Dynamics and epitope specificity of anti-human leukocyte antibodies following renal allograft nephrectomy.

BACKGROUND: A considerable proportion of patients awaiting kidney transplantation is immunized by previous transplantation(s). We investigated how allograft nephrectomy (Nx) and withdrawal of maintenance immunosuppression (WD-MIS) in patients with a failed renal allograft contribute to allosensitization. METHODS: HLA antibodies (HLAabs) were analyzed before and after Nx and/or WD-MIS using a single antigen bead assay. Patients were grouped as follows: (A) Nx and concomitant WD-MIS (n = 28), (B) Nx (n = 14) and (C) WD-MIS (n = 12). In a subgroup of patients, the epitope specificity of HLAabs was determined by adsorption and elution of sera with recombinant single HLA allele-expressing cell lines. RESULTS: Following Nx and/or WD-MIS, HLAabs were detectable in 100, 100 and 92% of patients in Groups A, B and C, respectively. In patients of all groups, de novo donor-specific HLAabs (DSAs) were found. After Nx, an increase in the breadth [percent panel reactive antibody (%PRA)] and mean fluorescence intensity of class I HLAabs was predominant. In contrast, an increase of class II HLAabs prevailed following WD-MIS. Experimental analysis of the epitope specificities revealed that 64% of the class I HLAabs classically denoted as non-DSA were donor epitope-specific HLAabs (DESA). CONCLUSIONS: Both Nx and WD-MIS contribute to alloimmunization with differing patterns concerning class I and II HLAabs. Nx preferentially increased class I HLAabs and most of the observed class I HLAabs were DESA. Considering that class I, but not class II, HLA molecules are constitutively expressed, our results support the hypothesis that the increase of HLAabs following Nx might have been caused by removal of the adsorbing donor tissue (sponge hypothesis).

Antibodies to HLA-E in nonalloimmunized males: pattern of HLA-Ia reactivity of anti-HLA-E-positive sera.

Natural anti-HLA Abs found in sera of healthy nonalloimmunized males recognize HLA-Ia alleles parallel to those recognized by anti-HLA-E mAbs (MEM-E/02/06/07). Therefore, some of the HLA-Ia Abs seen in healthy males could be due to anti-HLA-E Abs cross-reacting with HLA-Ia. If anti-HLA-E Abs occur in healthy nonalloimmunized males, it can be assessed whether they evoke HLA-Ia reactivity as do mouse HLA-E mAbs. IgG and IgM Abs to HLA-E and HLA-Ia alleles are identified in sera of healthy males using microbeads coated with recombinant denatured HLA-E or a panel of rHLA-Ia alleles. The pattern of allelic recognition is comparable to that of anti-HLA-E mAbs. Sixty-six percent of the sera with HLA-E IgG have a high level of HLA-Ia IgG, whereas 70% of those with no anti-HLA-E Abs have no HLA-Ia Abs. HLA-E IgM/IgG ratios of sera are divided into four groups: IgM(Low)/IgG(Low), IgM(High)/IgG(Low), IgM(High)/IgG(High), and IgM(Low)/IgG(High). These groups correspond to anti-HLA-Ia IgM/IgG ratio groups. When HLA-E IgM and IgG are absent or present in males, the IgM or IgG of HLA-Ia are similarly absent or present. The mean fluorescent intensity of HLA-Ia Abs correlates with that of anti-HLA-E Abs. Most importantly, HLA-E and HLA-Ia reactivities of the sera are inhibited by the shared, but cryptic, peptide sequences (117)AYDGKDY(123) and (137)DTAAQIS(143). Therefore, Abs to the H chain of HLA-E may be responsible for some of the HLA-Ia allele reactivity of the natural HLA-Ia Ab in human sera. Absence of any anti-HLA-Ia Abs in 112 nonvegans and the presence of the same in vegans suggest that dietary meat proteins might not have induced the natural allo-HLA Abs.

HLA epitopes - Empirically defined as conformational amino acids sequences of the HLA antigen and are likely to be part of the binding sites of anti-HLA antibodies.

Antibodies against HLA antigens are ubiquitous in the sera of transplant patients. Analysis of anti-HLA antibodies specificity has gone through a long history of development using assays like agglutination and lymphocytotoxicity, which utilize lymphocytes, and flow cytometry, which utilize multiplex beads coupled with single antigens. Hundreds of HLA antigens are identified to date, and the realization that antibody reactivity against the antigens is multispecific presented difficulties in accurately defining antibody specificity. Although Cross Reacting Groups (CREG), describing cross reactivity among HLA antigens, were helpful with determining specificity, they proved to be inadequate for the highly sensitized patients. Amino acid sequencing and three-dimensional modeling of the HLA molecules significantly advanced our understating of the HLA antigens and their epitopes. Although sensitive assays for antibody testing advanced analysis, they unmasked additional specificities undetectable by traditional methods, and the presence of naturally occurring anti-HLA antibodies in sera further complicated analysis and underscored the need to understand antibody reactivity and their epitopes. Hundreds of HLA class-I and class-II epitopes were defined by the Tekasaki and Duquesnoy groups and their usefulness in organ transplants were further advanced by a great number of transplant centers. Alloantibody specificities, CREGs, and nondonor specific antigens (NDSA) are now explained by public epitopes.

Human leukocyte antigen class II DQ alpha and beta epitopes identified from sera of kidney allograft recipients.

BACKGROUND: Epitopes are the sites to which antibodies bind. Both alpha and beta peptide chains of the human leukocyte antigen-DQ heterodimers (DQA1 and DQB1, respectively) contain polymorphic regions. We can identify DQA1 and DQB1 epitopes by DQ single antigen beads assay of the antibodies, correlating the beads' reaction patterns with either DQA1 or DQB1 alleles. METHODS: Sera from 74 transplant patients and 35 mouse DQB1 monoclonal antibodies were tested with DQ single antigen beads for their DQ allelic and serological specificities. Epitopes were defined by amino acids shared by the positive antigens of the antibodies. Unique amino acids were identified as potential epitope sites by comparing the peptide sequences of all human leukocyte antigen class II alleles. For the absorption or elution, patient's serum sample was absorbed by a homozygous B-lymphoblast cell line of specific DQ typing, the eluted antibody then tested with single antigen beads to demonstrate that the antibody reacted to a single epitope shared by multiple DQ antigens. RESULTS: Three DQA1 and 15 DQB1 epitopes were identified. We found that 21 patients produced antibodies against one of the DQA1 epitopes; 27 patients produced antibodies against one of the DQB1 epitopes. CONCLUSION: The DQA1 and DQB1 epitopes identified here seem to be immunogenic and to elicit DQ antibodies. For the DQB1 epitopes, multiple DQ serological specificities that were detected in the serum of a transplant patient could be explained as a single donor-specific DQ antibody reacting to a mismatched DQ epitope of the donor. Ten examples are shown here.

Human leukocyte antigen class I epitopes: update to 103 total epitopes, including the C locus.

BACKGROUND: Epitopes of human leukocyte antigen (HLA) are the sites to which the antibodies bind. We identify here 103 HLA class I epitopes shared by groups of class I antigens. In particular, our emphasis was on identifying epitopes exclusive to the C-locus antigens or interlocus epitopes among A, B, and C antigens. The use of monoclonal antibodies or alloantibodies eluted from HLA recombinant single antigen cell lines tested with a panel of single antigen beads have proved very useful in the identification of the epitopes. METHODS: Alloantibodies absorbed onto then eluted from HLA single antigen cell lines and monoclonal antibodies were tested with a panel of 95 A-, B-, and C- single antigen beads and the HLA specificities determined. Each epitope was defined by amino acids shared exclusively by the positive antigens for each antibody. RESULTS: In addition to the 58 A and B class I epitopes identified in an earlier study, we add 45 more new A, B, C epitopes including, for the first time, epitopes found on C locus antigens. CONCLUSION: Beads bearing single antigens tested with monoclonal or eluted alloantibodies proved very powerful in identifying epitopes shared among HLA antigens. These epitopes are the targets of the antibodies. Antibody specificities to non-donor-specific antigens, often found in sera of transplant patients, can now be understood as reactions to epitopes shared with the donor specific antigens. The importance of identifying these epitopes is that they may be the "transplantation antigens" responsible for antibody-mediated transplant rejection.

Analysis of HLA class I specific antibodies in patients with failed allografts.

BACKGROUND: The goals of this study are first to determine the epitope specificity of donor specific antibody (DSA) in the serum of alloimmunized transplant patients with a failed renal graft; and second to understand the correlation between the development of DSA and nondonor specific antibody (NDSA). METHODS: The sera of 35 pretransplant panel reactive antibody (PRA)-negative patients with failed allografts were examined with single-antigen (SA) luminex beads to identify human leukocyte antigen (HLA)-A and -B antibodies. Potential HLA antibody epitopes were identified by using computer software and verified by absorption and elution from single-antigen cell lines. RESULTS: Twenty-seven patients developed donor-specific HLA-A and/or -B antibodies, while the remaining eight patients had only nondonor-specific HLA-A and/or -B antibodies. The DSA-positive patients also had a long list of NDSA. Sixty-eight percent of the reactions found in 27 recipients with DSA were attributable to 66 epitopes on the mismatched donor HLA molecule. All 39 NDSA in eight patients with only NDSA shared 17 epitopes within positive allele specificities. By absorption and elution using recombinant cell lines having a single HLA specificity, we confirmed the epitopes involved in three patients. CONCLUSION: Development of most NDSA in patients with failed allografts is likely due to sharing epitopes with DSA and/or other NDSA.

HLA class I epitopes: recognition of binding sites by mAbs or eluted alloantibody confirmed with single recombinant antigens.

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.

HLA Epitopes: The Targets of Monoclonal and Alloantibodies Defined.

Sensitization to human leukocyte antigens (HLA) in organ transplant patients causes graft rejection, according to the humoral theory of transplantation. Sensitization is almost ubiquitous as anti-HLA antibodies are found in almost all sera of transplant recipients. Advances in testing assays and amino acid sequencing of HLA along with computer software contributed further to the understanding of antibody-antigen reactivity. It is commonly understood that antibodies bind to HLA antigens. With current knowledge of epitopes, it is more accurate to describe that antibodies bind to their target epitopes on the surface of HLA molecular chains. Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope). 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. Since 2006, we defined and reported 194 HLA class I unique epitopes, including 56 cryptic epitopes on dissociated HLA class I heavy chains, 83 HLA class II epitopes, 60 epitopes on HLA-DRB1, 15 epitopes on HLA-DQB1, 3 epitopes on HLA-DQA1, 5 epitopes on HLA-DPB1, and 7 MICA epitopes. In this paper, we provide a summary of our findings.

Detection of antibodies to HLA-DP in renal transplant recipients using single antigen beads.

With the development of single DP-antigen beads, antibodies to DP could clearly be segregated from other HLA antibodies. We studied 323 sera of patients from four different centers with functioning or rejected kidney grafts. DP antibodies were found in 5.1% of 138 patients with functioning grafts, and 19.5 % of 185 patients with rejected grafts (P<0.001)). 42.9% and 63.9% of the DP antibody positive patients had DR/DQ antibodies among the functioning and rejected group. Among patients who did not have Class I and DR/DQ antibodies, 13% of those who rejected a graft had DP antibodies, compared to 3.5% of patients with functioning grafts (P<0.05). DP 0301 had the highest specificity frequency in the rejected group. In conclusion, HLA-DP antibodies were detected at a higher frequency in patients who have rejected their grafts than those with functioning grafts. For regrafts, DP tissue typing is recommended to avoid presensitized DP alleles.

HLA class II DQA and DQB epitopes: recognition of the likely binding sites of HLA-DQ alloantibodies eluted from recombinant HLA-DQ single antigen cell lines.

Donor-specific antibodies (DSA) in sera of sensitized transplant patients are often produced against the specific epitopes on mismatched HLA antigens. In this study, we selected sera from 30 kidney transplant patients with DSA and AMR to define DQ epitopes. Using adsorption and elution assays, we identified 18 antibody reaction patterns to define 6 new epitopes and to confirm 12 previously defined epitopes. In one patient case, one mismatched antigen produced 3 different antibodies and, in another, antibodies were produced against the alpha and beta chains of the same antigen. For some sera, a single epitope can explain reactions for 27 of the 29 DQ beads in the single antigen panel. Several studies highlighted the prevalence of anti-DQ antibodies. In 2011, Almeshari et al. observed DQ DSA in 34/46 (74%) of rejection episodes - 44 patients had DSA and 20 lost their graft due to AMR. Other studies have shown a high prevalence of anti-DQ antibodies and an association with adverse effects on the graft. We conclude that analysis of the epitopes of the DQ antibodies using Adsorption/Elution and testing on single antigen DQ beads helps to better understand the specificities and cross-reactions of DQ antibodies in transplant patients.

Clinically irrelevant circulating human leukocyte antigen antibodies in the presence of ventricular assist devices.

INTRODUCTION: Identification of anti-human leukocyte antigen (HLA) antibodies by single-antigen beads (SAB) allows for prediction of donor-specific crossmatches (virtual crossmatches), thus facilitating the allocation of organs from deceased donors. However, the clinical relevance of HLA antibodies identified by SAB has been less than clear. This study demonstrates that sera from cardiac transplant candidates with a ventricular assist device (VAD) or infection may contain clinically irrelevant antibodies that bind to the beads but not to lymphocytes. METHODS: Investigated were 5 cardiac transplant candidates (3 with VAD, all with infections, and 1 retransplant) with positive HLA antibodies detected by SAB, but negative by cytotoxicity. To determine clinical relevance of the antibodies, flow cytometric crossmatches (FCXM) were performed. Untreated beads and elution buffer-treated beads to dissociate the ß-2 microglobulin and the peptide from the heavy chain were used. RESULTS: The virtual crossmatch data were compared with data from actual FCXMs. Of 40 T-cell and B-cell FCXM, SAB-identified HLA antibodies were predictive for only 1 T-cell and 9 B-cell FCXM outcomes. Patients' sera contained a mixture of antibodies directed against cryptic epitopes on the heavy chain and exposed epitopes. The mean fluorescence intensity of antibodies varied from 1,040 to 11,000. CONCLUSIONS: Sera from cardiac transplant candidates with or without VAD may contain natural antibodies that do not bind to intact antigens on the cell surface. Therefore, great care must be exercised before denying a life-saving transplant to these patients simply on the basis of SAB results.

Effect of amino acid mismatch in the UNOS dataset.

This study began with the 2010 UNOS data-set of 181,653 deceased donor kidney transplant cases and 92,577 living donor cases. Cases with ambiguous or missing HLA typing were excluded, and the remaining cases were split into subgroups by the number of previous transplants and ethnic groups of donor-patient pairs. 41,128 Caucasian donor-patient pairs that were primary living-donor transplant cases were used as the pilot population to identify potential epitope groups that have a negative effect on graft outcome. Sixty four of the most common HLA-A and -B antigens were selected. Amino acid sequences of the most frequently corresponding allele in the Caucasian population were used to build the starting theoretical epitope table. Amino acids of the 115 polymorphic positions, analyzed in one, two or three positions, resulted in 15,801,920 combinations. After eliminating combinations shared by no allele or by all 64 alleles, the table was trimmed to 1,635,044. Grouping combinations according to their antigen list (antigens that share the combinations/epitopes), 40,830 epitope groups were left. Based on the distances between amino acid positions of each epitope, and the requirement that each epitope must be shared by at least one allele, but not all 64, the number of theoretical epitopes was reduced to 39,670 and 3,703 epitope groups of unique antigen lists. The pilot population was composed of 41,128 primary living-donor transplants with Caucasian donor-patient pairs. For each of the 40,830 epitope groups in the non-distance-restricted table, 15-year death-censored graft survival was computed for epitope-group mismatches--i.e., cases with a BMQ0001 mismatch, with a BMQ0002 mismatch, etc.. Results were compared, using the log rank test, with average graft survival. Of the 3,703 epitope groups, 2,487 appeared in over 1000 cases, but only 88 of them had significant p-values, which ranged from 0.006 to 0.049, with 76 of the 88 significantly below average, 12 above average (Fig. 1). We then ran survival analyses taking the 76 epitope groups that were below average two at a time--i.e., cases with mismatches of the first and second epitope group, the first and third, first and fourth, etc. Of more than 2,500 pairs, 148 resulted in significantly (p < 0.01) lower than average survival in those primary living-donor cases. The effect of the 76 epitope-group mismatches that showed below-average results was then analyzed for other transplant populations--Caucasian donor-patient pair cases with deceased donors , Caucasian donor-patient pair cases with re-transplant living donors, Caucasian donor-patient pair cases with re-transplant deceased donors, and African-American donor-patient pair cases with primary living donors. None of these four populations exhibited any significant effect due to the 76 epitope- group mismatches. Likewise, the effect of the 148 epitope-group combination mismatches detailed in paragraph 5, above, was analyzed for the other four other transplant populations, detailed in paragraph 6. That significant effect was absent in all four. The analyses were repeated on the 40,830 epitope groups without the 27 angstrom distance constraint. Of the 40,830 epitope groups, 439 exhibited a significantly lower 15-year graft survival, with p-value ranging from 0.0053 to 0.0498. Again, none of these had any negatively significant effects on graft survival for the other four transplant populations. In the pilot population, the negative effect of the epitope group mismatches was clearly seen, but the significant differences did not carry across to the other four populations. That absence may be partially explained by the allele level differences in the HLA-A and -B typing of the donors and patients. Past studies indicate that the appearance of DSA has a negative effect on the graft outcome, so the mismatch of epitopes recognized by these DSA could also have similar negative effects. With the data available at present, and with the currently available assays for antibody detection, we are not able to analyze the impact of specific epitope mismatches. We will need the development of new methods of antibody detection that specifically indicate the exact epitope to which an antibody binds before we can continue this effort to determine the negative impact on graft survival due to epitope mismatches.

Anti-HLA-E mAb 3D12 mimics MEM-E/02 in binding to HLA-B and HLA-C alleles: Web-tools validate the immunogenic epitopes of HLA-E recognized by the antibodies.

HLA-E shares several peptide sequences with HLA-class Ia molecules. Therefore, anti-HLA-E antibodies that recognize the shared sequences may bind to HLA-class Ia alleles. This hypothesis was validated with a murine anti-HLA-E monoclonal antibody (mAb) MEM-E/02, which reacted with microbeads coated with several HLA-B and HLA-C antigens. In this report, the hypothesis was reexamined with another mAb 3D12, considered to be specific for HLA-E. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex Multiplex Flow-Cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The results showed that mAb 3D12 simulated MEM-E/02 in recognizing several HLA-B and HLA-C antigens. Both 3D12 and MEM-E/02 did not bind to HLA-A, HLA-F and HLA-G molecules. As observed with MEM-E/02, binding of 3D12 to HLA-E is inhibited by the peptides sequences (115)QFAYDGKDY(123) and (137)DTAAQI(142). Decrease in binding of mAb 3D12 to HLA class Ia, after heat treatment of antigen coated microbeads, supports the contention that the epitope may be located at the outside of the "thermodynamically stable" α-helix conformations of HLA-E. Several sequence and structure-based web-tools were employed to validate the discontinuous epitopes recognized by the mAbs. The scores obtained by these web-tools distinguished the shared peptide sequences that inhibited the mAb binding to HLA-E. Furthermore, ElliPro web tool points out that both mAbs recognize the conformational discontinuous epitopes (the shared inhibitory peptide sequences) in the secondary structure of the HLA-E molecule. The study favors the contention that the domain of the shared inhibitory peptide sequences may be the most immunogenic site of HLA-E molecule. It also postulates and clarifies that amino acid substitution on or near the binding domains may account for the lack of cross reactivity of 3D12 and MEM-E/02 with HLA-A, HLA-F and HLA-G molecules.

New HLA class I epitopes defined by murine monoclonal antibodies.

This study defines 10 epitopes by murine monoclonal antibodies, of which seven are new and three were previously defined by alloantibodies. Of particular interest, three antibodies reacted with almost all Bw4-associated antigens except that each was negative with one or two of the antigens. One was negative with B13, one negative with A25, and another negative with B13 & A24 antigens. These monoclonal antibodies exhibited reactivity contrary to Bw4 allosera, which typically react with all Bw4-associated antigens. All monoclonal antibodies were tested with a panel of 97 human leukocyte antigen (HLA) class I (A, B, and Clocus) rHLA single antigens (SA) individually coupled to different microsphere beads. Identifying HLA antigens sharing distinct epitopes can be helpful when selecting patient-donor transplantation pairs, explaining antibodies against rare specificities, or against non-donor-specific antigens (NDSA). This study adds seven new HLA class I epitopes to 103 already defined epitopes and provides more evidence that a monoclonal antibody and alloantibody can target the same epitope. The fact that mAbs can target the same epitopes targeted by allosera, makes mAbs useful in studies of cross-reactivity in HLA.

HLA-E monoclonal antibodies recognize shared peptide sequences on classical HLA class Ia: relevance to human natural HLA antibodies.

The non-classical HLA-Ib molecule, HLA-E share several peptide sequence similarities with the heavy chains of classical HLA class Ia (-B and -C) molecules. Therefore, the antibodies to HLA-E, that recognize shared sequences, may bind to HLA-Ia alleles. This hypothesis is tested by examining the affinity of HLA-E monoclonal antibodies (HLA-E-MAbs) to HLA-Ia molecules and by inhibiting the antibody binding to both HLA-E and HLA-Ia with the shared peptide sequence(s). Single recombinant HLA molecule-coated beads are used for antibody binding. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex multiplex flow-cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The number of HLA-Ia alleles recognized by the HLA-E-MAbs varies with the density of the antigen (quantity of antigen-coated beads) and dilution of MAb. Binding of HLA-E-MAbs to beta2 microglobulin (beta(2)m)-free HLA-Ia antigens confirms the location of the epitopes on the heavy chain (HC) of the antigens. Strikingly, the nature of alleles of HLA-Ia recognized by different HLA-E-MAbs is identical. The binding of HLA-E-MAbs to the HLA-Ia is inhibited dosimetrically by the adjacent peptides, (115)QFAYDGKDY(123) and (137)DTAAQI(142), but not by (126)LNEDLRSWTA(135), another closer shared peptide sequence. The inhibitory peptide sequences in HLA-E are at the alpha2-helix terminal facing beta(2)m. The HLA-Ia alleles recognized by HLA-E-MAb (e.g., MEM-E/02) are similar to those recognized by the natural anti-HLA antibodies found in the sera of healthy non-alloimmunized males. This study postulates that some, if not all, of the natural HLA-Ia antibodies seen in healthy males could be anti-HLA-E antibodies cross-reacting with HLA-Ia alleles.

Epitopes of HLA-A, B, C, DR, DQ, DP and MICA antigens.

This chapter presents lists of HLA epitopes that have been defined to date. It also presents examples of reactions of mAb and eluted allosera with the class I, class II and MICAsingle antigen beads. To date, we have identified 110 class I epitopes, of which 47 were defined by mAbs and 63 by alloantibodies that were eluted from rHLA class I single antigen cell lines. We listed 34 epitopes shared by the HLA-A locus antigens, 44 epitopes shared by HLA-B locus antigens, 4 epitopes shared by HLA-C locus antigens, 20 inter-locus epitopes shared by HLA-A-B locus antigens, 5 inter-locus epitopes shared by HLA-B-C locus antigens and 3 inter-locus epitopes shared by HLA-A-B-C locus antigens. Sixty HLA-DR epitopes have been defined mostly by one amino acid (aa) residue on the HLA-DR beta chain. Eighteen HLA-DQ epitopes have been defined on the HLA-DQB chain and on the HLA-DQA chain of the HLA-DQ antigens. A few DQ epitopes were defined by one aa residue. However, most can be defined by several alternative combinations of aa residues. DQA and DP epitopes--few in number at this time--were identified. Only seven MICA epitopes have been defined to date. All epitopes can be defined by an exclusive amino acid.

Epitopes of human leukocyte antigen class I antibodies found in sera of normal healthy males and cord blood.

This study defines 96 epitopes targeted by human leukocyte antigen (HLA) antibodies reported in the sera of normal healthy males with no history of deliberate alloimmunizations and in cord blood. These epitopes are accessible for antibody binding on either the intact or the dissociated forms of recombinant HLA class I single antigens. Sixty percent of the epitopes are accessible on dissociated antigens, are defined mostly by hidden amino acids, and are designated as cryptic epitopes. All 96 epitopes are located exclusively on A-, B-, or C-locus antigens except for one interlocus epitope. All sera in this study were tested in parallel, using single antigen beads that bear either intact or dissociated HLA antigens and antibodies with nearly identical specificities were identified in all tested sera. Because the specificities of these naturally occurring antibodies are unavoidably detected when testing for specificities of alloantibodies, it may be necessary to clearly differentiate the two forms of antibody. To date, the relevance of these antibodies in transplantation is unknown, but even if they are determined to be irrelevant to graft rejection, awareness of the newly identified epitopes could prove useful in avoiding the unnecessary exclusion of potential transplant donors.

Immunogenic HLA class I epitopes identified by humoral response to pregnancies.

The main objective of this study was to understand the humoral immunity against HLA so that this knowledge can be applied clinically. We investigated the various factors resulting in antibody production by 128 mothers against the child's inherited paternal alleles. Among 128 mother-child pairs, 39 different mismatch antigens were observed. Of these, 19 resulted in antibody production against the specific mismatched antigen. The 20 mismatched antigens that did not result in a humoral immune reaction were excluded from this study. We performed epitope analysis by testing 45 allo-antisera from mothers. We compared the amino acids that define these epitopes in the mother's HLA with those in the immunogen's HLA. The positions of the mismatched amino acids between them were determined to be the immunogenic amino acid positions of mismatched HLA. We found that the immunogenic epitopes were located mainly on the alpha 1 helix. Mothers who have different immunogens were shown to have different class II alleles. Epitope analysis of this study indicated the possibility that immunogenic amino acid positions exist for each of the different mismatching alleles. Mismatching at these immunogenic positions did not always result in the antibody production, and it is thought that the differences are due to the efficiency of class II alleles in presenting the mismatched alleles.

"Natural" human leukocyte antigen antibodies found in nonalloimmunized healthy males.

BACKGROUND: Human leukocyte antigen (HLA) antibodies were normally not found in subjects who have not been immunized by pregnancies, transfusions, or transplants. But with new methodology, we now see that HLA antibodies are often found in nonalloimmunized males. METHODS: The sera of 424 healthy male donors were tested with single antigen Luminex beads. RESULTS: Human leukocyte antigen antibodies were detected in 63% of 424 male blood donors when a fluorescent value of more than 1000 was used as the cutoff. Antibodies to class I was found in 42%, class II in 11% and both in 12%. Five males who were tested eight times over a 6-month period consistently had the same specificity at similar strength levels at each testing. The antibodies reacted with specificities that are rare in the general population: 18.9% had antibodies to A*3002; more than 10% had antibodies to A*3101, B76, B*8201, and Cw*1701. About half of the donors with antibodies had one or two specificities; the other half had three or more specificities. Among those with class II specificities, 20.5% had antibodies to DPA1*0201/DPB1*0101, and 10.8% to DQA1*0503/DQB1*0301. Because the above data were obtained by testing sera of 424 Mexican donors, as a check, 29 males in Los Angeles were tested and shown to have similar specificities at roughly similar frequencies. CONCLUSIONS: Normal males were found to have HLA antibodies to infrequent HLA specificities. It is likely that these HLA antibodies are produced to cross-reactive epitopes found in microorganisms, ingested proteins and allergens-making them natural antibodies.

Epitopes of HLA antibodies found in sera of normal healthy males and cord blood.

This chapter defines epitopes targeted by antibodies in the sera of two populations of healthy normal males and in cord blood samples from a third population. These epitopes are accessible for antibody binding on either the intact or dissociated forms of recombinant HLA class I single antigens. Sixty percent of these epitopes are defined by hidden amino acids, and are therefore designated as cryptic epitopes. All sera were tested in parallel, using single antigen beads that bore either intact or dissociated recombinant HLA antigens. Ninety-six HLA class I epitopes were characterized as epitopes of these antibodies. More than half were private epitopes, and the rest were shared by two-to-18 HLA antigens. Fifty-eight (60%) epitopes were accessible on dissociated HLA antigens. Of these, 41 were defined by hidden amino acids, 13 by at least one hidden amino acid in addition to exposed amino acids, and four were defined by exposed amino acids. Almost all epitopes were found exclusively on either A-, B-, or C-locus antigens--except for one inter-locus epitope. Antibodies with nearly identical specificities were found in all three of the tested populations. Most of these antibodies target epitopes that are accessible only on the dissociated forms of the HLA class I antigens. Specificities of such antibodies are unavoidably detected when testing for specificities of alloantibodies so it may be necessary to clearly differentiate the two forms of antibody. The relevance of these antibodies in transplantation is not yet known. But even if they are shown to be irrelevant to graft rejection, awareness of the newly identified epitopes could prove useful in avoiding unnecessary exclusion of potential transplant donors.