Name: personalized peptide arrays for detection of hla alloantibodies in organ transplantation
Uploaded: 2017-09-06T15:00:00.000+00:00
Duration: 8 min 7 s
Description: Watch this Scientific Journal Video about Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation at JoVE.com

We thank Drs. Shawn Li and Xing Li of Western University in Canada for their kind assistance with SPOT array production. We are grateful to the staff members at the Histocompatibility Core and at the Comprehensive Transplant Center of Northwestern University for providing sample services. This work has been partly supported by the Auxiliary Board of Northwestern Memorial Hospital, and by a faculty startup fund provided by Northwestern University to J.J..

All methods described here have been approved by the Northwestern University Institutional Review Board (IRB protocol#: STU00104680). An overall workflow of the protocol is illustrated in Figure 1.
1. Bioinformatic Analysis of Donor and Recipient HLA Sequences
<ol>
	<li>Retrieve sequences from IMGT/HLA database15.
	<ol>
		<li>Obtain HLA typing reports of both the organ donor and his/her recipient. 
		NOTE: Ensure proper procedures to protect confidential medical records are utilized. Typically, an Institutional Review Board (IRB) approval of the study protocol or the experimental test is required per institutional IRB guidelines. If HLA reports were from PCR-based typing (of either high or low resolution) or sequencing-based typing (SBT), go directly to step 1.1.3. If sequences were obtained from genomic/HLA sequencing, and if they are complete, use these sequences directly instead. Occasionally, when only incomplete typing or sequencing results are available, follow the additional step 1.1.2. to assign the &#34;missing&#34; alleles via the web tool described in step 1.1.2.</li>
		<li>Open the Ambiguous Allele Combinations following web link - http://www.ebi.ac.uk/ipd/imgt/hla/ambig.html, and search using the &#8220;Ambiguous Allele Combinations Search Tool&#8221; function to retrieve the hypothetical alleles. Then, proceed to step 1.1.3. to enter the allele name.</li>
		<li>Open the IMGT/HLA Allele Query Form at the following the web link - http://www.ebi.ac.uk/ipd/imgt/hla/allele.html, and input subject&#8217;s allele names (each individually, as shown in Figure 2) into the &#8220;Search for&#8221; box. Click on the &#8220;Search for alleles now&#8221; button. HLA allele names should use standard naming systems (i.e., A*, A*01, A*01:01:01:01, and any previous designations like A*01010101).</li>
		<li>Find the matching allele name displayed on the screen. Click on the allele button.</li>
		<li>Copy the &#34;Protein sequence&#34;, and paste it into a text document underneath a header line of &#34;&#62;allele name&#34;. Effectively, the text document is in a FASTA (FAST-All) format of the allele name and sequence.</li>
	</ol>
	</li>
	<li>Perform gene-based multiple alignments of donor and recipient/patient alleles.
	<ol>
		<li>One gene (i.e., DQB1*03:01:01:01) at a time, copy and paste all four alleles of the donor and patient FASTA sequences (two from the donor and two from the patient) into a combined text document. At this point, it is important to differentially denote donor allele names from patient allele names (Options include differential use of upper vs. lower cases; or create a distinguishing prefix or suffix extension to each allele name to separately mark donor vs. patient alleles). The input order of the sequences is not important because following alignment the order will be shuffled according to the levels of similarity between any pair of sequences.</li>
		<li>Copy and paste the sequences in FASTA format (as seen in Figure 3) from above into the &#8220;Enter your input sequences&#8221; box on the Cluster Omega website: http://www.ebi.ac.uk/Tools/msa/clustalo/.</li>
		<li>Click on &#34;Submit your job&#34; using standard settings of parameters: such as &#34;PROTEIN&#34;; and &#34;Clustal w/ numbers&#34;. Perform alignment by clicking on &#34;Submit&#34;. An alignment of the four sequences is displayed on the screen (Figure 4).</li>
	</ol>
	</li>
	<li>Mark donor-specific mismatches. 
	NOTE: It should be specially noted that the allelic sequences given here were based on the results of HLA typing, not sequencing. Therefore, there is a risk that relevant sequence variation in the particular donor-recipient pair may be missed. This potential problem will be mitigated as more and more transplant centers adopt accurate high-resolution typing and direct DNA sequencing.
	<ol>
		<li>Copy and paste the alignment text into a text document and create a new document. If the format of the aligned file is disturbed, fix the format by changing font style and size: Always use &#34;Courier New&#34; font in small type size to fit the rows. Save the document after the formatting issues are solved.</li>
		<li>Manually inspect the aligned sequences to identify all mismatched residues that belong uniquely to the donor. Mark each of these donor-specific residues using a distinguishing font color.</li>
		<li>Underline all letters in the donor&#39;s sequences that extend 14 residues both up- and downstream of the marked donor-specific mismatches (calculated as 15 amino acid peptide minus 1 residue of the mismatch).</li>
	</ol>
	</li>
	<li>Derive 15-mer peptide sequences in an overlapping series. 
	NOTE: The reason for choosing 15-mer peptides was based on the general knowledge of typical epitopes being 4-9 amino acids in length. Therefore, when we applied a &#34;moving window&#34; procedure with a step size of 4 amino acids ( Figure 1), our selection of a 15 amino acid length left a 15-4=11 amino acid overlap between any neighboring peptides in a series. In theory, this 11 amino acid overlap is sufficient to cover all immune epitopes of up to 9 amino acids in length, so that no epitope will be inadvertently &#34;split&#34; in half with only partial sequences on the array.
	<ol>
		<li>Use the underlined sequences as templates to sequentially derive short 15 amino acid sequences in a series that overlap by 4 residues between any two immediately adjacent sequences in the series.</li>
		<li>Copy and paste these 15 amino acid sequences into a spreadsheet( Figure 5) in a column format accompanied by notation columns that include the corresponding names of the donor alleles. An additional column to include aa residue positions may be useful.</li>
	</ol>
	</li>
	<li>Repeat the steps from 1.1.3. to 1.3. for each HLA allele (i.e., A, B, C, DQA1, DQB1, DRB1 and DP) of the donor and the recipient pair to derive the 15 aa short sequences of the donor.</li>
	<li>Copy and paste all columns into a master spreadsheet to create a long continuous column of all the 15 aa sequences from all the alleles of the donor. Write down the total number of rows (for the peptide sequences) in the document.</li>
</ol>
2. Design of Custom Array Layout and Production
<ol>
	<li>Generate a spreadsheet of peptide sequences with corresponding allele callings.The array has 20 rows and 30 columns and can hold up to 20x30=600 spots for peptides. Depending on the total number of peptides recorded in step 1.5 from one particular donor, and based our past experience with two examples that we had tried in Liu et al.17, the 600 spot array can hold all the sequences generated from ~2 separate transplant cases.
	<ol>
		<li>Rationally plan the most efficient way to fit the sets of peptides within the 600-spot format of the array.</li>
		<li>If more than one subject can fit into one whole array, insert empty rows after the first donor&#39;s sequences to match the total number of rows that can be divided by 30 (the number of spots in a row on the array).</li>
		<li>Immediately following the last empty row for the first set of sequences, paste in the entire column of sequence contents from the second transplant case.</li>
		<li>Repeat these steps until the array is filled and no more cases can be added without exceeding the 600 spots. If entire empty rows are left to fill at the bottom, &#34;move&#34; the empty row to be positioned between the two donor sets. This wide space left between cases makes cutting of the membrane after completion of the synthesis easier - following step 3.2. below.</li>
	</ol>
	</li>
	<li>Program the peptide sequences in the context of array layout. The program of the SPOT synthesizer takes a text format of the sequences (one row one peptide sequence), which can be directly obtained by saving the resulting spreadsheet from step 2.1.4 as a simple text document (without the extra column(s) for allele names, aa positions etc.).</li>
	<li>Run automated peptide array synthesis via the SPOT synthesizer. The entire operation of the synthesis is detailed in Kudithipudi et al.21. Note that Fmoc synthesis of two arrays takes ~4-5 days.</li>
</ol>
3. Probe and Reprobe Antisera from a Time Series of an Individual Transplant Recipient.
NOTE: The 600-spot membrane array has the dimensions ~7 cm x 13 cm. After synthesis, the arrays can be stored at room temperature as dry membranes for at least two years when shielded from direct light. Avoid excessive folding of the membrane to preserve its longevity for repeated use.
<ol>
	<li>Rehydrate the membrane array with ethanol and visualize peptide spots stained with Ponceau S.
	<ol>
		<li>Rehydrate the membrane array carrying the peptides following a stepwise procedure optimized in Li et al.24.
		<ol>
			<li>Immerse the array membrane in 20 mL of 100% ethanol.</li>
			<li>Add 20 mL distilled water to dilute the solution to 50% ethanol and incubate at room temperature for 15 min.</li>
			<li>Change the immersion solution to 40 mL of 100% water three times and incubate 15 min each time.</li>
			<li>Wash in a proper working buffer, e.g., 20 mL of TBST (Tris-buffered saline with 0.1%), three times for 5 min each.</li>
		</ol>
		</li>
		<li>Ponceau S staining of the array to visualize synthetic peptides
		<ol>
			<li>Add 20 mL of pre-formulated Ponceau S solution directly to the hydrated array and incubate for ~30 s with shaking.</li>
			<li>Run distilled water continuously over the membrane to de-stain background Ponceau S color. During the process, peptide spots of red color may become visible.</li>
			<li>Separate the portions of the array for each donor by carefully cutting between the sections of the array for individual cases. Mark the orientation of each array.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Pre-block array.
	<ol>
		<li>Block the membrane in 20 mL of 5% non-fat milk dissolved in TBST buffer. This milk-based solution will further de-stain Ponceau S color. Replace the milk buffer several times in order to achieve the clearest peptide images.</li>
		<li>For record-keeping purposes, take a photo of the Ponceau S image of the array using a hand-held camera (example in Figure 6).</li>
		<li>Continue blocking of the membrane in 5% non-fat milk at 4 &#176;C overnight or at room temperature for 2 h with rocking.</li>
	</ol>
	</li>
	<li>Incubate array with transplant antiserum. 
	NOTE: It should be noted that a phenomenon known as prozone or the Hook effect may result from complement-dependent interference in serum antibody analysis. To circumvent this problem, an optional serum pretreatment step can be considered with either EDTA or heat inactivation of serum samples.
	<ol>
		<li>Remove blocking buffer and wash the membrane with 20 mL of TBST three times the next day for 5 min each time.</li>
		<li>Add 20 &#181;L of the crude serum of the recipient to 20 mL of 2.5% milk in TBST buffer and incubate with the membrane for 2-3 h at room temperature. Note that in this initial round of probing, it is recommended that the last post-transplant serum (or likely most sensitized serum) in the time series is firstly used. This is based on the assumption that earlier specimens in the series, particularly from pre-transplant time points, have less variety of alloantibodies that tend to develop over time. This way, any possibility of signal interference from &#34;carry-over&#34; between probing rounds can be clearly distinguished from truly developed alloantibody reactivity due to immune responses against the graft.</li>
	</ol>
	</li>
	<li>Wash the array using 20 mL of TBST and incubate with secondary antibody.
	<ol>
		<li>Wash the membrane three times for 10 min each time.</li>
		<li>Incubate with goat anti-human IgG-HRP (Horseradish peroxidase) secondary antibody at 1:10,000 dilution in TBST buffer supplemented with 1% milk for another 2 h.</li>
	</ol>
	</li>
	<li>Wash and develop the blot.
	<ol>
		<li>Wash the membrane three times with TBST for 10 min each time.</li>
		<li>Perform enhanced chemiluminescence (ECL) using 5 mL of luminol solution freshly mixed with 5 mL of peroxide solution to develop the membrane (for 1 min).</li>
		<li>Visualize ECL signals using a suitable imager (i.e., ChemiDoc Imaging Systems or Azure C600).</li>
	</ol>
	</li>
	<li>Scan and quantify the blot.
	<ol>
		<li>Save the developed images (Figure 7, lower image) and perform quantification of spot intensity.</li>
	</ol>
	</li>
</ol>
4. Compare Antiserum Reactivity across a Clinical Time Series.
<ol>
	<li>Strip the array.
	<ol>
		<li>At this point, keep the membrane wet at all times.</li>
		<li>Strip the membrane by incubating with 20 mL of commercial stripping buffer at 37 &#176;C for 20 min, and then wash the membrane with TBST three times for 10 min each. Then repeat the blocking (step 4.2) and probing (step 4.3) steps using a different serum of the patient taken at a different time point.</li>
	</ol>
	</li>
	<li>Block stripped membrane. 
	Note: Following stripping, the membrane can be reused for another round of probing of a different serum from the same patient in a time series.
	<ol>
		<li>Block the membrane using 5% milk buffer as before (see step 3.2).</li>
	</ol>
	</li>
	<li>Reprobe a different antiserum from the same time series (repeat steps from 3.3-3.6; example in Figure 7, upper image). 
	NOTE: The stripped array can then be used to reprobe another serum specimen. Since the peptides are covalently conjugated to the supporting matrix of the membrane, we showed that the array can be reused for up to 20 rounds of stripping and reprobing cycles without losing its performance.</li>
	<li>Long-term storage of arrays.
	<ol>
		<li>Store the membrane in TBS buffer supplemented with 0.02% w/w sodium azide as preservative. Use a sealed plastic bag for long-term storage. At 4 &#176;C under protection from direct light, the wet membrane can be stored for at least 2 years.</li>
	</ol>
	</li>
</ol>
5. Data Acquisition and Analysis
<ol>
	<li>Manually annotate positive antigen peptides.
	<ol>
		<li>Locate spots showing positive antibody signals and determine each of their grid positions.</li>
		<li>Highlight the corresponding rows in the master worksheet for the positive peptides.</li>
	</ol>
	</li>
	<li>Retrieve peptide sequences listed in the master worksheet ( Figure 7, bottom list). According to the design principle outlined in step 1.4.1., neighboring serial spots share significantly overlapping sequences (15-4=11), therefore reactive epitopes may be shared by peptides in a consecutive series.
	<ol>
		<li>Highlight any positive spots occurring in succession.</li>
	</ol>
	</li>
	<li>Determine minimum epitope length for each peptide in a series.
	<ol>
		<li>Highlight any potentially shared epitope sequences based on overlapping segments of reactive peptides.</li>
	</ol>
	</li>
	<li>Structurally model antigen epitopes.
	<ol>
		<li>Obtain prototype HLA crystal structures from the Protein Data Bank found at the following the web link: http://www.rcsb.org/pdb/home/home.do.</li>
		<li>Display the prototype HLA structure in Pymol (download from www.pymol.org).</li>
		<li>Map&#34; or model discovered anti-donor epitopes to the 3D structures of the prototype HLA molecules by highlighting the reactive peptide sequences ( Figure 8). Click on &#34;Display&#34;, then &#34;Background&#34;, and select &#34;White&#34;. To save the image, go to &#34;File&#34; and &#34;Save Image As&#34; and select the file format &#34;png&#34;.</li>
	</ol>
	</li>
	<li>Report results and assign reactive epitopes to the corresponding alleles.</li>
	<li>Compare array results to the single-antigen beads (SAB) results, if available. 
	NOTE: The SAB test using single allelic antigens measures antibody reactivity towards a fixed panel of HLA proteins. Individual peptides that show antibody reactivity belong to certain HLA alleles that, if also included in the SAB panel, allow direct comparison between the array and the SAB results. Our previous study17 showed a high level of correlation between the results, suggesting that the peptide epitopes significantly contribute to the overall reactivity of SAB. In addition, the array results also reveal amino acid-levels of specificity.
	<ol>
		<li>Obtain SAB results from an HLA lab which provides information about whether and which donor alleles are reactive to post-transplant sera of the patient. Results from step 5.5. based on the array analysis also provide information about corresponding donor alleles positive for alloantibody reactivity.</li>
		<li>Compare SAB and array results.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+circulating+donor-specific+HLA+antibodies.">Clinical relevance of circulating donor-specific HLA antibodies.</a> Curr Opin Organ Transplant. 15 (4), 462-466 (2010).</li><li>Brown, N. K., Kheradmand, T., Wang, J., Marino, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+HLA+alleles:+Utility+of+next-generation+sequencing+methods.">Identification and characterization of novel HLA alleles: Utility of next-generation sequencing methods.</a> Hum Immunol. 77 (4), 313-316 (2016).</li><li>Cino, E. A., Choy, W. Y., Karttunen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Biases+of+Linear+Motifs.">Conformational Biases of Linear Motifs.</a> Journal of Physical Chemistry B. 117 (50), 15943-15957 (2013).</li><li>Duquesnoy, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+leukocyte+antigen+epitope+antigenicity+and+immunogenicity.">Human leukocyte antigen epitope antigenicity and immunogenicity.</a> Curr Opin Organ Transplant. 19 (4), 428-435 (2014).</li><li>Lavinder, J. 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+and+characterization+of+the+constituent+human+serum+antibodies+elicited+by+vaccination.">Identification and characterization of the constituent human serum antibodies elicited by vaccination.</a> Proc Natl Acad Sci U S A. 111 (6), 2259-2264 (2014).</li><li>Kloetzel, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+processing+by+the+proteasome.">Antigen processing by the proteasome.</a> Nat Rev Mol Cell Biol. 2 (3), 179-187 (2001).</li><li>Filippone, E. J., Farber, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Humoral+Theory+of+Transplantation:+Epitope+Analysis+and+the+Pathogenicity+of+HLA+Antibodies.">The Humoral Theory of Transplantation: Epitope Analysis and the Pathogenicity of HLA Antibodies.</a> J Immunol Res. 2016, 5197396 (2016).</li><li>Claas, F. H., Witvliet, M. D., Duquesnoy, R. J., Persijn, G. G., Doxiadis, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acceptable+mismatch+program+as+a+fast+tool+for+highly+sensitized+patients+awaiting+a+cadaveric+kidney+transplantation:+short+waiting+time+and+excellent+graft+outcome.">The acceptable mismatch program as a fast tool for highly sensitized patients awaiting a cadaveric kidney transplantation: short waiting time and excellent graft outcome.</a> Transplantation. 78 (2), 190-193 (2004).</li></ol>

No conflicts of interests declared.

The design of the SPOT array described here is for experimental study of alloantibody specificity in transplantation against an organ donor's HLA antigens. In contrast to the existing SAB assay broadly used in clinic, the antigen array method has a major advantage in its flexible design that can accommodate the true HLA sequences of the individual donor. The new platform exploits the potentials of the rapidly advancing DNA sequencing technology that will soon be able to produce accurate HLA allelic sequence readings with...

Organ replacement therapy that is routinely conducted across the world has saved millions of lives. Solid organ transplantation occurs in approximately 100 patients per million people in the USA annually, while a greater number still are on waitlists to receive donor organs due to a severe shortage of supply (according to information provided by the Organ Procurement and Transplantation Network - OPTN/UNOS: optn.transplant.hrsa.gov). Organ transplant is highly regulated in order to reduce organ waste and save lives, but the scientific tools used to inform these regulations are limited in effectiveness. For instance, the scientific community fully recognizes the highly polymorphic states of HLA molecules and accurate genetic tests of DNA using high-resolution typing and sequence-based typing (SBT) have been developed in recent years1,2. However, alloantibody testing methods have not yet been able to produce the vast variety of individual HLA sequences as antigen probes. The standard test nowadays uses an invariable panel of ~100 allelic antigens that are comprised of common variants of HLA, A, B, C, DQ, DP and DR sequences in human populations3,4,5,6. Frequently, the actual donor&#39;s HLA sequences are not included in the test panel, forcing transplant physicians and surgeons to infer donor-specific reactivity based on shared similarities between donor&#39;s actual sequences and corresponding &#34;standards&#34; in the test set7,8. Consequently, it is sometimes challenging to make a reliable estimation of rejection risk based on antibody test results9,10,11,12. Therefore, new personally customizable tests for alloantibodies are urgently needed13,14.
The HLA genes encode the major histocompatibility complex (MHC) receptors that have a key function in immune responses6. HLA genes are known to be the most polymorphic genes of the human genome6. Due to the rapid advancements in DNA sequencing strategies for the HLA genes, new allelic variants (or simply referred to as alleles) are being discovered at an explosive rate15,16. By March 2017, 16,755 validated alleles had been deposited to the IMGT/HLA Database (http://www.ebi.ac.uk/ipd/index.html), of which 12,351 were of class I and 4,404 were of class II groups. In stark contrast, only a little over 100 distinct alleles are represented in the standard single-antigen beads (SAB) assay, which is routinely used to detect alloantibodies in organ transplantation. The SAB method is built on a Luminex platform using flow cytometry. Since the assay utilizes an invariable set of antigens, apart from minor batch to batch variabilities in production, the antiserum test can be robustly standardized across individuals and across laboratories5. However, this test is unable to capture all alloantibodies developed specifically against the donor alleles, particularly when the donor sequences are absent from the SAB set. Although custom production of donors&#8217; antigens based on true sequences are desirable, there remain technical challenges in streamlining the necessary production and testing procedures.
We recently described an alternative methodology in a feasibility study of renal transplant subjects17. The method used peptide antigens in an array format for probing pre- and post-transplant sera of individual subjects. Each array was custom built using the SPOT synthesis method18,19,20,21,22,23 that produces peptide antigens, each 15 amino acids in length, entirely based on the respective organ donor&#39;s HLA alleles of A, B, C, DQA1, DQB1 and DRB1. SPOT synthesis is operated on a cellulose membrane using standard Fmoc-chemistry22 and can produce hundreds of custom peptides in parallel with a fully automated robotic system19,21. The membrane array can withstand multiple rounds of stripping and reprobing cycles. In our retrospective study17, we detected changes in antigen patterns with stored transplant antisera collected in a time series (i.e., before and after transplantation). Herein we describe the technical protocol for the workflow including array design, manufacturing, antiserum probing and result analysis. The method is intended for detecting alloantibodies against specific linear epitopes on transplant donors&#39; HLA molecules.

<table><tbody><tr><td>Peptide array</td><td>INTAVIS Bioanalytical Instruments</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Ponceau S solution</td><td>Sigma-Aldrich</td><td>P7170</td><td></td></tr><tr><td>Non-fat milk</td><td>Bio Rad Laboratories</td><td>1706404</td><td></td></tr><tr><td>TBST</td><td>Santa Cruz Biotechnology</td><td>10711454001</td><td></td></tr><tr><td>Goat anti-human IgG&amp;ndash;HRP&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A18811</td><td></td></tr><tr><td>Clarity Western ECL Substrate</td><td>Bio Rad Laboratories</td><td>1705061</td><td></td></tr><tr><td>Restore Western Blot Stripping Buffer</td><td>Thermo Scientifics</td><td>21059</td><td></td></tr><tr><td>ChemiDoc gel imaging system</td><td>Bio Rad Laboratories</td><td>1708265&amp;nbsp;</td><td></td></tr></tbody></table>

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.

In the original study using the array screening method17, we enrolled a total of 5 kidney transplant subjects. We obtained the HLA typing results of our cohort and of their respective donors. Their medical history and allelic antibody titers from SAB tests were also available to us. In our pilot study of these 5 patients, we devised two different methodologies: a standard array comprised of a fixed panel of peptides and personalized arrays that were custom made for...

Watch this Scientific Journal Video about Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation at JoVE.com

Personalized Peptide Arrays for Detection of HLA Alloantibodies 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 ...

personalized-peptide-arrays-for-detection-hla-alloantibodies-organ

Research

JoVE Journal

Biochemistry

10.0K Views.  Northwestern University Feinberg School of Medicine. Incongruenties in menselijke leukocyten antigeen (HLA) sequenties tussen organ donor en ontvanger paren zijn de belangrijkste oorzaak van antilichaam-gemedieerde afwijzing in orgaantransplantatie. Hier presenteren we het gebruik van aangepaste antigeen arrays die zijn gebaseerd op individuele donoren HLA sequenties sonde anti-donor HLA alloantibodies orgel geadresseerden.

Video: Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

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 ...

Immunology and Infection

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

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 ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Bioengineering

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

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 ...

Biology

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

Source: Liu, P., et al. <a href="https://app.jove.com/v/56278">Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation.</a> J. Vis. Exp. (2017).
This video demonstrates an assay to detect anti-donor HLA alloantibodies in organ recipients. A peptide array is synthesized to represent potential epitopes for antibody-mediated organ rejection. The array is incubated with the recipient&#39;s serum, containing alloantibodies that bind to their target epitopes. Antibody binding is detected using chemiluminescence to identify donor-specific HLA epitopes that incite alloantibody reactivity.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Design of Custom Array Layout and ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

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