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Mismatches outside exons 2 and 3 do not alter the peptide motif of the allele group B*44:02P
Human Immunology 72 (2011) 1039-1044
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Mismatches outside exons 2 and 3 do not alter the peptide motif of the allele group B*44:02P Christina Bade-Doeding a,*, Pedro Cano b, Trevor Huyton a, Soumya Badrinath a, Britta Eiz-Vesper a, Oliver Hiller a, Rainer Blasczyk a a b
Institute for Transfusion Medicine, Hannover Medical School, 30625 Hannover, Germany The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030, USA
A R T I C L E
I N F O
Article history: Received 20 January 2011 Accepted 4 August 2011 Available online 10 August 2011
Keywords: Polymorphism Peptide-binding motif Stem cell transplantation Functional alleles
A B S T R A C T
Sequence variations outside exons 2 and 3 do not appear to affect the function of human leukocyte antigen (HLA) class I alleles. HLA-B*44:02:01:01 and -B*44:27 are considered functionally identical because they differ by a single amino acid substitution of Val ⬎ Ala at position 199, which is located in the ␣3 domain. To validate that HLA-B*44:02:01:01 and -B*44:27 represent functionally identical alleles that might reflect a permissive mismatch in hematopoetic stem cell transplantation (HSCT), we determined their peptidebinding features. B-lymphoblastic cells were lentivirally transduced with B*44:02 and B*44:27 constructs and soluble recombinant molecules were purified by affinity chromatography. Peptides were isolated and sequencing of single peptides was performed using liquid chromatography– electrospray ionization–tandem mass spectrometry (LTQ-Orbitrap) technology. We demonstrate that the peptide motif of B*44:02199Val and B*44:27199Ala is identical. Both variants feature E at P2 and Y, F, or W at P⍀ in their ligands. Most of the identified peptides are 9 to 11 amino acids in length and approximately 20% of these ligands are shared between the alleles. Our results lead to the conclusion that B*44:02:01:01 and B*44:27 might have the same immune function, validating a theory that is now being used in deciding which donors to select in HSCT when there is no identical donor available. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The risks associated with the immune reactions characteristic of both allogeneic hematopoietic stem cell transplantation and solid organ transplantation depend on amino acid (AA) sequence differences between human leukocyte antigen (HLA) alleles. The systematic study of the effect of the AA sequence polymorphism on the function of the HLA molecule and on the immune response to transplantation is therefore necessary. Effector cytotoxic T lymphocytes target cells displaying allogeneic peptides and are able to detect even single-AA exchanges between peptides; for that reason the comparison of allelic peptide-binding features helps to weigh the importance of individual AA substitutions within the peptide-binding region (PBR) of certain HLA allelic variants. AA substitutions might have an impact not only on the binding of peptides to the individual HLA molecule but also on the conformation of the respective peptide–HLA (pHLA) complex. These subtle changes can then be recognized by T cells, resulting in an immunologic response.
* Corresponding author. E-mail address: [email protected] (C. Bade-Doeding).
Forty-seven AA positions within the PBR of HLA molecules have been attributed a role in peptide binding [1] and are subdivided into 6 specificity pockets (A–F) [2]. Previous studies demonstrated that even a single-AA exchange at a given position within the PBR can restrict the features of the presented peptides if it occurs as part of a specific pocket [3–7]. However, the magnitude of a given polymorphism is dependent not only on its position but also on the nature of the exchanged AAs, as well as their neighboring AAs; therefore, even a single mismatch may impact the transplantation outcome. The question of whether a mismatch is permissive or nonpermissive is critical in deciding which person is the bestmatched donor and whether the selected donor exhibits an acceptable mismatch. Since even small differences between HLA allotypes can have dramatic effects on their function, the selection criteria for identifying acceptable mismatches when no matched donor is available remain poorly defined. When sequence analysis is carried out to find a matched donor, exons 2 and 3 (encoding for the ␣1 and ␣2 domain of the HLA heavy chain) are the regions of interest and it is currently believed that sequence variations outside exons 2 and 3 do not have any effect on the function of HLA class I alleles. According to this view, alleles with identical sequences in exons 2 and 3 are thought to be functionally identical, irrespective of any sequence differences outside
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.08.004
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exons 2 or 3. In this view, because the alleles have the same biologic function, they can be considered the same functional allele and not 2 different alleles. The impact of HLA mismatches is determined by several factors, of which the individual peptide-binding features are the most influential. If differences in peptide-binding features are determined, then an immune response would be expected. In contrast, if the peptide-binding features between mismatched alleles are similar or identical, then immune responses cannot be excluded, but would be thought to be less probable. A measure of similarity based upon the peptide-binding profiles of different alleles therefore gives us insight into their functional similarity and can guide us toward those alleles that require further investigation on the cellular level. The difficulties of establishing the selection criteria for identifying acceptable mismatches are exemplified in the molecular differences between HLA-B*44:02:01:01 and -B*44:27. These 2 alleles differ at position 199 in the ␣3 domain of the heavy chain (Fig. 1). In this study we hypothesized that B*44:02:01:01 and -B*44:27 can be considered the same functional allele. Although B*44:02:01:01 is much more common, it must be taken into account that B*44:27 is always associated with Cw*07:04, whereas B*44:02:01:01 is mostly associated with Cw*05:01 and rarely with Cw*07:04. That means that B*44:27 came as a mutation or gene conversion from the uncommon block B*44:02:01:01–Cw*07:04. 2. Subjects and methods 2.1. Design of lentiviral vectors For expression of truncated B*44:02 and B*44:27 molecules, coding DNA from an HLA-B*44:02-positive donor (exons 1 through 4) was amplified by polymerase chain reaction (PCR) using the primers HLA-B3-TAS (5= gag atg cgg gtc acg gca c 3=) and HLA-E4WAS (5= cca tct cag ggt gag ggg ct 3=). The PCR product was ligated
in the eukaryotic expression vector pcDNA3.1V5/His using the pcDNA3.1V5/His TA cloning kit (Invitrogen, Karlsruhe, Germany). The recombinant pcDNA3.1V5/His/B*44:02 vector was used as a template for the lentiviral pRRL.PPT.SFFV.mcs.pre/B*4402 vector. The B*44:02 insert was ligated in the pRRL.PPT.SFFV.mcs.pre vector and the insertion of the B*44:02 construct was verified by sequencing (forward and reverse direction) using a 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). The B*44:27 allele was produced by side-direct mutagenesis using the QuikChange multi site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). The pRRL.PPT.SFFV.mcs.pre/ B*44:02 vector was used as template. For expression of full-length HLA-B*44 variants, cDNA (exons 1 through 7) from an HLA-B*44:02-positive donor was amplified by PCR using the primers HLA-B3-TAS (5= gag atg cgg gtc acg gca c 3=) and HLA-B-TAAS-E7 (5= tca agc tgt gag aga cac atc ag 3=). Lentiviral pRRL.PPT.SFFV.mcs.pre vector was generated by following the cloning procedure as described above. 2.2. Production of lentiviral particles in HEK293T cells HEK293T cells have been transfected by adding 1 mg/mL polyethyleneimine (Sigma–Aldrich Chemie Gmbh, Munich, Germany) in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) followed by an incubation of 20 minutes with 5 g plasmid/6 ⫻ 106 HEK293T cells (packaging plasmid, psPAX2; envelope plasmid, pDM2G). After 24 hours of incubation the medium was changed; 48 hours posttransfection, virus-containing supernatant was removed, passed through a 0.45-m filter (Millipore GmbH, Schwalbach, Germany), and concentrated overnight by centrifugation at 16⬚C at 10,000 rpm. The lentiviral pellet was then dissolved in RPMI 1640 (Invitrogen). 2.3. Transduction of B-lymphoblastoid cell lines (B-LCLs) The lentiviral transduction of the B-LCLs was performed by adding the dissolved lentiviral pellet in the presence of 8 g/mL protamine sulfate (Sigma–Aldrich) to the cells, followed by incubation for 8 hours. Transduced B cells were then cultured in complete RPMI 1640 medium containing 10% fetal calf serum. 2.4. Analysis of HLA-B44 surface expression on B-LCLs Cell surface expression of HLA-B*44 in the presence (LCL 721.221 cells) or absence (LCL 721.220 cells) of tapasin (TPN) was assessed by flow cytometry using an anti-Bw4 –FITC labeled antibody (One Lamda, BmT GmbH, Meerbusch-Osterath, Germany) and W6/32–PE labeled antibody. The cells were washed twice with phosphate-buffered saline containing 0.5% bovine serum albumin and then incubated with 10 L of the antibody stock for 20 minutes at 4⬚C. The cells were washed twice with phosphate-buffered saline/0.5% bovine serum albumin and then analyzed using a FacsCanto flow cytometer (BD Biosciences, Heidelberg, Germany). Real-time PCR was also performed to determine the levels of B*44specific mRNA in transduced cells. 2.5. Verification of sHLA expression
Fig. 1. Location of residue 199 in the ␣3 domain of the heavy chain. A ribbon representation of a B*44:02 peptide–HLA complex (PDB ID: 3KPL) [16] to illustrate the location of amino acid position 199. The bound peptide is colored magenta, 2m is colored green, heavy chain is colored blue, and position 199 in the ␣3 domain of the heavy chain is highlighted in red.
Expression of V5-tagged, truncated HLA-B*44 (sHLA) molecules was quantified using a sandwich enzyme-linked immunosorbent assay in which anti-HLA-A-B-C W6/32 (Serotec, Du¨sseldorf, Germany) [8,9] or anti-V5 (Invitrogen) monoclonal antibodies (mAb) were employed as capture antibodies. Horseradish peroxidase– conjugated anti-2m mAb (Dako, Hamburg, Germany) served as the detection antibody. B-LCL 721.221 clones with the highest recombinant protein expression rates were used for large-scale production.
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2.6. Large-scale production of sHLA molecules and affinity purification Highly producing clones were cultured and expanded in bioreactors (CELLine, Integra, Fernwald, Germany). Harvests were centrifuged for 20 minutes at 1,200 rpm to remove cellular material and then filtered through a 0.45-m filter (Sartorius, G×ttingen, Germany) and stored at ⫺20⬚C. Supernatants of each HLA-B*44 variant were pooled, adjusted to pH 8.0, and affinity purified using N-hydroxysuccinimide (NHS)-activated HiTrap columns, precoupled with mAb W6/32. All purification was performed using a BioLogic DuoFlow system (Bio-Rad, Hercules, CA). Trimeric complexes were eluted using 0.1 M glycine/HCl buffer (pH 2.7); 2.5 mg sB*44:27 protein and 3.0 mg sB*44:02 protein complexes were subsequently used for peptide analysis. 2.7. Characterization of sHLA-B*44-derived peptides Peptides were eluted from trimeric sHLA-B*44 complexes by treatment with 0.1% trifluoroacetic acid. Peptides were separated by filtering them through a YM membrane with a 10-kDa cutoff (Millipore, Schwalbach, Germany). The flowthrough containing the peptides was then subjected to an Eksigent nano-LC Ultra 2D highperformance liquid chromatograph coupled to an Orbitrap ion trap (Thermo Fischer, Waltham, MA), resulting in a high mass accuracy (⬍5 ppm). Database queries were performed using Mascot software[10] implementing the IPI human and the respective decoy databases. 3. Results 3.1. Surface expression of B*44/199 variants Flow cytometric analysis indicated that the B*44/199 variants are exclusively expressed on the cell surface of LCL 721.221 cells in the presence of TPN (Fig. 2). For LCL 721.220 cells lacking TPN, no surface expression could be determined on the cell surface, suggesting that both B*44:02 and B*44:27 are dependent on TPN for loading and peptide presentation. We then sequenced by liquid chromatography– electrospray ionization–tandem mass spectrometry (LTQ-Orbitrap) the peptides bound by 2 HLA alleles, B*44:02 and B*44:27, which were expressed and secreted by B-LCL 721.221 cells. Analysis of the identified peptide data is presented below. 3.2. Peptide features The mass spectrometer used for this study (LTQ-Orbitrap) has a high mass accuracy in the range of 10 ppm. To obtain the highest probability of peptide identification we selected only those peptides that had a ␦ value of 0.0 Da and excluded all other peptides. For example, even a ␦ value of 0.1 Da would mean for a peptide with a mass of 1,000 Da that there would be an error of 100 parts per million associated with the measurement. A total of 194 unique peptides (nonduplicates) were identified following elution from purified recombinant sHLA-B*44:02 molecules (Suppl. Table 1). By comparison we identified 208 unique peptides from purified recombinant sHLA-B*44:27 molecules (Suppl. Table 2). The MS/MS spectra of 3 long peptides are presented as representative examples (Suppl. Fig. 1). Most of the peptides identified were of the canonical 9 to 11 AAs in length with 164/194 (84.5%) for B*44:02 and 161/208 (77.4%) for B*44:27 (Fig. 3). A larger percentage of peptides (15.5%, 30/194) from the B*44:02 subtype were longer than 11 AA compared with those identified in B*44:27 (13.9%, 29/208). This frequency may be reflective of the different number of peptides identified between the 2 alleles and our stringent cutoff values for selecting peptides. Interestingly, as with our previously published data on other HLA-B group peptides[11], several peptides of extraordinary length, ⬎15
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residues, were identified from the B*44:02 subtype, including two 16-mer peptides and one 19-mer peptide, all of which have a ␦ score of 0.0 and fulfill the correct charge mass of the single ions. Further analysis of the peptide sequences demonstrated clear anchor motifs for HLA-B*44:02 with E at the P2 position and W, F, or Y at the P⍀⬎ position of the respective peptide. Our data therefore expand the previously published peptide motif for B*44:02 peptides [6,12,13] by including W as a bona fide P⍀ anchor. Indeed, W was the most frequent AA at this position, being reported in 80/194 (41.2%) of the isolated peptides compared with F in 47/194 (24.2%) and Y in 46/194 (23.7%; Fig. 4). The peptide anchor motifs derived from the eluted B*4427 peptides are identical to that of B*4402 with E at the P2 position and W, F, or Y at the P⍀ position of the respective peptide. Likewise, W was the most frequent P⍀ amino acid (73/208; 35%) compared with F (51/208; 24.5%) and Y (69/208; 33.1%). 3.3. Shared peptides A total of 402 peptides, 194 from B*44:02 and 208 from B*44:27 alleles (Suppl. Tables 1 and 2), were identified containing 49 shared peptides and 353 unique peptides. Taken together with 19 previously published peptide sequences for B*44:02 (http://www. syfpeithi.de/) that are shared with B*44:27, this gives 68/353 (19.3%) of the sequenced peptides that are shared and preferentially selected from the available peptide pool by both alleles. These 68 shared peptides are shown in Suppl. Tables 3 and 4 describing 19 previously published B*44:02 peptide sequences and 49 newly identified peptide sequences eluted from both alleles. 3.4. Differential selection of peptides Some of the B*44-derived ligands originated from different regions of the same protein and had overlapping sequences (Suppl. Table 5). These peptides differed both in their length (9 –12 AA) and in their C-terminal anchor residues. The shorter peptide versions were N- and/or C-terminally truncated, and related peptides were detected in distinct B*44 alleles. 4. Discussion The selection and features of those peptides that are able to be bound by a certain HLA variant and subsequently presented to the immune system remain questionable for most alleles. In the HLA heavy chain, variations within the PBR can affect (1) the peptidebinding motif [3–5,8,11], (2) the feature and length of the peptides [11], and (3) the individual conformation of a pHLA complex [11,12]. Consequently, every AA substitution at a given position within the HLA molecule can impact the immunologic outcome; this can be highly dependent on the influence of its neighboring AA. In current transplantation scenarios the decision for choosing the best-matched donor is often made on available functional data for matched alleles at the ␣1 and the ␣2 domain (PBR) that is encoded by exons 2 and 3. It is currently believed that sequence variations outside the exon 2/3 regions do not have any significant effect on the function of HLA class I alleles. In this study we considered the alleles B*44:02 and B*44:27 to have the same immune function (i.e., to not be recognized as foreign by each other), by virtue of the fact that they differ in a single AA substitution at position 199 (V ⬎ A), which lies outside the exon 2/3 regions (Fig. 1). Our aims were to use peptide sequencing to investigate if this polymorphic difference influences the peptide-binding features of these class I alleles and to assess their functional disparity or similarity. To enable a meaningful comparison of peptide selection, we ensured that the peptides presented through B*44:02 and B*44:27 are selected and loaded by the peptide-loading complex (PLC). The function of TPN, a dedicated part of the PLC, is to stabilize the PBR of the HLA molecule against irreversible denaturation and to maintain it in a peptide-receptive state before peptides are selected and
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Fig. 2. Surface expression of HLA-B*44/199 variants on LCL 721.220 (TPN⫺) and LCL 721.221 (TPN⫹) cells. Flow cytometric analysis for cells stained with anti Bw4 –FITC and w6/32–PE labeled monoclonal antibodies. (A) FACs plots for nontransduced LCL 721.221 (left) and LCL 721.220 (right) cells, respectively (negative controls). (B and C) FACs plots for the B*44:02 and B*44:27 in LCL 721.221 (left) and LCL 721.220 (right) cells, respectively. Both HLA B*44:02 and B*44:27 were expressed only on the surface of LCL 721.221 cells.
loaded [14]. We here demonstrate that the polymorphic difference at position 199 does not affect the association of B*44:27 with TPN, illustrating that peptides for B*44:02 and B*44:27 are selected, optimized, and loaded through the same pathway. Additionally, we ensured that the peptide repertoire of each variant was selected from a single peptide pool (LCL 721.221 cells) and that it was determined under near identical conditions. Our data illustrate that both allotypes select a similar set of peptides with identical peptide-binding motifs, with E at the P2 position and W, F, or Y at the P⍀ position of the respective peptide. Furthermore, 19.3% of the peptides are shared peptides, highlighting the fact that they select identical peptides from the vast peptide
pool available and providing further evidence that they might be considered functionally similar. Our data provide a representative analysis of B*44-derived peptides; however, it must be noted that the identified ligands specifically reflect the peptide pool available to sHLA molecules in transduced LCL 721.221 cells. We anticipate that some differences in the peptide repertoire might be observed from different cell types expressing exogenous HLA or from endogenous HLA in B*44-positive patient cells. The questions that arise are as follows: Can all alleles with identical sequences in exons 2/3 be considered functionally identical? Alternatively, what mismatches outside exons 2/3 can be con-
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Fig. 3. Frequency of peptide length (AA) for HLA-B*44:02 and B*44:27. Peptides bound to HLA-B*44:02 and B*44:27. The length (9 to ⬎12 residues) of eluted peptides specific for each of the individual B*44 alleles is represented. Most of the peptides identified were of the canonical 9 to 11 amino acids (AAs) in length with 84.5% for B*44:02 and 77.4% for B*44:27. A total of 15.5% of peptides derived from the B*44:02 subtype were longer than 11 AA compared with 13.9% of those derived from B*44:27. The 1 identified 8-mer peptide for B*44:27 is not shown.
represented by a vector in a vector space in which each dimension is a peptide sequence property. In this vector space each allele is represented by a vector and the distance between 2 vectors is a measure of histocompatibility between alleles [16]. If the known peptide-binding profile is limited (i.e., if only a small number of peptides have been eluted from the selected allele), then to fully characterize the allele in terms of peptide features, a compatibility comparison can be performed that takes into account the overlap between 2 peptide-binding profiles (sets of peptides binding to a particular allele.) This method is more opaque because it does not show why or how a unique peptide may bind to 1 allele but not to another, although they share the same peptidebinding motif. However, this method has 2 advantages: first, it requires peptide-binding sets of smaller size, and second, it reveals the asymmetric nature of the compatibility relation. The relation of histocompatibility between alleles X and Y (HC(X,Y)) is not symmetric (HC(X,Y) ⫽ HC(Y,X) or HC(X,Y) ⫽ HC(Y,X)) [17]. The immune reaction elicited by transplanting an organ carrying allele X to a patient carrying allele Y may be different if it is the donor who carries allele Y and the patient who carries allele X. We define the histocompatibility function as follows: HC(P, D) ⫽
sidered functionally identical? When considering these questions the described binding site for the T cell coreceptor CD8 on the ␣3 domain of the HLA heavy chain [15] may need to be considered. Three clusters of residues appear to contribute to binding (220 – 232, 233–235, 245–247), with a loop region (223–239) playing the predominant role. In addition, several individual functional mutations have also been described that can influence CD8 HLA class I interactions, including residues 227 and 245. Because ⬎70% of the alleles in the Anthony Nolan database are not sequenced outside exons 2/3, it remains possible that any 2 alleles with identical sequences in their exon 2/3 regions could still contain mismatches that influence CD8 binding and thus the “avidity threshold” of the T-cell receptor, thereby changing the subsequent immune response. For such a transplantation scenario one would also anticipate an obvious directionality. To the extent that (1) histocompatibility and transplantation outcome depend on T-cell recognition and (2) T-cell recognition of different alleles depends on the ability of these alleles to bind peptides with distinct different AA sequence features, we can therefore define histocompatibility in terms of the repertoire of peptides that alleles bind. According to this logic, when 2 alleles bind peptides with the same amino acid sequence features and present them to T cells, they might be considered compatible. Therefore, matching patients and donors is effectively an exercise of comparing the repertoire of peptides bound by the alleles that patients and donors carry and present to T cells. In some cases the second assumption does not hold, and it is possible for 2 alleles to have the same peptide-binding profile and yet show different T-cell recognition. In fact, B*44:02 and B*44:03 bind peptides with the same sequence properties (E at P2 and F, Y, W, or L at P⍀), but can be recognized as foreign by each other [6]. Upon further analysis, however, although they share a large set of peptides they can both bind, there are peptides that bind to B*44:02 and not to B*44:03 and vice versa. Such known examples are rare and the individual sequence properties that distinguish them are in many cases undetermined. This example provides evidence supporting our claim that T-cell recognition of 1 allele can be predicted by means of analyzing its peptide-binding profile. In such a transplantation scenario, we face the task of comparing 2 peptide-binding profiles to estimate and predict histocompatibility. If peptide-binding profiles could be fully characterized in terms of their sequence properties, allele peptide-binding profiles can be
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Card(PBP(P 艚 D)) Card(PBP(D))
where P is the allele carried by the patient and D is the allele carried by the donor, Card ( ) is a function that returns the cardinality of a set or the number of elements in it, PBP (X) is the peptide-binding profile or set of peptides known to bind to X, and PBP (X 艚 Y) is the set of peptides that bind to both X and Y. For instance, we have that HC (B*44:02, B*44:03) ⫽ 0.67 and HC (B*44:03, B*44:02) ⫽ 0.80 [6]. In evaluating the host-versus-graft or graft rejection reaction, the relation to consider is HC (P,D). In evaluating the graft-versushost reaction, by contrast, the HC (D,P) must be taken into account instead. Therefore, a transplant with a B*44:02 donor and a B*44:03 patient can be expected to result in a stronger graft-versus-host reaction because of a lower level of histocompatibility (HC (B*44: 02, B*44:03) ⫽ 0.67) than a transplant with a B*44:03 donor and a B*44:02 patient with a higher measure of histocompatibility (HC (B*44:03, B*44:02) ⫽ 0.80). In regard to graft rejection or hostversus-graft reaction, it would be the opposite, assuming that the difference between an HC ⫽ 0.67 and HC ⫽ 0.80 is functionally significant, which may not be the case. As for the present study concerning B*44:02 and B*44:27, we have HC (B*44:02, B*44:27) ⫽ 49/159 ⫽ 0.31 and HC (B*44:27,
Fig. 4. Frequency of C-terminal amino acids for B*44:02 and B*44:27. Peptide sequences demonstrated as anchor motifs for HLA-B*44:02 and B*44:27 W, F, or Y at the P⍀ position of the respective peptide. W was the most frequent amino acid at P⍀, with 41.2% for B*44:02 and 35% for B*44:27. F could be detected with a frequency of 24.2% for B*44:02 and 24.5% for B*44:27 and Y with a frequency of 23.7% for B*44:02 and 33.1% for B*44:27.
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B*44:02) ⫽ 49/145 ⫽ 0.34, meaning that these 2 alleles are very close to full compatibility with only minor and probably insignificant differences. The future of bone marrow transplantation does not depend so much on our ability to match patients and donors as on our understanding of how to mismatch patients and donors and still have successful clinical outcomes. This understanding can only take the form of a measure of histocompatibility. The similarity of allele peptide-binding profiles is such a measure because B*44:02 and B*44:27 have similar peptide-binding profiles and therefore might be considered compatible. Further analysis on other exon 4 mismatches is needed to validate the theory of functional alleles. It must also be taken into account that the complexity of alloreactive T-cell responses restricted to distinct pHLA complexes is sometimes unpredictable [18]. Given that in this study we identified 80% of the peptide repertoire to be different, alloreactivity cannot be fully excluded. Of course this theoretic position must be validated by means of proper clinical trials. Nevertheless, the success of current practices in bone marrow transplantation overlooking non-peptide-binding regions of both class I and class II HLA alleles provides indirect informal evidence in support of this position. The meticulous and systematic characterization of peptidebinding profiles for each and every allele, as well as the evaluation of T-cell response in the context of 1-allele mismatches, will open the door to a new scoring system for HLA mismatches in bone marrow transplantation. Acknowledgments The technical assistance of Susanne Aufderbeck is grateful acknowledged. This work was supported by a grant from the German Federal Ministry of Education and Research (reference number 01E00802). Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.humimm.2011.08.004. References [1] Chelvanayagam G. A roadmap for HLA-A, HLA-B, and HLA-C peptide binding specificities. Immunogenetics1996;45:15–26.
[2] Saper MA, Bjorkman PJ, Wiley DC. Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 A resolution. J Mol Biol 1991;219:277–319. [3] Elamin NE, Bade-Doeding C, Blasczyk R, Eiz-Vesper B. Polymorphism between HLA-A*0301 and A*0302 located outside the pocket F alters the POmega peptide motif. Tissue Antigens 2010;76:487– 490. [4] Bade-Doeding C, DeLuca DS, Seltsam A, Blasczyk R, Eiz-Vesper B. Amino acid 95 causes strong alteration of peptide position Pomega in HLA-B*41 variants. Immunogenetics 2007;59:253–9[CrossRef]. [5] Bade-Doeding C, Elsner HA, Eiz-Vesper B, Seltsam A, Holtkamp U, Blasczyk R. A single amino-acid polymorphism in pocket A of HLA-A*6602 alters the auxiliary anchors compared with HLA-A*6601 ligands. Immunogenetics 2004;56: 83– 8[CrossRef]. [6] Macdonald WA, Purcell AW, Mifsud NA, Ely LK, Williams DS, Chang L, et al. A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition. J Exp Med 2003;198:679 – 91[CrossRef]. [7] Prilliman KR, Jackson KW, Lindsey M, Wang J, Crawford D, Hildebrand WH. HLA-B15 peptide ligands are preferentially anchored at their C termini. J Immunol 1999;162:7277– 84[PubMed]. [8] Barnstable CJ, Bodmer WF, Brown G, Galfre G, Milstein C, Williams AF, et al. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 1978;14:9 – 20[CrossRef]. [9] Brodsky FM, Parham P, Barnstable CJ, Crumpton MJ, Bodmer WF. Monoclonal antibodies for analysis of the HLA system. Immunol Rev 1979;47:3– 61[CrossRef]. [10] Hirosawa M, Hoshida M, Ishikawa M, Toya T. MASCOT: multiple alignment system for protein sequences based on three-way dynamic programming. Comput Appl Biosci 1993;9:161–7[PubMed]. [11] Bade-Doeding C, Theodossis A, Gras S, Kjer-Nielsen L, Eiz-Vesper B, Seltsam A, et al. The impact of human leucocyte antigen (HLA) micropolymorphism on ligand specificity within the HLA-B*41 allotypic family. Haematologica 2011; 96:110 – 8. [12] Fleischhauer K, Avila D, Vilbois F, Traversari C, Bordignon C, Wallny HJ. Characterization of natural peptide ligands for HLA-B*4402 and -B*4403: implications for peptide involvement in allorecognition of a single amino acid change in the HLA-B44 heavy chain. Tissue Antigens 1994;44:311–17. [13] Lemmel C, Weik S, Eberle U, Dengjel J, Kratt T, Becker HD, et al. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling. Nat Biotechnol 2004;22:450 – 4[CrossRef]. [14] Wearsch PA, Cresswell P. The quality control of MHC class I peptide loading. Curr Opin Cell Biol 2008;20:624 –31[CrossRef]. [15] Salter RD, Benjamin RJ, Wesley PK, Buxton SE, Garrett TP, Clayberger C, et al. A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2. Nature 1990;345:41– 6[CrossRef]. [16] Cano P, Fan B. A geometric and algebraic view of MHC-peptide complexes and their binding properties. BMC Struct Biol2001;1:2[CrossRef]. [17] Cano P, Fan B, Stass S. A geometric study of the amino acid sequence of class I HLA molecules. Immunogenetics 1998;48:324 –34[CrossRef]. [18] Gras S, Kjer-Nielsen L, Chen Z, Rossjohn J, McCluskey J. The structural bases of direct T-cell allorecognition: implications for T-cell-mediated transplant rejection. Immunol Cell Biol 2011;89:388 –95.
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Mismatches outside exons 2 and 3 do not alter the peptide motif of the allele group B*44:02P Christina Bade-Doeding a,*, Pedro Cano b, Trevor Huyton a, Soumya Badrinath a, Britta Eiz-Vesper a, Oliver Hiller a, Rainer Blasczyk a a b
Institute for Transfusion Medicine, Hannover Medical School, 30625 Hannover, Germany The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030, USA
A R T I C L E
I N F O
Article history: Received 20 January 2011 Accepted 4 August 2011 Available online 10 August 2011
Keywords: Polymorphism Peptide-binding motif Stem cell transplantation Functional alleles
A B S T R A C T
Sequence variations outside exons 2 and 3 do not appear to affect the function of human leukocyte antigen (HLA) class I alleles. HLA-B*44:02:01:01 and -B*44:27 are considered functionally identical because they differ by a single amino acid substitution of Val ⬎ Ala at position 199, which is located in the ␣3 domain. To validate that HLA-B*44:02:01:01 and -B*44:27 represent functionally identical alleles that might reflect a permissive mismatch in hematopoetic stem cell transplantation (HSCT), we determined their peptidebinding features. B-lymphoblastic cells were lentivirally transduced with B*44:02 and B*44:27 constructs and soluble recombinant molecules were purified by affinity chromatography. Peptides were isolated and sequencing of single peptides was performed using liquid chromatography– electrospray ionization–tandem mass spectrometry (LTQ-Orbitrap) technology. We demonstrate that the peptide motif of B*44:02199Val and B*44:27199Ala is identical. Both variants feature E at P2 and Y, F, or W at P⍀ in their ligands. Most of the identified peptides are 9 to 11 amino acids in length and approximately 20% of these ligands are shared between the alleles. Our results lead to the conclusion that B*44:02:01:01 and B*44:27 might have the same immune function, validating a theory that is now being used in deciding which donors to select in HSCT when there is no identical donor available. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The risks associated with the immune reactions characteristic of both allogeneic hematopoietic stem cell transplantation and solid organ transplantation depend on amino acid (AA) sequence differences between human leukocyte antigen (HLA) alleles. The systematic study of the effect of the AA sequence polymorphism on the function of the HLA molecule and on the immune response to transplantation is therefore necessary. Effector cytotoxic T lymphocytes target cells displaying allogeneic peptides and are able to detect even single-AA exchanges between peptides; for that reason the comparison of allelic peptide-binding features helps to weigh the importance of individual AA substitutions within the peptide-binding region (PBR) of certain HLA allelic variants. AA substitutions might have an impact not only on the binding of peptides to the individual HLA molecule but also on the conformation of the respective peptide–HLA (pHLA) complex. These subtle changes can then be recognized by T cells, resulting in an immunologic response.
* Corresponding author. E-mail address: [email protected] (C. Bade-Doeding).
Forty-seven AA positions within the PBR of HLA molecules have been attributed a role in peptide binding [1] and are subdivided into 6 specificity pockets (A–F) [2]. Previous studies demonstrated that even a single-AA exchange at a given position within the PBR can restrict the features of the presented peptides if it occurs as part of a specific pocket [3–7]. However, the magnitude of a given polymorphism is dependent not only on its position but also on the nature of the exchanged AAs, as well as their neighboring AAs; therefore, even a single mismatch may impact the transplantation outcome. The question of whether a mismatch is permissive or nonpermissive is critical in deciding which person is the bestmatched donor and whether the selected donor exhibits an acceptable mismatch. Since even small differences between HLA allotypes can have dramatic effects on their function, the selection criteria for identifying acceptable mismatches when no matched donor is available remain poorly defined. When sequence analysis is carried out to find a matched donor, exons 2 and 3 (encoding for the ␣1 and ␣2 domain of the HLA heavy chain) are the regions of interest and it is currently believed that sequence variations outside exons 2 and 3 do not have any effect on the function of HLA class I alleles. According to this view, alleles with identical sequences in exons 2 and 3 are thought to be functionally identical, irrespective of any sequence differences outside
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.08.004
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exons 2 or 3. In this view, because the alleles have the same biologic function, they can be considered the same functional allele and not 2 different alleles. The impact of HLA mismatches is determined by several factors, of which the individual peptide-binding features are the most influential. If differences in peptide-binding features are determined, then an immune response would be expected. In contrast, if the peptide-binding features between mismatched alleles are similar or identical, then immune responses cannot be excluded, but would be thought to be less probable. A measure of similarity based upon the peptide-binding profiles of different alleles therefore gives us insight into their functional similarity and can guide us toward those alleles that require further investigation on the cellular level. The difficulties of establishing the selection criteria for identifying acceptable mismatches are exemplified in the molecular differences between HLA-B*44:02:01:01 and -B*44:27. These 2 alleles differ at position 199 in the ␣3 domain of the heavy chain (Fig. 1). In this study we hypothesized that B*44:02:01:01 and -B*44:27 can be considered the same functional allele. Although B*44:02:01:01 is much more common, it must be taken into account that B*44:27 is always associated with Cw*07:04, whereas B*44:02:01:01 is mostly associated with Cw*05:01 and rarely with Cw*07:04. That means that B*44:27 came as a mutation or gene conversion from the uncommon block B*44:02:01:01–Cw*07:04. 2. Subjects and methods 2.1. Design of lentiviral vectors For expression of truncated B*44:02 and B*44:27 molecules, coding DNA from an HLA-B*44:02-positive donor (exons 1 through 4) was amplified by polymerase chain reaction (PCR) using the primers HLA-B3-TAS (5= gag atg cgg gtc acg gca c 3=) and HLA-E4WAS (5= cca tct cag ggt gag ggg ct 3=). The PCR product was ligated
in the eukaryotic expression vector pcDNA3.1V5/His using the pcDNA3.1V5/His TA cloning kit (Invitrogen, Karlsruhe, Germany). The recombinant pcDNA3.1V5/His/B*44:02 vector was used as a template for the lentiviral pRRL.PPT.SFFV.mcs.pre/B*4402 vector. The B*44:02 insert was ligated in the pRRL.PPT.SFFV.mcs.pre vector and the insertion of the B*44:02 construct was verified by sequencing (forward and reverse direction) using a 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). The B*44:27 allele was produced by side-direct mutagenesis using the QuikChange multi site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). The pRRL.PPT.SFFV.mcs.pre/ B*44:02 vector was used as template. For expression of full-length HLA-B*44 variants, cDNA (exons 1 through 7) from an HLA-B*44:02-positive donor was amplified by PCR using the primers HLA-B3-TAS (5= gag atg cgg gtc acg gca c 3=) and HLA-B-TAAS-E7 (5= tca agc tgt gag aga cac atc ag 3=). Lentiviral pRRL.PPT.SFFV.mcs.pre vector was generated by following the cloning procedure as described above. 2.2. Production of lentiviral particles in HEK293T cells HEK293T cells have been transfected by adding 1 mg/mL polyethyleneimine (Sigma–Aldrich Chemie Gmbh, Munich, Germany) in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) followed by an incubation of 20 minutes with 5 g plasmid/6 ⫻ 106 HEK293T cells (packaging plasmid, psPAX2; envelope plasmid, pDM2G). After 24 hours of incubation the medium was changed; 48 hours posttransfection, virus-containing supernatant was removed, passed through a 0.45-m filter (Millipore GmbH, Schwalbach, Germany), and concentrated overnight by centrifugation at 16⬚C at 10,000 rpm. The lentiviral pellet was then dissolved in RPMI 1640 (Invitrogen). 2.3. Transduction of B-lymphoblastoid cell lines (B-LCLs) The lentiviral transduction of the B-LCLs was performed by adding the dissolved lentiviral pellet in the presence of 8 g/mL protamine sulfate (Sigma–Aldrich) to the cells, followed by incubation for 8 hours. Transduced B cells were then cultured in complete RPMI 1640 medium containing 10% fetal calf serum. 2.4. Analysis of HLA-B44 surface expression on B-LCLs Cell surface expression of HLA-B*44 in the presence (LCL 721.221 cells) or absence (LCL 721.220 cells) of tapasin (TPN) was assessed by flow cytometry using an anti-Bw4 –FITC labeled antibody (One Lamda, BmT GmbH, Meerbusch-Osterath, Germany) and W6/32–PE labeled antibody. The cells were washed twice with phosphate-buffered saline containing 0.5% bovine serum albumin and then incubated with 10 L of the antibody stock for 20 minutes at 4⬚C. The cells were washed twice with phosphate-buffered saline/0.5% bovine serum albumin and then analyzed using a FacsCanto flow cytometer (BD Biosciences, Heidelberg, Germany). Real-time PCR was also performed to determine the levels of B*44specific mRNA in transduced cells. 2.5. Verification of sHLA expression
Fig. 1. Location of residue 199 in the ␣3 domain of the heavy chain. A ribbon representation of a B*44:02 peptide–HLA complex (PDB ID: 3KPL) [16] to illustrate the location of amino acid position 199. The bound peptide is colored magenta, 2m is colored green, heavy chain is colored blue, and position 199 in the ␣3 domain of the heavy chain is highlighted in red.
Expression of V5-tagged, truncated HLA-B*44 (sHLA) molecules was quantified using a sandwich enzyme-linked immunosorbent assay in which anti-HLA-A-B-C W6/32 (Serotec, Du¨sseldorf, Germany) [8,9] or anti-V5 (Invitrogen) monoclonal antibodies (mAb) were employed as capture antibodies. Horseradish peroxidase– conjugated anti-2m mAb (Dako, Hamburg, Germany) served as the detection antibody. B-LCL 721.221 clones with the highest recombinant protein expression rates were used for large-scale production.
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2.6. Large-scale production of sHLA molecules and affinity purification Highly producing clones were cultured and expanded in bioreactors (CELLine, Integra, Fernwald, Germany). Harvests were centrifuged for 20 minutes at 1,200 rpm to remove cellular material and then filtered through a 0.45-m filter (Sartorius, G×ttingen, Germany) and stored at ⫺20⬚C. Supernatants of each HLA-B*44 variant were pooled, adjusted to pH 8.0, and affinity purified using N-hydroxysuccinimide (NHS)-activated HiTrap columns, precoupled with mAb W6/32. All purification was performed using a BioLogic DuoFlow system (Bio-Rad, Hercules, CA). Trimeric complexes were eluted using 0.1 M glycine/HCl buffer (pH 2.7); 2.5 mg sB*44:27 protein and 3.0 mg sB*44:02 protein complexes were subsequently used for peptide analysis. 2.7. Characterization of sHLA-B*44-derived peptides Peptides were eluted from trimeric sHLA-B*44 complexes by treatment with 0.1% trifluoroacetic acid. Peptides were separated by filtering them through a YM membrane with a 10-kDa cutoff (Millipore, Schwalbach, Germany). The flowthrough containing the peptides was then subjected to an Eksigent nano-LC Ultra 2D highperformance liquid chromatograph coupled to an Orbitrap ion trap (Thermo Fischer, Waltham, MA), resulting in a high mass accuracy (⬍5 ppm). Database queries were performed using Mascot software[10] implementing the IPI human and the respective decoy databases. 3. Results 3.1. Surface expression of B*44/199 variants Flow cytometric analysis indicated that the B*44/199 variants are exclusively expressed on the cell surface of LCL 721.221 cells in the presence of TPN (Fig. 2). For LCL 721.220 cells lacking TPN, no surface expression could be determined on the cell surface, suggesting that both B*44:02 and B*44:27 are dependent on TPN for loading and peptide presentation. We then sequenced by liquid chromatography– electrospray ionization–tandem mass spectrometry (LTQ-Orbitrap) the peptides bound by 2 HLA alleles, B*44:02 and B*44:27, which were expressed and secreted by B-LCL 721.221 cells. Analysis of the identified peptide data is presented below. 3.2. Peptide features The mass spectrometer used for this study (LTQ-Orbitrap) has a high mass accuracy in the range of 10 ppm. To obtain the highest probability of peptide identification we selected only those peptides that had a ␦ value of 0.0 Da and excluded all other peptides. For example, even a ␦ value of 0.1 Da would mean for a peptide with a mass of 1,000 Da that there would be an error of 100 parts per million associated with the measurement. A total of 194 unique peptides (nonduplicates) were identified following elution from purified recombinant sHLA-B*44:02 molecules (Suppl. Table 1). By comparison we identified 208 unique peptides from purified recombinant sHLA-B*44:27 molecules (Suppl. Table 2). The MS/MS spectra of 3 long peptides are presented as representative examples (Suppl. Fig. 1). Most of the peptides identified were of the canonical 9 to 11 AAs in length with 164/194 (84.5%) for B*44:02 and 161/208 (77.4%) for B*44:27 (Fig. 3). A larger percentage of peptides (15.5%, 30/194) from the B*44:02 subtype were longer than 11 AA compared with those identified in B*44:27 (13.9%, 29/208). This frequency may be reflective of the different number of peptides identified between the 2 alleles and our stringent cutoff values for selecting peptides. Interestingly, as with our previously published data on other HLA-B group peptides[11], several peptides of extraordinary length, ⬎15
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residues, were identified from the B*44:02 subtype, including two 16-mer peptides and one 19-mer peptide, all of which have a ␦ score of 0.0 and fulfill the correct charge mass of the single ions. Further analysis of the peptide sequences demonstrated clear anchor motifs for HLA-B*44:02 with E at the P2 position and W, F, or Y at the P⍀⬎ position of the respective peptide. Our data therefore expand the previously published peptide motif for B*44:02 peptides [6,12,13] by including W as a bona fide P⍀ anchor. Indeed, W was the most frequent AA at this position, being reported in 80/194 (41.2%) of the isolated peptides compared with F in 47/194 (24.2%) and Y in 46/194 (23.7%; Fig. 4). The peptide anchor motifs derived from the eluted B*4427 peptides are identical to that of B*4402 with E at the P2 position and W, F, or Y at the P⍀ position of the respective peptide. Likewise, W was the most frequent P⍀ amino acid (73/208; 35%) compared with F (51/208; 24.5%) and Y (69/208; 33.1%). 3.3. Shared peptides A total of 402 peptides, 194 from B*44:02 and 208 from B*44:27 alleles (Suppl. Tables 1 and 2), were identified containing 49 shared peptides and 353 unique peptides. Taken together with 19 previously published peptide sequences for B*44:02 (http://www. syfpeithi.de/) that are shared with B*44:27, this gives 68/353 (19.3%) of the sequenced peptides that are shared and preferentially selected from the available peptide pool by both alleles. These 68 shared peptides are shown in Suppl. Tables 3 and 4 describing 19 previously published B*44:02 peptide sequences and 49 newly identified peptide sequences eluted from both alleles. 3.4. Differential selection of peptides Some of the B*44-derived ligands originated from different regions of the same protein and had overlapping sequences (Suppl. Table 5). These peptides differed both in their length (9 –12 AA) and in their C-terminal anchor residues. The shorter peptide versions were N- and/or C-terminally truncated, and related peptides were detected in distinct B*44 alleles. 4. Discussion The selection and features of those peptides that are able to be bound by a certain HLA variant and subsequently presented to the immune system remain questionable for most alleles. In the HLA heavy chain, variations within the PBR can affect (1) the peptidebinding motif [3–5,8,11], (2) the feature and length of the peptides [11], and (3) the individual conformation of a pHLA complex [11,12]. Consequently, every AA substitution at a given position within the HLA molecule can impact the immunologic outcome; this can be highly dependent on the influence of its neighboring AA. In current transplantation scenarios the decision for choosing the best-matched donor is often made on available functional data for matched alleles at the ␣1 and the ␣2 domain (PBR) that is encoded by exons 2 and 3. It is currently believed that sequence variations outside the exon 2/3 regions do not have any significant effect on the function of HLA class I alleles. In this study we considered the alleles B*44:02 and B*44:27 to have the same immune function (i.e., to not be recognized as foreign by each other), by virtue of the fact that they differ in a single AA substitution at position 199 (V ⬎ A), which lies outside the exon 2/3 regions (Fig. 1). Our aims were to use peptide sequencing to investigate if this polymorphic difference influences the peptide-binding features of these class I alleles and to assess their functional disparity or similarity. To enable a meaningful comparison of peptide selection, we ensured that the peptides presented through B*44:02 and B*44:27 are selected and loaded by the peptide-loading complex (PLC). The function of TPN, a dedicated part of the PLC, is to stabilize the PBR of the HLA molecule against irreversible denaturation and to maintain it in a peptide-receptive state before peptides are selected and
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Fig. 2. Surface expression of HLA-B*44/199 variants on LCL 721.220 (TPN⫺) and LCL 721.221 (TPN⫹) cells. Flow cytometric analysis for cells stained with anti Bw4 –FITC and w6/32–PE labeled monoclonal antibodies. (A) FACs plots for nontransduced LCL 721.221 (left) and LCL 721.220 (right) cells, respectively (negative controls). (B and C) FACs plots for the B*44:02 and B*44:27 in LCL 721.221 (left) and LCL 721.220 (right) cells, respectively. Both HLA B*44:02 and B*44:27 were expressed only on the surface of LCL 721.221 cells.
loaded [14]. We here demonstrate that the polymorphic difference at position 199 does not affect the association of B*44:27 with TPN, illustrating that peptides for B*44:02 and B*44:27 are selected, optimized, and loaded through the same pathway. Additionally, we ensured that the peptide repertoire of each variant was selected from a single peptide pool (LCL 721.221 cells) and that it was determined under near identical conditions. Our data illustrate that both allotypes select a similar set of peptides with identical peptide-binding motifs, with E at the P2 position and W, F, or Y at the P⍀ position of the respective peptide. Furthermore, 19.3% of the peptides are shared peptides, highlighting the fact that they select identical peptides from the vast peptide
pool available and providing further evidence that they might be considered functionally similar. Our data provide a representative analysis of B*44-derived peptides; however, it must be noted that the identified ligands specifically reflect the peptide pool available to sHLA molecules in transduced LCL 721.221 cells. We anticipate that some differences in the peptide repertoire might be observed from different cell types expressing exogenous HLA or from endogenous HLA in B*44-positive patient cells. The questions that arise are as follows: Can all alleles with identical sequences in exons 2/3 be considered functionally identical? Alternatively, what mismatches outside exons 2/3 can be con-
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Fig. 3. Frequency of peptide length (AA) for HLA-B*44:02 and B*44:27. Peptides bound to HLA-B*44:02 and B*44:27. The length (9 to ⬎12 residues) of eluted peptides specific for each of the individual B*44 alleles is represented. Most of the peptides identified were of the canonical 9 to 11 amino acids (AAs) in length with 84.5% for B*44:02 and 77.4% for B*44:27. A total of 15.5% of peptides derived from the B*44:02 subtype were longer than 11 AA compared with 13.9% of those derived from B*44:27. The 1 identified 8-mer peptide for B*44:27 is not shown.
represented by a vector in a vector space in which each dimension is a peptide sequence property. In this vector space each allele is represented by a vector and the distance between 2 vectors is a measure of histocompatibility between alleles [16]. If the known peptide-binding profile is limited (i.e., if only a small number of peptides have been eluted from the selected allele), then to fully characterize the allele in terms of peptide features, a compatibility comparison can be performed that takes into account the overlap between 2 peptide-binding profiles (sets of peptides binding to a particular allele.) This method is more opaque because it does not show why or how a unique peptide may bind to 1 allele but not to another, although they share the same peptidebinding motif. However, this method has 2 advantages: first, it requires peptide-binding sets of smaller size, and second, it reveals the asymmetric nature of the compatibility relation. The relation of histocompatibility between alleles X and Y (HC(X,Y)) is not symmetric (HC(X,Y) ⫽ HC(Y,X) or HC(X,Y) ⫽ HC(Y,X)) [17]. The immune reaction elicited by transplanting an organ carrying allele X to a patient carrying allele Y may be different if it is the donor who carries allele Y and the patient who carries allele X. We define the histocompatibility function as follows: HC(P, D) ⫽
sidered functionally identical? When considering these questions the described binding site for the T cell coreceptor CD8 on the ␣3 domain of the HLA heavy chain [15] may need to be considered. Three clusters of residues appear to contribute to binding (220 – 232, 233–235, 245–247), with a loop region (223–239) playing the predominant role. In addition, several individual functional mutations have also been described that can influence CD8 HLA class I interactions, including residues 227 and 245. Because ⬎70% of the alleles in the Anthony Nolan database are not sequenced outside exons 2/3, it remains possible that any 2 alleles with identical sequences in their exon 2/3 regions could still contain mismatches that influence CD8 binding and thus the “avidity threshold” of the T-cell receptor, thereby changing the subsequent immune response. For such a transplantation scenario one would also anticipate an obvious directionality. To the extent that (1) histocompatibility and transplantation outcome depend on T-cell recognition and (2) T-cell recognition of different alleles depends on the ability of these alleles to bind peptides with distinct different AA sequence features, we can therefore define histocompatibility in terms of the repertoire of peptides that alleles bind. According to this logic, when 2 alleles bind peptides with the same amino acid sequence features and present them to T cells, they might be considered compatible. Therefore, matching patients and donors is effectively an exercise of comparing the repertoire of peptides bound by the alleles that patients and donors carry and present to T cells. In some cases the second assumption does not hold, and it is possible for 2 alleles to have the same peptide-binding profile and yet show different T-cell recognition. In fact, B*44:02 and B*44:03 bind peptides with the same sequence properties (E at P2 and F, Y, W, or L at P⍀), but can be recognized as foreign by each other [6]. Upon further analysis, however, although they share a large set of peptides they can both bind, there are peptides that bind to B*44:02 and not to B*44:03 and vice versa. Such known examples are rare and the individual sequence properties that distinguish them are in many cases undetermined. This example provides evidence supporting our claim that T-cell recognition of 1 allele can be predicted by means of analyzing its peptide-binding profile. In such a transplantation scenario, we face the task of comparing 2 peptide-binding profiles to estimate and predict histocompatibility. If peptide-binding profiles could be fully characterized in terms of their sequence properties, allele peptide-binding profiles can be
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Card(PBP(P 艚 D)) Card(PBP(D))
where P is the allele carried by the patient and D is the allele carried by the donor, Card ( ) is a function that returns the cardinality of a set or the number of elements in it, PBP (X) is the peptide-binding profile or set of peptides known to bind to X, and PBP (X 艚 Y) is the set of peptides that bind to both X and Y. For instance, we have that HC (B*44:02, B*44:03) ⫽ 0.67 and HC (B*44:03, B*44:02) ⫽ 0.80 [6]. In evaluating the host-versus-graft or graft rejection reaction, the relation to consider is HC (P,D). In evaluating the graft-versushost reaction, by contrast, the HC (D,P) must be taken into account instead. Therefore, a transplant with a B*44:02 donor and a B*44:03 patient can be expected to result in a stronger graft-versus-host reaction because of a lower level of histocompatibility (HC (B*44: 02, B*44:03) ⫽ 0.67) than a transplant with a B*44:03 donor and a B*44:02 patient with a higher measure of histocompatibility (HC (B*44:03, B*44:02) ⫽ 0.80). In regard to graft rejection or hostversus-graft reaction, it would be the opposite, assuming that the difference between an HC ⫽ 0.67 and HC ⫽ 0.80 is functionally significant, which may not be the case. As for the present study concerning B*44:02 and B*44:27, we have HC (B*44:02, B*44:27) ⫽ 49/159 ⫽ 0.31 and HC (B*44:27,
Fig. 4. Frequency of C-terminal amino acids for B*44:02 and B*44:27. Peptide sequences demonstrated as anchor motifs for HLA-B*44:02 and B*44:27 W, F, or Y at the P⍀ position of the respective peptide. W was the most frequent amino acid at P⍀, with 41.2% for B*44:02 and 35% for B*44:27. F could be detected with a frequency of 24.2% for B*44:02 and 24.5% for B*44:27 and Y with a frequency of 23.7% for B*44:02 and 33.1% for B*44:27.
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B*44:02) ⫽ 49/145 ⫽ 0.34, meaning that these 2 alleles are very close to full compatibility with only minor and probably insignificant differences. The future of bone marrow transplantation does not depend so much on our ability to match patients and donors as on our understanding of how to mismatch patients and donors and still have successful clinical outcomes. This understanding can only take the form of a measure of histocompatibility. The similarity of allele peptide-binding profiles is such a measure because B*44:02 and B*44:27 have similar peptide-binding profiles and therefore might be considered compatible. Further analysis on other exon 4 mismatches is needed to validate the theory of functional alleles. It must also be taken into account that the complexity of alloreactive T-cell responses restricted to distinct pHLA complexes is sometimes unpredictable [18]. Given that in this study we identified 80% of the peptide repertoire to be different, alloreactivity cannot be fully excluded. Of course this theoretic position must be validated by means of proper clinical trials. Nevertheless, the success of current practices in bone marrow transplantation overlooking non-peptide-binding regions of both class I and class II HLA alleles provides indirect informal evidence in support of this position. The meticulous and systematic characterization of peptidebinding profiles for each and every allele, as well as the evaluation of T-cell response in the context of 1-allele mismatches, will open the door to a new scoring system for HLA mismatches in bone marrow transplantation. Acknowledgments The technical assistance of Susanne Aufderbeck is grateful acknowledged. This work was supported by a grant from the German Federal Ministry of Education and Research (reference number 01E00802). Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.humimm.2011.08.004. References [1] Chelvanayagam G. A roadmap for HLA-A, HLA-B, and HLA-C peptide binding specificities. Immunogenetics1996;45:15–26.
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