R.Qa-1b cells can be generated by transducing the C1R cells with the Qa-1 lentiviral vector.
<ol>
	<li>Add 100 &#956;L/well of a capture anti-IFN-&#947; antibody diluted in coating buffer (PBS) into the wells of an ELISPOT plate. Seal and incubate the plate at 4 &#176;C overnight.</li>
	<li>On the second day, discard the coating buffer containing the capture anti-IFN-&#947; antibody and add 200 &#956;L/well-blocking solution (serum-free medium) and incubate the plate for 2 h at room temperature.</li>
	<li>Irradiate the C1R and C1R.Qa-1b cells with 9,600 Rads.
	<ol>
		<li>Alternatively, treat the C1R and C1R.Qa-1b cells with mitomycin C as described in the Step 2.2 except that the treatment time will be 30 min.</li>
	</ol>
	</li>
	<li>Adjust the C1R and C1R.Qa-1b cells in the serum-free medium to 4 x 106 cells/mL.</li>
	<li>Discard the blocking solution in the plate and add the C1R and C1R.Qa-1b cells at 50 &#956;L/well (200,000 cells/well) and mix.</li>
	<li>Add 50 &#956;L/well of 100x&#160;diluted OLP_pool stock (in the serum-free medium) and mix properly. Incubate the plate at room temperature for 2 - 3 h.</li>
	<li>Adjust the OLP_pool-restimulated CD8+ T cells to 1 - 2 x 106 cells/mL in the serum-free medium containing 150 U/mL of IL-7 and add 50 &#956;L/well (50,000 - 100,000 cells/well) to the plate. Centrifuge the plate (58 x g) for 5 min.</li>
	<li>Incubate the plate at 37 &#176;C and 5% CO2 overnight.</li>
	<li>Aspirate cell suspension. Wash wells 2 times with deionized (DI) water (250 &#956;L/well). Allow wells to soak for 5 min at each wash step.</li>
	<li>Wash wells 3 times with Wash Buffer I (PBS containing 0.05% Tween20). Allow wells to soak for 1 min at each wash step. Discard wash buffer.</li>
	<li>Add 100 &#956;L of detection antibody diluted in a dilution buffer (PBS containing 10% fetal bovine serum (FBS)).</li>
	<li>Incubate the plates at room temperature for 2 h.</li>
	<li>Discard detection antibody solution. Wash wells 3 times with 250 &#181;L/well Wash Buffer I. Allow wells to soak for 1 min at each wash step.</li>
	<li>Add 100 &#956;L/well of enzyme conjugate (Streptavidin-HRP) diluted in the dilution buffer at 1:100 dilution.</li>
	<li>Incubate the plates for 1 h at room temperature.</li>
	<li>Discard enzyme conjugate solution. Wash wells 4 times with 250 &#181;L/well Wash Buffer I. Allow wells to soak for 1 min at each wash step.</li>
	<li>Wash wells 2 times with 250 &#181;L/well Wash Buffer II (PBS).</li>
	<li>Add 100 &#181;L of Substrate Solution (mix 1 drop=20 &#181;L of AEC Chromogen with 1 mL of AEC substrate) to each well. Monitor spot development for 5 to 60 min.</li>
	<li>When desired results start to appear, stop the substrate reaction by washing wells with distilled water.</li>
	<li>Air-dry the plates at room temperature for 2 h or overnight until it is completely dry. Removal of the plastic tray under the plates will facilitate drying. Store plates in a sealed plastic bag in the dark until it is analyzed.</li>
	<li>Enumerate spots manually by inspecting under a dissecting microscope or using an ELISPOT plate reader. See the representative result in Figure 2.</li>
</ol>
5. Determination of Individual Peptides in the OLP_pool which Stimulate to the Qa-1 Restricted IFN-&#947; Secretion in an OLP_pool-specific CD8+ T Cell Line
<ol>
	<li>Determine the individual peptides that stimulate the OLP_pool-specific, Qa-1-restrictred IFN-&#947; secretion in an OLP_pool-specific CD8+ T cell line using the above described IFN-&#947; ELISPOT assay (final concentration of each individual peptide used for the ELISOPT assay is 10 &#956;g/mL). See representative results in Figure 3. 
	NOTE: An OLP-specific, Qa-1-restircted response is defined as a response that is at least three times of the response in the presence of C1R.Qa-1b cells but without the OLP. In the MOG OLP study, OLP68, OLP96, and OLP105 all met this criterion. Therefore, all the three OLPs potentially contain Qa-1 epitopes12 (Figure 3). However, the OLP105 consistently gave the strongest response; we hence performed a detailed analysis of the OLP105 (Figure 4 and Figure 5)12.</li>
</ol>
6. Identification of the Optimal Qa-1 Epitope in a 15-mer OLP which Stimulates the Epitope-specific, Qa-1-restricted Response in an OLP_pool-specific CD8+ T Cell Line
<ol>
	<li>Synthesize C- and N-terminally truncated peptides of a 15-mer OLP as shown in Figure 4.</li>
	<li>Examine IFN-&#947; secretion in an OLP-specific CD8+ T cell line by stimulating the CD8+ T cells with each of the truncated peptides as well as the parental 15-mer OLP in the presence of C1R or C1R.Qa-1b cells using the above described IFN-&#947; ELISPOT assay. The final concentration of each individual peptide used for the ELISPOT assay is 10 &#956;g/mL. A peptide is defined as the optimal Qa-1 epitope if the peptide: 1) consistently gives similar or stronger IFN-&#947; response, as compared to the original 15-mer peptide, in the presence of C1R.Qa-1b cells; 2) is between 8 - 10-mer (9-mer peptide preferred) which are the peptide lengths bound to MHC-I molecules15,17. See Representative Results in Figure 5.</li>
</ol>

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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>

Authors declare no conflict of interest.

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>K&lt;sup&gt;b-/-&lt;/sup&gt;D&lt;sup&gt;b-/-&lt;/sup&gt; mice</td><td>The Lackson Laboratory</td><td>Stock#: 019995</td><td>We used Taconic H2-K&lt;sup&gt;b&lt;/sup&gt;H2-D&lt;sup&gt;b&lt;/sup&gt; 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-&amp;gamma; 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-1&lt;sup&gt;b&lt;/sup&gt;</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-&amp;gamma; 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 ...

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

Research

JoVE Journal

Immunology and Infection

6.9K Views.  Loma Linda University. QA-1 (HLA-E in mens) behoort tot een groep van niet-klassieke grote histocompatibility complex 1b moleculen. Immunisatie met QA--1-bindende epitopes heeft aangetoond dat weefsel-specifieke immuunregulatie vergroten en verbeteren van verschillende auto-immuunziekten. Hierin beschrijven we een overlappende peptide bibliotheek strategie voor de identificatie van QA--1 epitopes in een eiwit.

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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Biochemistry

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Genetics

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Bioengineering

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Synthetic Antigen Controls for Immunohistochemistry

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 &#176;C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities.
This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires ...

Medicine

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Source: Cornett, E. M., et al. <a href="https://app.jove.com/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...

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

Samenvatting

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