Recently we identified 58 epitopes shared among A and B human leukocyte antigen (HLA) class I antigens by monoclonal antibodies (mAbs) or allo antibodies that were absorbed and eluted from HLA recombinant single antigen cell lines (rHLA) (1). Testing with a panel of single HLA antigen beads (2) was crucial in the identification of the epitopes. With the recent development of single antigen C locus beads, it has now become possible to expand investigations into the epitopes for the C locus. Of special interest is that the C locus epitopes are often shared with the A and B locus. In fact, two of the previously described AB locus epitopes have now been determined to also contain C locus specificities.
As noted earlier (1), the epitopes explain the previously observed serologic cross-reactivity often described as cross-reactive groups (CREGs) (3, 4). The epitopes, which are based on the amino acid structure of the HLA molecule, underlie the observed cross-reactions of antibodies. They explain why immunization to a single antigen can result in antibodies against a series of antigens. These antibodies, often described as nondonor specific antibodies (NDSA), can now be understood as reactions to HLA antigens sharing the same epitope. The importance of identifying these epitopes is that, according to the humoral theory of transplantation (5, 6), they are the “transplantation antigens” responsible for antibody- mediated transplant rejection.
MATERIALS AND METHODS
Monoclonal Antibodies and Allosera
Twenty-two mouse mAbs, supernatants or aliquots of ascites, diluted 1:10 to 1:20,000 were used in this study. Because most of the mAbs were not purified, protein concentrations of the final dilutions were not determined. Their specificity has been previously characterized by both serological and immunobinding assays. Because mAbs by definition are monospecific, adsorption experiments were not generally conducted.
Twenty-one anti-HLA alloantibody samples were obtained from multiparous women, placentas, or patients undergoing platelet transfusions or transplants. Sera were adsorbed by an appropriate rHLA SA cell line derived from the LCL712.2 B host cell line. The rHLA single antigen cell line used for adsorption of the antibody was selected based on the known serological specificity of each sample. Forty microliters of serum (diluted 1:3) was mixed with 3–5×106 cells and subsequently incubated for 30 min at room temperature (RT). The cells were then centrifuged to remove the adsorbed sera for testing. Some sera were adsorbed using a purified rHLA single antigen (SA) attached to microsphere beads. 0.5×106 beads were used per adsorption and the antibody was eluted as described below.
After adsorbing the allosera with the cell lines or beads, the cells/beads were washed with phosphate-buffered saline. The adsorbed antibody was then eluted by mixing 60 μl of ImmunoPure IgG Elution Buffer (Pierce, Rockford, IL; catalog 21004) with the cells or beads, and then incubating for 10 min at room temperature. After incubation, the eluates were separated by centrifugation, removed, and neutralized by 3 μl of 1 M TRIS-HCl pH 9.5. Most eluates were from an initial adsorption/elution step. However, in some cases the initial eluate was adsorbed by another recombinant cell line and the second eluate was tested with the single-antigen beads.
Single Antigen Beads Assays
Monoclonal antibodies, or antibody eluates were tested with 95 HLA class I (A, B, and C-locus) rHLA single antigens individually coupled to different microsphere beads, and with negative and positive control beads (LABScreen beads: LS1A01, LS1A02 and LS1A03, One Lambda Inc. Canoga Park, CA) (2). The HLA alleles represented in the SA bead panel are listed in Table 1. LABScreen assays were performed according to the manufacturer's protocol.
Data generated from the LABScan 100 were analyzed using computer software. Trimmed mean fluorescence values for the SA beads reactions were obtained from the output (.csv) file generated by the flow analyzer and were adjusted for background signal using the formula ([sample #N bead– sample negative control bead]–[negative control #N bead– negative control negative control bead]). The adjusted reaction values were then normalized by multiplying each value by a corresponding normalization factor derived from the results of the mAb W6/32 with the same beads. Normalization factors were calculated by dividing the average value of all mAb W6/32 reactions by the adjusted fluorescence value for each bead. The data were then graphed using either Excel spread sheets or HLA Visual software (One Lambda Inc. Canoga Park, CA). All normalized reactions that were above zero were considered as potential positive reactions for this study, but values for the epitope-specific reactions were rarely below 400.
HLA Amino Acid Sequences, Epitopes, and Distances Between Residues
Amino acid sequences of the HLA antigens or alleles were downloaded from the Anthony Nolan internet website (7). Based on the data analysis mentioned above, we determined the positive antigens for each mAb or eluted alloantibody. An epitope search program was then utilized to identify distinguishing amino acids (aa) that are exclusively shared by the positive antigens at particular sequence positions. The program searched for one, two, three, or four common unique aa positions. Among the many possibilities generated, we selected for consideration the positions that are exposed to the surface of the molecule and that are within the antibody binding span estimated at 494Å2 (19×26Å) (8) or 750Å2 (9). Approximate distances in angstroms between two amino acids were calculated using the Cn3D Viewer software (10) and the three-dimensional structure of an HLA-A0201 molecule 1QEW (11). Amino acids that were exclusively unique to a group of antigens that were reactive with a mAb or an eluted alloantibody, preferably exposed to the surface of the molecule, and within the binding span of the antibody were considered a distinguishing characteristic of the epitope. Epitope ID numbers were assigned depending upon the number of unique aa sites involved (1–200 for one aa, 201–400 for two aa, or 401–600 for three or four aa positions).
A total of 49 HLA class I epitopes were recognized by the series of monoclonal antibodies and eluted alloantibodies used in this study. Four epitopes from our previous study (1), where only A and B loci antigens were analyzed, are now redefined (epitope 10 and 11) or the C-locus antigens are included (epitopes 205 and 222). For example, epitope 10 (Table 2) was earlier defined by the aa arginine (R) at position 69. However, when aa sequences for the C-locus were included in the analysis, all C-locus antigens were found to have the aa arginine at this position; therefore 69(R) is not exclusively unique to the B46 antigen. However, the amino acids alanine (A) and lysine (K) at positions 46 and 66 respectively are uniquely exclusive to B46 and therefore they identify the epitope. Epitope 222 (Table 3) is now shown to be shared by the C-locus antigens (Cw2 and Cw17) in addition to A6602, B7, B13, B27, B47, B48, B60, B61, B73, and B81 antigens.
Overall, we found 45 new epitopes which include 11 shared by A locus antigens, 17 shared by B locus antigens, 4 shared by C locus, and 13 interlocus epitopes of which 6 shared by A and B, 5 shared by B and C, and 2 shared by A, B, and C antigens.
The mAbs selected for this study recognized epitopes with one to four unique aa at certain position(s) (Tables 2 and 3). The combination aa positions were not contiguous but were within a conformational distance allowing antibody binding. The interlocus epitope 238 (Table 2) is an example of an epitope defined by two uniquely shared aa positions. The reactive HLA antigens A1, A2, A3, A11, A25, A26, A29, A32, A33, A34, A36, A43, A66, A68, A69, A74, B57, B58, and B63 were all found to have glycine (G) at position 56 and arginine (R) at position 65. Although several other HLA class I antigens have 56(G) and 65(R), none of these antigens share both amino acids at these two positions, making the aa combination unique for this mAb-reactive group of HLA antigens.
For the allosera, eluates recovered from the rHLA SA cell lines were found to recognize epitopes consisting of unique aa residues at 1, 2, 3, or 4 sequence positions (Tables 2 and 3). Epitope 241 in Table 2 is an example of a two aa epitope that was defined by serum AS264 adsorbed with a recombinant cell line expressing only the HLA-A3601 antigen. The eluted antibody reacted with A1, A11, A25, A26, A34, A36, A43, A6601, and A80 antigens. All nine antigens share one epitope defined by any of the three two-aa combinations listed. For example, the combination of the two amino acids arginine (R) at position 65 and aspartic acid (D) at position 90, located in the alpha-1 domain of the HLA alpha molecule, can define this epitope.
Absorptions and elutions were not needed (nn) for most of the allosera listed in Table 2. However, most allosera that defined epitopes present on C-locus antigens were absorbed by rHLA single antigen cell lines and the eluted antibody was tested with the A, B, and C SA beads (Table 3).
The actual fluorescence data for several of the immuno-binding reactions are presented graphically (Figs. 1–3) for the positive (epitope-specific) reactions for three allosera. Only the highest negative reactions are included to show the marked drop in signal from the epitope positive antigens; the other SA beads had little or no fluorescent signal. The figures also depict the relevant aa residues at the pivotal antibody-binding positions, as defined by their unique sequence composition for the reactive HLA alleles on the SA beads.
Figure 1 shows the results of the first eluate, from the B3501 rHLA cell line, illustrating an epitope that is shared by three C and 20 B locus antigens. Cw9, Cw0302 (Cw10), and Cw0304 (Cw10), B4005, B46, B63, B53, B49, B75, B35, B62, B77, B50, B56, B71, B5703, B52, B5701, B78, B72, B5102, B58, and B5101 all share the amino acids leucine (L) and tryptophan (W) at positions 163 and 167, respectively. Combined, these amino acids, which are exclusive to these antigens at the two positions, define the epitope (no. 245, Table 3).
Figure 2 illustrates the SA beads reactions to the B46, B73, and Cw0302 (Cw10), Cw1601, Cw0702, Cw0801, Cw0303 (Cw9), Cw0304 (Cw10), Cw1402, Cw0102, and Cw1203 antigens that share the aa valine (V) at position 76 and asparagine (N) at position 80. Both amino acids are located in the alpha 1 domain of the HLA antigen and combined define the epitope (no. 246, Table 3).
Figure 3 shows an example of an epitope shared by A, B, and C class I antigens. Epitope 38 is shared by 19 A locus, one B locus, and two C locus antigens. Antigens A3303, A6801, Cw0702, Cw1701, A7401, A3301, A6901, A6802, A3401, A2901, A2902, A2501, A0203, A2601, A3101, A3201, A0201, A6601, A4301, A6602, A0206, and B7301 all share the aa glutamine (Q) at position 253. Position 253 is located in close proximity of the cellular membrane on the alpha chain of the HLA antigen. Although the aa is exposed to the surface of the molecule, it is uncertain that it is accessible for antibody binding due to its location. However, glutamine is the only aa we found that is exclusively common to all positive antigens, listed above, and may therefore play a role in defining this epitope (no. 38, Table 3).
In this study, we used recombinant single antigens to determine the specificities of every serum and monoclonal antibody tested. All recombinant single antigens were developed in a mammalian cell expression system of HLA transfected cells. This system provides all of the crucial elements, including posttranslational modification, to produce mature glycosylated HLA antigens that are indistinguishable from native HLA class I antigens (2).
We have described earlier (1) the unique ability of class-I A and B SA beads to identify 58 HLA class I epitopes recognized by mAbs and alloantibodies. Here we add 34 more epitopes shared by A, B, or A and B antigens (Tables 1 and 2). Thus, in combination with the previously published table, there are now 92 A and B locus epitopes. We estimate that these epitopes will be the common ones encountered in the American population. The total permutations of amino acid combinations which can make up epitopes are immense, but in practice, we can hope that the eventual number that will function as immunogenic epitopes will be rather limited.
We concentrated here on finding the epitopes of the C-locus antigens. We describe 13 epitopes which were either found exclusively on C locus or shared with other antigens. Two epitopes which we had described earlier as AB epitopes when tested with new C locus single antigen beads were found here to also contain C locus antigens. Epitopes 205 and 222 (Table 3) identified by a mAb and an alloantibody eluted from Cw0202 rHLA SA cell line respectively, when retested with A, B, and C loci SA beads; all C locus antigens sharing the same amino acids with the A and B antigens were also positive.
For most A, B, and C antigens, the epitope is often located in the alpha 1 and alpha 2 domains on the alpha chain of the HLA antigen. We note here that three epitopes (37, 38, 41) are identified by a single amino acid at positions located in the alpha 3 domain, and in the case of epitope 38 (253Q) the amino acid is in close proximity to the cell membrane. It is therefore uncertain, at least for epitope 38, whether it is accessible to bind the antibody. However, it is the only amino acid that is unique to the group of antigens sharing epitope 38, and the role that 253Q may play in defining the epitope can not be determined in this study. Several epitopes were observed more frequently than others epitopes among the list of 13 C locus defined. In particular, epitopes 38, 222, and 244 were found in several other sera.
The cell surface expression of the C locus antigens has been estimated at 10% that of the A and B antigens (12). Payne et al. studied the reactivity of the B46 antigen with alloantisera specific to the Cw1 and Cw3 HLA antigens (13). The molecular basis of reactivity of anti Cw1 and anti Cw3 alloantisera with the HLA-B46 haplotypes was reported by Zemmour et al. (12, 14). We note here that B46, Cw1, and Cw3 also share epitopes 32, 246, and 421 with other antigens (Table 3). For example, epitope 246 is shared by Cw7, Cw8, Cw12, Cw14, Cw16, and B73 in addition to B46, Cw1, Cw9 (Cw3), and Cw10 (Cw3). The expansion to all of the antigens sharing one epitope was made possible by the use of the SA beads.
Most of the alloantibodies in this study are from pregnancies. However, sera from transplant patients, recently tested, were also found to have C-locus specificities (data not shown). The epitopes identified by the antibodies were probably the targets of antibody attack (6). According to the humoral theory (5, 6), these epitopes can also be the key factors that need to be matched for transplants as was noted earlier (15). Matching for the C locus antigens, as advocated for bone marrow transplants, may be indicated for organ transplants. The relative strengths of the epitopes as immunogenicity will be of importance in selecting mismatches for unrelated bone marrow transplants when a complete match cannot be found.
Duquesnoy has proposed triplet epitopes taking three amino acids around variable positions on the HLA molecule (16) and recently proposed “patches” of amino acids within 3.0 to 3.5 angstroms of a variable position where one or two patches (epilets) identify epitopes that are conformational in nature (17). In contrast to these theoretic epitopes, the epitopes described here are based on actual antibody reactions identified by SA beads. In many instances, several antibodies were found which produced identical reactions. Hopefully, others will also find antibodies that react to the exact epitopes described here.
Epitopes identified here explain many of the complex antibodies previously found in the sera of multiparous women, multitransfused patients, and patients who had rejected an organ transplant. We have previously called attention to the difficulty in finding all the antibodies in a serum by conventional methods, and the need for testing with SA technology (18). The complex series of antibodies identified by the SA beads can be best understood when they are defined by the epitopes actually recognized. Screening for the presence of donor-specific antibodies in solid organ transplant patients often reveals that even a single “antigen” mismatch can result in the production of antibodies to many other “antigens.” This phenomenon can now be understood when considering the epitope that was mismatched and the corresponding immune response to the epitope. Thus, rather than describing the series of antigens reacting with an antibody, we propose to assign the antibody specificity to the target epitope(s).
We thank Dr. Daniel Cook for helpful suggestions and Mike Chen, Mamie Lias, and Nori Sasaki for their excellent technical assistance.
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