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HLA Epitopes: The Targets of Monoclonal and Alloantibodies Defined.

1. Introduction

Sensitization to HLA antigens in organ transplant patients causes graft rejection, according to the humoral theory of transplantation [1]. Sensitization is almost ubiquitous as it is evident in the detection of anti-HLA antibodies in the sera of recipients--in one study, almost all patients waiting for regraft of a kidney transplant have anti-HLA antibodies [2]. Determining specificity of the anti-HLA antibody has advanced in recent years using recombinant HLA single antigens (SA) coated on color-coded Luminex beads [3]. The reactivity of anti-HLA antibodies with HLA antigens and the phenomenon of cross-reactivity has been the subject of investigation for decades. Amino acid sequences of the HLA molecules which greatly contributed to our understanding of antibody and antigen reactivity has been introduced since 1987 [4-12]. Antibodies are commonly described as binding to HLA antigens; however, it is more accurate to describe the reactivity of the antibody as binding to specific epitopes on the surface of HLA antigens--epitopes are conformational amino acid arrangements and are the targets of antibodies. Some epitopes are private, found exclusively on one antigen; others are public epitopes shared by two or more antigens. The phenomenon of cross-reactivity in HLA testing, often explained as cross-reactive groups (CREGs), of antigens with antibody can be clearly explained now by public epitopes--an antibody targeting a public epitope shows positive reaction with all antigens sharing the epitope.

Since 2006, we defined and reported on 194 HLA class I unique epitopes, including 138 epitopes on intact HLA class I (heavy chain + [beta]2m +peptide), and 56 cryptic epitopes on dissociated HLA class I (heavy chain only) [13-18]. 110 epitopes on intact HLA class I were defined using murine monoclonal and human alloantibodies, and the remaining 28 epitopes were defined with naturally occurring anti-HLA antibodies. Naturally occurring (natural) HLA antibodies found in cord blood and healthy males were used to define the 56 cryptic epitopes on dissociated HLA class I. In addition, 83 HLA class II unique epitopes were defined and reported, including 60 epitopes on HLA-DRB1, 15 epitopes on HLA-DQB1, 3 epitopes on HLA-DQA1, and 5 epitopes on HLA-DPB1 [15, 19-22]. All HLA-DRB1 epitopes were defined using solely amino acid sequence data, in contrast to HLA-DQA1, HLA-DQB1, and HLA-DPB1 epitopes that were defined using human alloantibodies. Lastly, we defined and reported on 7 MICA epitopes using human alloantibodies [15, 22]. In this paper, we provide a summary of our findings.

2. Materials and Methods

The principle we used to define HLA epitopes is summarized in (Figure 1). Briefly, if an antibody is determined to test positive with certain HLA antigens and negative with others, it is reasonable to assume that the antibody is targeting a specific epitope on the positive antigens. Epitopes are conformational arrangements of amino acids (aa) at sequence positions on the surface of antigens that must be within the binding span of the antibody. To define an epitope, a computer search, in published aa sequences of tested antigens, was performed to identify exclusively shared aa at one or more sequence positions among the positive antigens--these amino acids define the epitope.

Murine monoclonal antibodies or transplant recipient and healthy male HLA antibodies isolated from sera and cord blood by first adsorbing them onto appropriate recombinant HLA (rHLA) single antigen cells, then eluted with an acidic buffer (ImmunoPure IgG elution buffer, Pierce, Rockford, IL), and neutralized with 1M TRIS-HCl pH 9.5 (Figure 2) were all tested with the single antigen beads (One Lambda Inc., Canoga Park, CA) to determine the specificity of the antibodies [14]. HLA class I SA beads treated with a buffer that dissociates the peptide and the beta-2-microglobulin ([beta]2m) from the heavy chain of the intact HLA antigens on the beads [17] were used to reveal the specificity of antibodies targeting epitopes on dissociated class I heavy chains. MFI values of 1000 or above were considered positive except when the overall reactions of an eluted antibody were weak, a cutoff of MFI 400 was used.

Computer software was utilized to search for exclusive amino acids in the structure of antigens showing positive reactions with an antibody. Searches were performed within sequences of HLA class I heavy chains, MICA antigens, DR beta chains, DQ beta and alpha chains, and DPB chains. All amino acid sequences were obtained from the HLA Informatics Group at the Anthony Nolan website [23]. One or more amino acids found exclusively at the same sequence positions in the chains of positive antigens, but not in the sequence positions of negative antigens, were designated as the defining amino acids for an epitope. The defining amino acid(s) had to be within the antibody binding span [24, 25]--estimated at 494A2-750A2 area (Figure 3) and the aa(s) must be exposed at the surface of the antigen--exceptions are noted between parentheses (Table 1).

The efficacy of isolating HLA antibody from HLA sera with adsorption and elution assays, testing the eluted antibody with the SA beads to determine specificity and the definition of the epitope on the surface of positive antigens (corresponding to antibody specificity) are shown in (Figure 4). Alloserum with determined specificity A2, A68, A69, B57, and B58 was adsorbed separately with SA rHLA A6901 and B5801 cells. Eluted antibodies tested with the SA beads showed specificity A2, A68, and A69 and A2, B57, and B58, respectively. HLA antigens A2, A68, and A69 share an epitope defined by glycine (G) at position 62; therefore, 62G defines the epitope. Similarly, HLA antigens A2, B57, and B58 share an epitope defined by threonine (T) at position 142 or histidine (H) at position 145; therefore 142T or 145H define the epitope.

3. Results

3.1. Class I Epitopes on Intact Antigens. 138 unique epitopes were defined for one or a group of two or more intact HLA class I antigens. 110 unique epitopes were defined by using SA beads (Table 1, partial list; complete table in the supplemental information available online at https://doi.org/10.1155/2017/3406230) assays to test eluted alloantibodies that were adsorbed from human sera onto the surface of mammalian rHLA single antigen cells then eluted, and murine monoclonal antibodies to determine specificity of each antibody. Epitopes were defined by identifying exclusively unique amino acids among the positive antigens. Also defined were 28 unique epitopes targeted by naturally occurring anti-HLA antibodies found in sera of healthy males and in cord blood (Table 2). All epitopes were defined by identifying exclusively unique amino acids among the positive antigens. Here, we present partial lists in tables and example figures of epitopes--complete tables and other figures can be found in the supplemental information document.

The number of epitopes defined for each antigen, using human alloantibodies, varied from 4 to 23 (Table 3). In general, there was no correlation between the number of epitopes and the frequency of antigen in the population. For example, for HLA A2, the most frequent antigen (f=30.3% to 54%), we defined 16 epitopes while for A25, with a frequency of f = 0.0% to 6.1%, we defined 19 epitopes. Class I epitopes were found to be shared by antigens of the same locus or by inter-locus antigens--BC or ABC. Epitopes are defined by 1, 2, 3, or 4 aa's. For example, 17 A-locus epitopes were defined by 1 aa, two B-locus epitopes by 4 aa's, or two ABC loci epitopes defined by 2 aa's. In addition, amino acids and positions on the HLA class I heavy chain epitopes were found at varying frequencies in epitope definitions. The most frequent was position 163 located in the alpha 2 domain, and the aa threonine (T) was found to the most frequent in our studies.

The following examples illustrate HLA class I epitopes for the A-locus, B-locus, and C-locus and AB-, BC-, and ABC-loci antigens. Illustration shows SA beads specificity, antigens sharing the epitopes, and their position on the HLA class I heavy chain.

A-locus: Epitope 422 is shared by A-locus antigens A2, A3, A11, A24, A68, and A69 defined by 149A+150A + 151H combined. Three amino acids at three positions are necessary to define this epitope; indeed, HLA-A*02 : 01 and A* 02 : 06 are positive while A*02 : 03 is negative. A* 02 : 01 and A* 02 : 06 share epitope 422 defined as 149A+ 150A+ 151H, while negative antigen A* 02 : 03 does not share the epitope. A*02 : 03 has 150A+151H but has threonine (T) at position 149 instead of alanine (A)--one amino acid difference in the epitope renders the antibody to be nonreactive with A* 02 : 03. The aa defining epitope 422 are exposed at the surface of the heavy chain and are within the binding span of the HLA antibody. The furthest amino acids are 7.88 [Angstrom] apart (Figure 5).

B-locus: Epitope 21 is shared by B-locus antigens B13, B4005, B41, B44, B45, B47, B49, B50, B60, and B61 and defined by 41T. Threonine (T) is exclusively unique to the antigens at position 41 located in the alpha 1 domain of the HLA class I heavy chain (Figure 6).

C-locus: Epitope 40 shared by the C-locus antigens Cw*0801 and Cw*0501 and defined by 177K located in the alpha 2 domain of the HLA heavy chain (Figure 7).

AB-Loci: Epitope 205 shared by the AB-loci A32, A74, B7, B8, B4005, B41, B42, B48, B60, B61, B73, and B81 and defined by 109L + 131R--the two positions are 11.8A apart and therefore within the binding span of the antibody. Also, the C-locus antigens Cw1, Cw2, Cw4, Cw5, Cw6, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, Cw15, Cw16, Cw17, and Cw18 share the same amino acids at positions 109 and131 but were not tested with the C-locus beads at the time of the study.

Epitope 24 is shared by the AB-loci Bw4-associated antigens A23, A24, A25, A32, B13, B2705, B37, B38, B44, B47, B49, B51, B52, B53, B57, B58, B59, B63, and B77 and defined by either 82L or 83R located in the alpha 1 domain of the HLA class I heavy chain.

Epitope 423 is shared by the AB-loci Bw4-associated antigens A23, A25, A32, B2705, B37, B38, B44, B47, B49, B51, B52, B53, B57, B58, B59, B63, and B77 (A24, B13 negative) and defined by 83R + 144Q + 145R. This epitope was defined using a monoclonal antibody and seems to be a variant of epitope 24 shared by all Bw4-associated antigens. Other variants of the BW4-associated antigens epitope (epitopes 249 and 250) also defined with monoclonal antibodies (Table 1).

BC-Loci: Epitope 246 is shared by BC-loci antigens B46, B73, Cw1, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, and Cw16 and defined by 76V + 80N. The two amino acids are 8.69 A apart which is within the binding span of the HLA antibody.

ABC-loci: Epitope 38 is shared by the ABC-loci antigens A2, A25, A26, A29, A31, A32, A33, A34, A43, A66, A68, A69, A74, B73, Cw7, and Cw17 and defined by the amino acid glutamine (Q) at position 253 of the HLA class I heavy chain located in the alpha 3 domain proximal to the cell membrane (Figure 8).

3.2. Cryptic Epitopes on Dissociated HLA Class I Antigens. Naturally occurring anti HLA antibodies were detected in nonalloimmunized healthy males [26], and 96 of their target epitopes were defined [16]. 58 natural antibodies are only reactive with dissociated HLA class I antigens, heavy chain only (Table 4). 56 unique epitopes on dissociated HLA class I defined [16].

Epitope 5007 is shared by the HLA class I A-locus antigens A31 and A33 and defined by isoleucine (I) at the cryptic position 73. Antibody reactivity with the intact antigen is obstructed because position 73 is located under the peptide. It is slightly reactive with the intact HLA class I antigens. Reactivity increased by up to 10-fold with the dissociated antigens (heavy chain only)--when (32m and the peptide are dissociated from the heavy chain (Figure 9).

Epitope 5024 is shared by the HLA class I B-locus antigens B7, B42, B54, B55, B56, B67, B81, and B82 and defined by 66I + 70Q. Reactions strength of the antibody is stronger with the unobstructed epitope after dissociation of the peptide from the heavy chain.

Epitope 5037 is shared by the HLA C-locus antigens Cw4, Cw6, Cw17, and Cw18 and defined by 73A + 77N. Antibody reaction strength increases with the unobstructed epitope after removal of the peptide.

3.3. MICA Epitopes. MICA or MHC class I polypeptide-related sequence A antigens have similar aa structure as the HLA class I ABC heavy chains. However, MICA antigens are not associated with a peptide and beta 2 microglobulin. Seven epitopes were defined for MICA antigens (Table 5).

Epitope 6002 is shared by MICA antigens MICA* 001, 002, 004, 007, 009, 012, 018, and 027 and defined by glutamine (Q) at position 91; therefore, 91Q defines the epitope (Figure 10).

Epitope 6004 is shared by MICA antigens MICA* 04, 009, and 027 and defined by (36Y), 129V, or 173E.

3.4. Class II Epitopes

3.4.1. HLA-DRB1 Epitopes. Unlike class I epitopes, the 60 HLA class IIB1 epitopes were defined based solely on amino acid sequence of the DR antigen beta chain where all epitopes can be defined by one single amino acid at one position (Table 6). The number of epitopes for each DR antigen was from 8 to 21 epitopes.

For example, epitope 1028 is shared by class II DR antigens DR1, DR4, DR7, DR9, DR10, DR11, DR12, DR13, DR14, DR15, DR16, DR51, DR53, and DR103 and defined by threonine (T) at position 77 (Figure 11).

3.4.2. HLA-DQA1 and HLA-DQB1 Epitopes. Eighteen HLA class II DQB1 and DQA1 epitopes are defined using the adsorption and elution assays described in the Materials and Methods above. Fifteen of the epitopes are located on the beta chain of the DQ antigen and three on the alpha chain (Table 7). The number of epitopes for DQB chains was 4-8 and only one for DQA chains (Table 8).

Sera from allosensitized patients can be expected to have anti-HLA antibodies to class I and II antigens. As illustrated in (Figure 12), this serum has antibodies directed against DR, DQ, and DP antigens. The serum was adsorbed with DQA1*02 : 01/DQB1*04 : 01 rHLA cells and the eluted antibody reacted with DQ4, DQ5, and DQ6 antigens which share epitope 2007 (Table 7).

One antigen mismatch can elicit an immune response to several epitopes on an HLA antigen. A serum from renal transplant patient with DQA1* 02 : 01/DQB1*02 : 02 mismatch has two antibodies. One antibody targets epitope 2017 (defined by histidine (H) in position 52) on the DQA1*02 : 01 alpha chain and the other antibody targets epitope 2001 (defined by leucine (L) in position 52) on the DQB1*02 : 02 beta chain (Table 7). As shown in the table, alternative epitope definitions are separated by "/."

The efficacy of adsorption and elution assays is demonstrated where one serum with DQ specificity, including DQA1*02 : 01, underwent four separate adsorptions and elutions with rHLA DQ cells. Two of the cells have the relevant DQA1*02 : 01 chain, and the eluted antibodies show positive reactions with all heterodimers that contain the DQA1*02 : 01chain (red and green bars). However, eluents from adsorptions with irrelevant cells (no DQA1*02 : 01 chain) showed negative reactions (yellow and blue bars) (Figure 13).

The following examples illustrate HLA-DQA1 and HLADQB1 epitopes. Illustration shows SA beads specificity, antigens sharing the epitopes and their position on the DQA1 and DQB1 chains.

HLA-DQA1 epitopes: epitope 2018 is shared by the alpha chains of the DQ4, DQ5, and DQ6 antigens and defined by glutamine (Q) at position 53.

HLA-DQB1 epitopes: epitope 2002 is shared exclusively by the beta chains of the DQ4 antigen and defined by leucine (L) in position 56.

Epitope 2010 is shared by the beta chains of DQ antigens DQ4, DQ5, DQ6, DQ8, and DQ9 and defined by 45G + 46V.

Epitope 2022 is exclusive to DQB1* 05 : 01 chain on the DQ5 antigen and defined by 125S + 126Q.

Epitope 2006 is shared by DQB1*03 : 01 (DQ7), DQB1*03 : 02 (DQ8), and DQBT03 : 03 (DQ9) and defined by proline (P) at position 55 on the beta chains of the DQ antigens.

3.4.3. HLA-DPB1 Epitopes. Five HLA class II DPB epitopes were defined. Four of the epitopes required 3-4 amino acids for definition, and one was defined by a single amino acid/position (Table 9).

Epitope 4001 is shared by the HLA class II DPB1 chains of the DP antigens DPB1*01 : 01, DPB1*03 : 01, DPB1*05 : 01, DPB1*09 : 01, DPB1*10 : 01, DPB1*11 : 01, DPB1* 13 : 01, DPB1* 14 : 01, DPB1*17 : 01, and DPB1*19 : 01 and defined by 84D + 85E + 86A + 87V. All four amino acids needed to define the epitope. Negative antigens that did not share epitope 4001 are shown as gray bars (Figure 14).

HLA class II DP epitope 4003 is shared by DPB1 chains DPB1*02 : 01, DPB1*04 : 02, DPB1*10 : 01, and DPB1*18 : 01 (red bars) and defined by 84D + 85E + 86A + 87V. Negative antigens that did not share epitope 4003 are shown in gray bars (Figure 15).

4. Discussion

Cross-reactivity of antibodies with HLA antigens has been investigated for decades [4, 5, 7-9, 27]. Studies to identify HLA epitope, the target of antibodies, started more than 50 years ago [10], and numerous other studies followed since then [5, 6, 11, 28-34]. The amino acid structure of HLA antigen chains was reported for the HLA A2 in 1987 [12], and now, complete sequences of all known HLA antigen chains are readily available online [23]. Single antigens expressed on a mammalian cell line allowed us to simplify adsorption/elution assays and isolate one antibody from multispecific allosera, with multiple antibodies, and test the isolated antibody with the single antigen beads to more accurately determine antibody specificity. Isolated antibodies tested with large panels of HLA class I or class II single antigen beads were shown to be positive with certain antigens of the bead panels and negative with others. It is, therefore, reasonable to assume that the positive antigens share a public epitope which can easily be confirmed by looking at the amino acid sequences of these antigens.

HLA single antigen bead assays are simplified assigning anti-HLA antibody specificities by simply listing all antigens that are positive with the serum or antibody. Because a positive antibody-antigen reaction indicates binding of antibody to the single antigen on the bead, we postulate that the antibody must be specific to the antigen. However, the single antigen beads assay often reveals more antibody specificities than other antibody detection assays. This is clearly seen when an immunological response to a mismatched antigen produces antibody specificity to nondonor antigens and in some cases unexpectedly to rare antigens. HLA antigens share public epitopes; therefore, the extra antibody specificity of non-donor-specific and rare antigens can now be explained as antibody binding to public epitopes located on the positive antigens. Defining epitopes of HLA gives us better understanding of the breadth of non-donor-specific specificities found in sera. For example, specificity of antibodies to rare antigens like A80 and B76 were unexpectedly higher than the antigens' frequency (<0.5%) in the general population. The two antigens have 9 and 13 epitopes, respectively (Table 3).

HLA epitopes were defined using computer software by searching, in published sequences of class I and class II antigens, for exclusive amino acids at the same position(s) that are shared by all positive-reacting antigens. Amino acid sequences and the 3D structures of available HLA antigens, used to ensure that aa's are exposed on the surface of the antigens, helped in defining close to 300 epitopes. Assay-positive antigens that share epitopes, defined by exclusively shared aa's, correspond to the antibody specificities. Although it is beyond the scope of assays used in our studies to determine the exact conformational arrangement of each epitope and all amino acids that constitute the epitope, the defining amino acids must be a focal part of the epitope. Public epitopes found exclusively on positive antigens and not on negative antigens are likely not coincidences. For several epitopes defined in our studies, the difference of one aa among alleles of the same antigen, at least one amino acid position can determine whether the allele is positive or negative with the antibody (Figure 5).

We have demonstrated that some antibodies target an epitope on one single antigen (private epitope) or an epitope on a group of two or more antigens (public epitopes). Furthermore, in anti-DQ antisera, immunological responses can produce antibodies to epitopes located on either or both polymorphic chains of the DQ antigens.

The usefulness of epitopes beyond determining correct antibody specificity in sera of transplant patients has been the subject of study recently. Reports on matching donors and recipients or selecting organ donors based on epitope matching are numerous. For example, Duquesnoy reported on HLA epitope-based matching for organ transplantation [35, 36]. Wiebe reported on epitope matching to minimize de novo donor-specific antibodies to improve transplantation outcome [37] and Walton et al. reported on the usefulness of matching at the epitope level which protects against chronic lung allograft dysfunction [38].

https://doi.org/10.1155/2017/3406230

Conflicts of Interest

Nadim El-Awar has no conflict of interest regarding the publication of this paper (retired from One Lambda Inc., or now Thermo Fisher Scientific, September 11, 2014). Vadim Jucaud has no conflict of interest regarding the publication of this paper. Anh Nguyen has no conflict of interest regarding the publication of this paper.

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Nadim El-Awar, Vadim Jucaud, and Anh Nguyen Terasaki Foundation Laboratory, Los Angeles, CA, USA

Correspondence should be addressed to Nadim El-Awar; nelawar@terasakilab.org

Received 21 December 2016; Accepted 14 February 2017; Published 7 May 2017

Academic Editor: Senthami R. Selvan

Caption: Figure 2: Alloantibody adsorption/elution with recombinant single antigen cell line. The antibody is eluted with an acidic buffer, and the eluate is neutralized with TRIS buffer.

Caption: Figure 3: Top view of HLA class I heavy chain 1 and 2 domains. Rectangles show approximate binding span area of antibody.

Caption: Figure 4: Alloserum with specificity A2, A68, A69, B57, and B58. Antibody eluted from adsorption with A6901 recombinant cells has the specificity of A2, A68, and A69 (b). Antibody eluted from adsorption with B5801 recombinant cells has the specificity of A2, B57, and 58 (c).
Table 1: HLA class I epitope--partial list. Full list of 110 epitopes
in supplemental file.

Epitope                  Antigens that share epitope (a)
number

1                                     A1, 36
6                                       A3
4                              A25, 26, 34, 43, 66
7          B7, 8, 13, 18, 27, 35, 37, 38, 39, 4005, 41, 42, 44, 45, 46,
           47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 59, 60, 61, 62, 64,
                    65, 67, 71, 72, 73, 75, 76, 77, 78, 81, 82
8                                      B13
14                            Al, 23, 2402, 80, B76
16                  Al, 36, 11, 25, 26, 34, 43, 6601, 80, B73
17                                 A2, B57, 58
18                                  A2, 68, 69
40                                    Cw5, 8
222            A6602, B7, 13, 27, 47, 48, 60, 61, 73, 81, Cw2, Cwl7
223                       B7, 13, 27, 47, 48, 60, 61, 81
244                          Cw2, 4, 5, 6, 15, 17, 18
244         B35, 4005, 46, 49, 50, 51, 52, 53, 56, 57, 58, 62, 63, 71,
                            72, 75, 77, 78, Cw9, CwlO
246                   B46, 73, Cwl, 7, 8, 9, 10, 12, 14, 16

249          A*2301, A*2402, A*2403, A*2501, A*3201, B*1513, B*1516,
             B*27052, B*3701, B*3801, B*4402, B*4403, B*4701, B*4901,
             B*5101, B*5102, B*5201, B*5202, B*5301, B*5701, B*5703,
                                  B*5801, B*5901
                              B* 1301 & B* 1302 Neg.
250          A*2301, A*2402, A*2403, A*3201, B*1301, B*1302, B*1513,
             B*1516, B*27052, B*3701, B*3801, B*4402, B*4403, B*4701,
             B*4901, B*5101, B*5102, B*5201, B*5202, B*5301, B*5701,
                        B*5703, B*5801, B*5901 A2501 Neg.

Epitope    Amino acid(s) define epitope (b)    A/M
number

1                   44K/150V/158V/              M
6                        161D                   M
4            (9Y) + 149T/(74D) + 149T (b)       M
7                      65Q (c)                  M
8                   145L/41T + 46A              M
14                    166D/167G                 A
16                    [90D] (c)                 A
17                       62G                    A
18                    142T/145H                 A
40                       177K                   A
222              163E+166E/ 163E+167W          A4
223                    76E+163E                 A
244                   77N + 80K                 A
244                   163L+167W                 A
246           76 V + 80N/73T + 76V + 79R       A/A
249               82L+145R/83R+145R             M
250              82L + 90A/83R + 90A            M

Epitope    Adsorption rHLA cell line
number

1                     N/A
6                     N/A
4                     N/A
7                     N/A
8                     N/A
14                   A2402
16                   A8001
17                   B5801
18                   A6901
40                     nn
222                  Cw0202
223                  B0703
244                 Cwl 701
244             B62 (B1501)/B35
246                Cwl802/nn
249                  N/A
250                  N/A

M designates murine monoclonal antibody, A designates alloantibody,
and adsorption rHLA cell line indicates the cell line used to adsorb
then elute the antibody; aa: amino acids; nn: not needed; N-A: not
applicable; (a) serological antigens shown, alleles are shown when not
all alleles of an antigen are positive (i.e., share epitope); (b)
possible alternative epitopes are separated by "-"; plus sign "+"
indicates two or more positions-aa needed to define the epitope; amino
acids not exposed at the surface of the HLA molecule are between
parentheses; cepitope also shared by C-locus antigens (not shown here)
is between square brackets.

Table 2: Epitopes on intact antigens targeted by naturally occurring
antibodies--partial list. Complete list in supplemental file.

Epitope    Dissociated     Epitope site    Epitope
number      antigen(s)                      number

5059          A0101         158V+163R        5073
201             A2          43Q + 62G        5075
3            A23, A24          65G           5076
31           A30, 31           56R           5077
5064          A3002         17S + 76E        5078
5066          A6602            149T          5081
5068           A80             56E+          5085
406           B2705       65Q+69A + 80T      5086
236          B57, B58       43P + 62G

Epitope            Dissociated antigen(s)             Epitope site
number

5059                        B76                        163L+166D
201         Cw*0102, 0302, 0303, 0304, 1402,1802          219W
3                           Cw16                          193L
31                          Cw17                          170G
5064                        Cw7                           273S
5066                     Cw9, Cw10                     163L+173K
5068                         B8                       (67F) + 131R
406                         Cw6                     80K+90D + (114D)
236

Plus sign "+" indicates two or more positions/aa needed to define the
epitope; amino acids not exposed at the surface of the HLA molecule
are between parentheses.

Table 3: Partial list of epitopes on HLA class I antigens. Complete
list in supplemental file.

Antigen    Number of                    Epitope number
            epitopes

A1             11         1      12      13      14      15      16
A2             16         2      13      17      18      19      27
A25            19         4      12      16      23      24      27
                         247     249     423
A80            9         13      14      15      16      28      29
B13            16         7       8      21      22      24      32
B54            17         7      25      32      33      204     215
B76            13         7      14      22      25      33      43
Cw1            5         32      205     232     246     421
CW2            5         32      39      205     222     244
CW4            4         32      205     232     244
CW9            6         32      39      205     245     246     421
CW10           6         32      39      205     245     246     421

Antigen                       Epitope number

A1          208     238     241     242     248
A2          32      38      201     210     211     238     242
A25         32      38      209     211     213     214     233

A80         208     241     242     A80      9      13      14
B13         33      43      217     218     222     223     233
B54         216     224     226     228     229     232     233
B76         211     216     218     227     233     240     403
Cw1
CW2
CW4
CW9
CW10

Antigen           Epitope number

A1
A2          247     412     422
A25         238     241     243

A80         15      16      28      29
B13         235     250     418
B54         234     401     402     410
B76         B76     13       7      14
Cw1
CW2
CW4
CW9
CW10

Table 4: Cryptic (C) epitopes on dissociated class I HLA
antigen--partial list. Complete list in supplemental file.

Epitope    Dissociated         Epitope site        Epitope
number      antigen(s)                              number

5006          A3002               (152R)             5033
5007         A31, A33             (73I)              5036
5008          A3401           (63N) + (66K)          5038
5009          A3402       (63N) + (66K) + (156L)     5039
5010           A80                (31S)              5049
5027            B8                 (9D)              5052
5031           B82            (24S) + (99F)

Epitope    Dissociated    Epitope site (c)
number      antigen(s)

5006           Cw2             (211T)
5007           Cw17        (116F)+(143S)
5008           Cw6          (9D) + (97W)
5009           Cw7         (66K) + (99S)
5010          A6602        (114Q) + 163E
5027           B76          (70N) +166D
5031

(c) Amino acids and their positions on the HLA-dissociated antigens
define each epitope. In intact antigens, these amino acids are not
exposed at the surface (cryptic).

Table 5: MICA epitopes.

Epitope            MICA antigens sharing epitope
number

6001                    MICA* 001, 012, 018
6002        MICA*001, 002, 004, 007, 009, 012, 018, 027
6003                      MICA* 004, 009
6004                    MICA* 027, 004, 009
6005                         MICA* 017
6006                         MICA* 004
6007                         MICA* 027

Epitope    aa/position define epitope (a)
number

6001                   (24T)
6002                    91Q
6003                    122V
6004              (36Y)/129V/173E
6005                    91R
6006                    181R
6007                 213I/251R

Epitope    rMICA cells used for adsorption/elution
number

6001                       MICA*018
6002                          ND
6003                          ND
6004                       MICA*004
6005                          ND
6006                          ND
6007                          ND

ND: not done; amino acids not exposed on the surface of the MICA
antigen are shown between parentheses; (a) possible alternative
epitope definitions are separated by "/"; epitopes.

Table 6: Partial list of HLA class II DR epitopes defined based on aa
acid sequence of the beta chain of the antigens. Complete list in
supplemental file.

Epitope             DR antigens sharing epitope              Position/
number                                                      amino acid

1001                       DR7, DR9, DR53                       4Q
1004                         DR4, DR10                          11V
1008                            DR7                             25Q
1017                            DR11                            58E
1018       DR7, DR8, DR11, DR12, DR13, DR16, DR51, DR103        70D
1028        DR1, DR4, DR7, DR9, DR10, DR11, DR12, DR13,         77T
                DR14, DR15, DR16, DR51, DR53, DR103
1029                          DR7, DR9                          78V
1039       DR1, DR7, DR9, DR15, DR16, DR51, DR52, DR53,        140A
                               DR103
1032       DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14,         96H
                          DR17, DR18, DR52

Table 7: Fifteen HLA class II DQ[beta] epitopes and three Dq[alpha]
epitopes defined.

Epitope      DQ antigens sharing epitope      Position/amino acid (b)
number (a)

2001                     DQB2                   28S/30S/37I/52L/55L
2002                     DQB4                           56L
2003           DQB4, DQB5, DQB6, DQB7,              28T/46V/52P
                      DQB8, DQB9
2004                  DQB5, DQB6             84E/85V/86A/89G/90I/221Q
2005                     DQB7                           45E
2006               DQB7, DQB8, DQB9                     55P
2007               DQB4, DQB5, DQB6                  52P + 55R
2008         DQB2, DQB5, DQB7, DQB8, DQB9           (9Y + 11F)
2009           DQB2, DQB4, DQB5, DQB6,               34R + 45G
                      DQB8, DQB9
2010         DQB4, DQB5, DQB6, DQB8, DQB9            45G + 46V
2011                DQB5, DQB0601                   38 V + 46V
2012                  DQB8, DQB9                      45G+55P
2013         DQB2, DQB4, DQB7, DQB8, DQB9      84Q/85L/86E/87L/89T/
                                                     220H/221H
2014            DQB4, DQB7, DQB8, DQB9      77T + 84Q/77T + 85L/77T +
                                                86E/77T + 87L/182N
2015                     DQB5                   70G + 71A/116I/125S
2017                  DQA1*0201                     47K/52H/54L
2018           DQA1*04/DQA1*05/DQA1*06                40G/47C
2019                   DQA1*03                   26S/47Q/56R/187T

(a) Epitope 2008 defined using mAb; (b) possdefined by more than a
single position/aa are separated by are separated by "/"; epitopes
that are "+"; amino acids not exposed at the surface of the HLA
molecule are between parentheses.

Table 8: Number and epitopes on HLA class II DQA1 and DQB1 antigens.

Antigen      Number of                      Epitopes
              epitopes

DQA1*0201        1         2017
DQA1*03          1         2019
DQA1*04          1         2018
DQ2              4         2001     2008     2009     2013
DQ4              7         2002     2003     2007     2009     2010
DQ5              8         2003     2004     2007     2008     2009
DQ0601           6         2003     2004     2007     2009     2010
DQ7              6         2003     2005     2006     2008     2013
DQ8              8         2003     2006     2008     2009     2010
DQ9              8         2003     2006     2008     2009     2010

Antigen               Epitopes

DQA1*0201
DQA1*03
DQA1*04
DQ2
DQ4           2013     2014
DQ5           2010     2011     2015
DQ0601        2011
DQ7           2014
DQ8           2012     2013     2014
DQ9           2012     2013     2014

Table 9: HLA class II DP epitopes.

Epitope                    DP antigens sharing epitope
number

4001         DPB1*0101, DPB1*0301, DPB1*0501, DPB1*0901, DPB1*1001,
              DPB1*1101, DPB1*1301, DPB1*1401, DPB1*1701, DPB1*1901
4002                DPB1*0301, DPB1*0901, DPB1*1401, DPB1*1701
4003                DPB1*0201, DPB1*0402, DPB1*1001, DPB1*1801
4004                           DPB1*1101, DPB1*1501
4005                     DPB1*0201, DPB1*0401, DPB1*0402

Epitope     Position/amino acid (a)
number

4001         84D + 85E + 86A + -87V

4002            55D + 56E + -57D
4003            55D + 56E + -57E
4004                 (33Q)
4005         84G + 85G + 86P + 87M

(a) Epitopes defined by more than a single position/aa are separated
by "+"; amino acids not exposed at the surface of the HLA molecule are
between parentheses.

Figure 1: Main empirical testing steps to define HLA epitopes.

Main steps for epitope definition

(1) Antibody preparation and testing with single antigen beads

Monoclonal antibody or alloantibody isolated from sera by
adsorption--to then elution--from a single antigen recombinant cell
line (Figure 2).

In this case, the results in the figure show that A2, 68, and 69
beads/antigen are positive while the rest (90 beads, not all shown)
are negative--an indication that the antibody is targeting a unique
epitope on the positive antigens.

(2) Exclusively unique aa's at certain positions on positive
antigens

Computer software search in aa sequences of all 96 alleles shows
all 6 positive alleles share the aa's T and H at positions 142 and
145, respectively--the negative antigens/alleles (not all shown)
have different aa's at these positions.

(3) Epitope definition. Use of 3D HLA aa molecular structure
software to determine the following:

(i) Amino acids at the exclusively shared positions of the positive
beads are exposed to the surface of the antigens for antibody to
bind.

(ii) When more than one aa defines an epitope, the distance between
any two aa's is within the binding span of the antibody (Figure 3).

The empirical determination that one antibody reacts positively
with some single antigens and not with others, the positive
antigens that exclusively share aa's at certain sequence positions,
and the exclusive aa's that are at the surface of the antigens and
are within the binding span of the antibody allow for epitope
definition.

Amino acid sequences of
HLA class I alleles

                    Amino acid position

         138      142      143     144    145    147

A02       M        T        T       K      H      W
A02       M        T        T       K      H      W
A02       M        T        T       K      H      W
A68       M        T        T       K      H      W
A68       M        T        T       K      H      W
A69       M        T        T       K      H      W
B07       T        I        T       Q      R      W
B08       T        I        T       Q      R      W
B37       T        I        T       Q      R      W

Figure 5: Epitope 422 shared by A-locus antigens A2, A3, A11, A24,
A68, and A69 defined by the aa acid combination 149A + 150A + 151H.
One amino acid substitution at position 149 (aa T substituted for aa
a) could be the reason that A2 allele A* 0203 is negative while
alleles HLA A * 0201 and A * 0206 are positive.

                                  aa position

Antigen      Allele       149        150        151

A1           A*0101       A          V          H
A68          A*6802       A          A          H
A2           A*0206       A          A          H
A69          A*6901       A          A          H
A2           A*0201       A          A          H
A68          A*6801       A          A          H
A3           A*0301       A          A          H

A1           A*0101       A          V          H
A24          A*2402       A          A          H
A11          A*1102       A          A          H
A11          A*1101       A          A          H
A24          A*2403       A          A          H
A2           A*0203       T          A          H

Figure 6: Epitope 21 shared by the B-locus antigens B13, B4005, B41,
B44, B45, B47, B49, B50, B60, and B61 and defined by 41T.

Antigen                    Allele B0702        aa & position 41 A

B13                           B1301                    T
B4005                         B4005                    T
B41                           B4101                    T
B41                           B4102                    T
B44                           B4402                    T
B44                           B4403                    T
B45                           B4501                    T
B47                           B4701                    T
B49                           B4901                    T
B50                           B5001                    T
B60                           B4001                    T
B61                           B4002                    T
B48                           B4801                    A
B81                           B8101                    A

Figure 7: Epitope 40 shared by the C-locus antigens Cw*0801 and
Cw*0501 and defined by 177K.

                            aa & positions

                     173          177          178

A*01011               E            E            T
B*07021               E            D            K
Cw*010201             E            E            T
Cw*080101             E            K            T
Cw*050101             E            K            T
B*27052               E            E            T
B*2708                E            E            T

Figure 8: Epitope 38 shared by the ABC-loci antigens A2, A25, A26,
A29, A31, A32, A33, A34, A43, A66, A68, A69, A74, B73, Cw7, and Cw17
and defined by the amino acid glutamine (Q) at position 253.

              aa and                         aa and
             position                       position
                253                            253

A*0101           E            A*0101            E
B*0702           E            B*0702            E
Cw*0102          E            Cw*0102           E
A*3303           Q            A*0203            Q
A*6801           Q            A*2601            Q
Cw*0702          Q            A*3101            Q
Cw* 1701         Q            A*3201            Q
A* 7401          Q            A*0201            Q
A*3301           Q            A*6601            Q
A*6901           Q            A*4301            Q
A*6802           Q            A*6602            Q
A*3401           Q            A*0206            Q
B*2901           Q            B*7301            Q
A*2902           Q            A*3001            E
B*2501           Q            B*4403            E

Figure 9: Epitope 5007 shared by the HLA class I A-locus antigens A31
and A33 and defined by isolucine (I) at position 73. The epitope is
accessible on the dissociated antigens and show stronger reactivity
when the peptide has been dissociated from the heavy chain. Position
73 is not exposed in an intact HLA class I antigen. After acid buffer
treatment and neutralization of the eluate, epitope 5007 becomes
exposed and reacts with the antibody 10-fold.

                         aa & position

                   71           73           74

A*01011            S            T            D
A*31012            S            I            D
A*3301             S            I            D
A*3303             S            I            D
B*5801             A            T            Y

Figure 10: Epitope 6002 shared by MICA antigens MICA*001,002, 004,
007, 009, 012, 018, and 027 and defined by glutamine (Q) at position
91.

MICA
                    aa position
Antigen                 91

MICA*001                 Q
MICA*002                 Q
MICA*004                 Q
MICA*007                 Q
MICA*009                 Q
MICA*012                 Q
MICA*018                 Q
MICA*027                 Q
MICA*017                 R

Figure 11: Epitope 1028 shared by class II DR antigens DR1, DR4,
DR7, DR9, DR10, DR11, DR12, DR13, DR14, DR15, DR16, DR51,
DR53, and DR103 and defined by threonine (T) at position 77.

Antigen                     aa pos. 77

DR1                             T
DR103                           T
DR10                            T
DR11                            T
DR12                            T
DR15(2)                         T
DR16(2)                         T
DR4                             T
DR4                             T
DR13(6)                         T
DR14(6)                         T
DR7                             T
DR8                             T
DR9                             T
DR18(3)                         N
DR17(3)                         N

Figure 12: Unabsorbed serum has antibodies with specificity to DR, DQ,
and DP antigens (green bars). After adsorbing the serum with rHLA DQ
cells, the eluted antibody shows specificity to DQ antigens only (red
bars).

HLA DQ allele    Position/amino acid

                     52           55

DQB1*05:01           P            R
DQB1*04:01           P            R
DQB1*04:02           P            R
DQB1*05:01           P            R
DQB1*05:02           P            R
DQB1*06:01           P            R
DQB1*06:02           P            R
DQB1*06:03           P            R
DQB1*05:01           P            R
DQB1*06:04           P            R
DQB1*06:09           P            R
DQB1*02:01           L            L
DQB1*02:02           L            L
DQB1*03:01           P            P
DQB1*03:02           P            P
DQB1*03:03           P            P

Figure 13: Several DQ antigens (heterodimers) with the DQA1*02 : 01
chain are shown below. They all share epitope 2017 which is defined by
histidine (H) in position 52 of DQA1*02 : 01 chain. Eluted antibodies
from relevant DQ antigens are positive (red and green bars). Eluted
antibodies from irrelevant (no DQA1*02 : 01) are negative (yellow and
blue bars).

HLA DQ allele       Position/       HLA DQ allele       Position/
                  amino acid 52                       amino acid 52

DQA1*01:01              S            DQA1*01:01             S
DQA1*02:01              H            DQA1*03:03             R
DQA1*01:01              S            DQA1*04:01             R
DQA1*01:02              S            DQA1*05:01             R
DQA1*01:03              S            DQA1*05:03             R
DQA1*03:01              R            DQA1*05:05             R
DQA1*03:02              R            DQA1*06:01             R

Figure 14: HLA class II DP epitope 4001 shared by DPB chains
DPB1*0101, DPB1*0301, DPB1*0501, DPB1*0901, DPB1*1001, DPB1*1101,
DPB1*1301, DPB1*1401, DPB1*1701, and DPB1*1901 (red bars) and defined
by 84D + 85E + 86A + 87V. Negative antigens that did not share epitope
4001 are shown in (gray bars).

Allele                        Amino acids and position

                     84           85           86           87

DPB1*01:01           D            E            A            V
DPB1*01:01           D            E            A            V
DPB1*03:01           D            E            A            V
DPB1*05:01           D            E            A            V
DPB1*09:01           D            E            A            V
DPB1*10:01           D            E            A            V
DPB1*11:01           D            E            A            V
DPB1*13:01           D            E            A            V
DPB1*14:01           D            E            A            V
DPB1*17:01           D            E            A            V
DPB1*19:01           D            E            A            V
DPB1*02:01           G            G            P            M
DPB1*04:01           G            G            P            M
DPB1*04:02           G            G            P            M

Figure 15: HLA class II DP epitope 4003 shared by DPB chains
DPB1*0201, DPB1*0402, DPB1*1001, and DPB1*1801 (red bars) and
defined by 84D + 85E + 86A + 87V. Negative antigens that did not share
epitope 4003 are shown in (gray bars).

Allele                          Amino acids and position

                   53       54       55       56       57       58

DPB1*01:01         R        P        A        A        E        Y
DPB1*02:01         R        P        D        E        E        Y
DPB1*04:02         R        P        D        E        E        Y
DPB1*10:01         R        P        D        E        E        Y
DPB1*18:01         R        P        D        E        E        Y
DPB1*01:01         R        P        A        A        P        Y
DPB1*03:01         R        P        D        E        D        Y
DPB1*04:01         R        P        A        A        E        Y
DPB1*05:01         R        P        E        A        E        Y
DPB1*09:01         R        P        D        E        D        Y
DPB1*11:01         R        P        A        A        E        Y
DPB1*13:01         R        P        A        A        E        Y
DPB1*14:01         R        P        D        E        D        Y
DPB1*17:01         R        P        D        E        D        Y
DPB1*19:01         R        P        E        A        E        Y
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Title Annotation:Human leukocyte antigens
Author:Awar, Nadim El-; Jucaud, Vadim; Nguyen, Anh
Publication:Journal of Immunology Research
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2017
Words:8123
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