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.

The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes.

BACKGROUND: Anti-HLA-DQ antibodies are the predominant HLA class II donor-specific antibodies (DSAs) after transplantation. Recently, de novo DQ DSA has been associated with worse allograft outcomes. The aim of this study was to determine the further complement-binding characteristics of the most harmful DQ DSA. METHODS: Single-antigen bead technology was used to screen 284 primary kidney transplant recipients for the presence of posttransplantation DQ DSA. Peak DSA sera of 34 recipients with only de novo DQ DSA and of 20 recipients with de novo DQ plus other DSAs were further analyzed by a modified single-antigen bead assay using immunoglobulin (Ig)-G subclass-specific reporter antibodies and a C1q-binding assay. RESULTS: Compared with recipients who did not have DSA, those with de novo persistent DQ-only DSA and with de novo DQ plus other DSAs had more acute rejection (AR) episodes (22%, P=0.005; and 36%, P=0.0009), increased risk of allograft loss (hazards ratio, 3.7, P=0.03; and hazards ratio, 11.4, P=0.001), and a lower 5-year allograft survival. De novo DQ-only recipients with AR had more IgG1/IgG3 combination and C1q-binding antibodies (51%, P=0.01; and 63%, P=0.001) than patients with no AR. Furthermore, the presence of C1q-binding de novo DQ DSA was associated with a 30% lower 5-year allograft survival (P=0.003). CONCLUSIONS: The presence of de novo persistent, complement-binding DQ DSA negatively impacts kidney allograft outcomes. Therefore, early posttransplantation detection, monitoring, and removal of complement-binding DQ might be crucial for improving long-term kidney transplantation outcomes.

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.

The HLA-matching effect in different cohorts of kidney transplant recipients: 10 years later.

Almost all the HLA-matching effects found by the 2000 analysis were confirmed by this study. The only HLA-matching effect found in the 2000 analysis that disappeared were those of "small matching effect" found in sub-populations of type I diabetes (PRA < 10%, donor age 20-35). The 2000 analysis found a lack of HLA matching effect in non-African American kidney transplant patients with type I diabetes between 1987 and 2000. The 2000 analysis found that a patients' ethnic group was a factor in graft survival; African American patients were found to have a significantly lower 10-year graft survival in the 5 or 6 mismatched group (27%) compared to Caucasian patients (40%). In addition, Asian patients (42%) had higher graft survival compared to that of Caucasian patients. In this study, we observe a similar pattern with death-censored graft analysis for all ethnic groups with 10-year graft survivals at 72.9% for Asians, 69.5% for Caucasians, and 49.3% for African Americans. There was an overall lack of HLA-matching effect on patient survival in the 2000 analysis. In our current analysis, the patient survivals remained virtually the same despite moderate increase in graft survival over the same period of time. The HLA-C locus mismatch was found to have additive effect to the 10-year graft survival trends observed in A and B mismatch cases. HLA-DQ mismatch on the other hand, showed limited HLA-matching effect and did not show the same additive effect as C. There are various possible issues in the DQ mismatch analysis, from the consistency of DQ typing results, lack of diversity in the DQ antigen, to the possibility of DQ mismatch having little effect on the graft survival. Utilizing kidney transplant cases performed from 1995 through 2000, the 2000 analysis projected 10-year survivals of 64% and 47% for the 0 ABDR mismatch and 5 or 6 ABDR mismatched cases respectively; the 2000 projection only missed actual death-censored survivals by 9% lower for the 0 mismatch and 17% lower for the 5 or 6 mismatch cases. Utilizing the transplant cases of 2005 through 2009, we projected their 10-year graft survivals for year 2020. The 10-year graft survival for 0 ABDR mismatched patients is expected to be over 85% and nearly 70% for 5 or 6 ABDR mismatched patients. The general upward trend of graft survival we have observed in the last 10 years has been dependent upon the development of novel transplant protocols and use of novel immunomodulatory reagents. This trend is likely to continue given the promise of new drugs and personalized healthcare. The decreasing range of the differences in the 10-year graft survival between best matched and worst matched HLA groups is also likely to continue. One interesting trend that is clearly evident is the increasing difference between the best and worst HLA-matching in terms of the associated graft half-life. The positive HLA-matching effect on long-term graft survival is clearly evident and should be taken into consideration for all kidney transplants.

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.

Mimetic human leukocyte antigen epitopes: shown by monoclonal antibodies and extra antibodies produced on transplantation.

BACKGROUND: Transplant patients often produce human leukocyte antigen (HLA) antibodies against their donors and produce more specificities than can be accounted for by HLA antigen mismatches. We theorize that the presence of extra, otherwise unexplainable specificities could be accounted for if antibodies reacted to more than one epitope (primary and mimetic) on distinct HLA molecules. The theory states that mimetic epitopes consist of the same three amino acids that comprise the primary, sterically placed approximately the same distance apart as are the corresponding amino acids of the primary. METHODS: A mimetic epitope table containing all primary epitopes and corresponding mimetic epitopes was built. Then, the HLA specificities of monoclonal and single patient antibodies were determined. These specificities that could not be defined by unique position or amino acid epitopes alone were then used to query the mimetic epitope table. RESULTS: A single antibody from a transplant patient and three monoclonal antibodies produced reactions that can best be explained as the result of one antibody reacting to the same amino acids at two distinct sites on the molecule. Those position and amino acid combinations (pos/aa) are the primary and mimetic epitopes. Using computerized methods of searching, mimetic epitopes were found in five additional kidney transplant patients who produced nondonor-specific antibodies in addition to donor-specific antibodies. CONCLUSIONS: Epitopes on the HLA molecule that mimic the primary epitope have been found. We suggest that these mimetic epitopes explain the additional antibodies often found on HLA immunization resulting from allograft rejections, pregnancies, and transfusions.

Using the mimetic epitope concept to find a possible way to widen the graft-survival difference between matched and mismatched renal transplants.

1. Immunological responses cause antibodies to be produced, and transplant patients often produce HLA antibodies against their donors' HLA. But in many cases, more antibodies are produced than just those against mismatched HLA. Many patients with zero mismatched transplants also produce antibodies, and some of these grafts fail. This study used graft survival analysis that contemplated the primary epitopes against the donors' alleles and the corresponding mimetic epitopes, less those shared with the patients' alleles. 2. A table of primary epitopes and corresponding mimetic epitopes was built in a previous study. For each case studied, here, a subset of the table was selected for all of the donor's HLA alleles. The subset was reduced by eliminating all primary and mimetic epitopes shared by the patient's own HLA alleles. The remaining mimetic and primary epitopes-the maximum pool of epitopes (MaxPEpi)-produced epitope frequency (Efr) for each allele. Using a zero-HLA-AB-mismatch subset (n = 16) of the Greenville and Holland kidney transplant data set (n = 451), the Efr for each allele in the MaxPEpi of each case was tallied and compared with the corresponding observed antibody specificities. The average frequencies were 2,869 and 1,975, respectively, and the t-test was significant at the p-value of less than 0.00001, indicating that the higher the frequency, the more likely that the corresponding allele appears as a part of the specificity. 3. The trends were used to perform survival analysis on zero-mismatch kidney transplant cases in the 2007 UNOS data file. Different variables of MaxPEpi parameters and cutoff points were tried with half of the deceased-donor cases in the file, survival rates for those below and above each cutoff point were compared by the log-rank test. The average Efr with a cutoff of 3,700 yielded the best 10-year graft survival in this subgroup of the 2007 UNOS cases. The results were supported and verified by analyzing the second half of the deceased-donor cases and the living-donor cases in the 2007 UNOS data set. 4. With the 2007 file living-donor cases, 10-year graft survival rate was 86.2% for cases below the cutoff, and 85.4% for those above, and the difference was significant at a p-value less than 0.01. A larger difference was observed for 2007 living-donor cases, excluding identical sibling transplants, but the difference was not statistically significant due to the low number of cases. 5. The same trends were found for 10-year survivals with cases in the 2008 data, but were not statistically significant. With living donors, the rates below and above the cutoff were 85.7% and 85.2%, respectively, and with deceased donors, 72.7% and 72.1%, respectively, p < 0.125. As with the 2007 data, cases in the 2008 file with living donors--excluding identical sibling transplants--had survival rates of 91.7% and 79.0%, respectively, but the 12.7% difference was not statistically significant because of the low number of cases.

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.

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.

Effect of amino acid mismatch in the UNOS 2007 dataset.

1. The study began with the 2007 UNOS dataset of 270,690 kidney transplant, from which were selected post-1995 first transplants with Caucasian donor/patients and available ABDR typing data, yielding 87,616 cases. These were split into cadaver donor (n=46,927) and living donor (n=40,689) populations. 2. Cases with broad antigens such as A9, A10, B12 and B17 were eliminated, as were cases that had failed within 30 days. That left 28,264 cadaver donor and 26,211 living donor cases. 3. We looked at every theoretical mismatch between donors and patients in the living donor population. Overall, 405 single, 21,269 double, and 391,325 triple position/amino acid mismatches were included in the analysis. Two tallies for each mismatch were generated: "function" and "fail" based on patient's associated graft survival. 4. We generated a list of "fail" single, double, and triple position/amino acid mismatches and computed 10-year survival curves for each of the mismatches, comparing them with the average 10-year survival curve using the log-rank test. Based on the log-rank statistics, a ranking of the bad mismatches was established. 5. We looked at the long-term graft survival of additive single, double, and triple "fail" mismatches in cadaver donor transplants. 6. Survival curves of transplants with position/ amino acid mismatches were generated and compared with the survival curves of the traditional standards: 0 ABDR mismatch; 1 A, 1 B and 1 DR mismatch; and full 6 ABDR mismatches. 7. The greatest effect was seen in first transplants with a male recipient, but that were not Caucasian-to-Caucasian. Up to 125 double mismatches resulted in a 10-year survival 29% lower than 0 ABDR mismatches. 8. In first transplants that were not Caucasian-to-Caucasian 1154 out of 1177 (98%) pos/aa mismatches (single, double, or triple) had lower 10-year survival than cases with one mismatch, each, in A, B and DR. Looking at re-grafts, we see that 498 out of 499 with single, double and triple mismatches had lower 10-year survival than that of cases with one mismatch, each, in A, B and DR. 9. Overall, position/amino acid mismatches had consistently lower 10-year survival than 1 A, 1 B, 1 DR mismatches. We believe our selection of "fail" pos/aa mismatches provide a good starting point for establishing a list of mismatches to be looked for and avoided in future transplants in order to give them a better chance of survival.

HLA matching by amino acid/positions in double and triple combinations.

1. Began with the 2006 UNOS dataset and then selected for Caucasian donor/patients (post-1995 transplant and first transplant kidney only with full ABDR typing data). This was then split into cadaver donor (n = 20,542) and living donor (n = 21,890) populations. 2. Looked at every theoretical mismatch between donor and patients in the living donor population. Overall, 167 single, 4601 double, and 83,655 triple position/amino acid mismatches were included in the analysis. Two tallies for each mismatch were generated: "good" and "bad" based on patient's associated graft survival. 3. Generated a list of "bad" single, double, and triple position/ amino acid mismatches. 4. Looked at associated long-term graft survivals of patient/donor pairs with additive single, double, and triple "bad" mismatches in the cadaver donor population. 5. Survival curves of transplant pairs with associated mismatches were generated and compared with traditional standards of 0ABDR mismatch, 1 or more ABDR mismatch, and full 6ABDR mismatch survival curves. 6. The greatest effect was seen in re-grafted male patients where up to 50 double mismatches were associated with a 10-year survival 27% lower than 0ABDR mismatch. 7. Many of the "bad" position/amino acid mismatches identified were the same as those identified by generation of antibodies against Class I and Class II epitopes. This is evidence that antibody-defined epitopes and those functioning as transplantation epitopes influencing long-term graft survival are the same. 8. Overall, we are disappointed that position/ amino acid mismatches were not associated with more markedly lower survivals. It appears that current immunosuppression can largely negate the effect of mismatching for immunogenic epitopes.

Unexpected frequencies of HLA antibody specificities present in sera of multitransfused patients.

1. The resolution of single antigen beads is high enough to distinguish between a patient's own HLA phenotype and antibody specificity down to the allele level. 2. Mismatched antigens generally produce an antibody specific to that antigen. Therefore, one would naturally expect antibody specificity frequency to correlate with phenotype frequencies for any given population (e.g., rare phenotypes would rarely have an antibody generated against it). We called this the antigen mismatch model. 3. When testing the sera of 367 highly sensitized patients with single antigen beads, we found results which did not correlate with the antigen mismatch model. Antibodies specific for rare antigens occurred at surprisingly high levels within the study population. 4. We postulate that these high frequencies are due to mismatches on epitopes as defined by absorption/elution experiments (9) or NEpis. Thus, a mismatch on a common antigen could produce an antibody specific for a rare antigen as long as the two antigens share a NEpi (unique position/amino acid combination). 5. The probability of mismatching on 58 NEpis based on published phenotype frequencies was calculated. We called this the NEpi mismatch model. 6. Both the antigen mismatch model and the NEpi mismatch model were compared to observed antibody specificity frequencies in our study population. Overall, the NEpi mismatch model correlated with observed frequencies much more accurately than the traditional antigen mismatch model. 7. In conclusion, we present a probable explanation for higher-than-expected antibody specificity frequencies and propose that mismatching on NEpis has further potential clinical implications.

Unexpected frequencies of HLA antibody specificities in the sera of pre-transplant kidney patients.

1. We utilize single-antigen beads to test 103 pre-transplant patients to discern HLA antibody specificity frequencies. 2. Overall, we found much higher levels of immunization to HLA than anticipated, particularly rare specificities. We postulate that the presence of epitopes and mismatching on epitopes are responsible for these higher levels of immunization. 3. Two models were generated: (A) The first was an antigen mismatch model representing the traditional theory that higher rates of immunization to an antigen would occur with a higher incidence of the antigen within the population. This model was based on published gene frequencies (4), which were converted into antigen frequencies. (B) The second model was an epitope mismatch model based on published gene frequencies (4) as well as experimentally proven epitopes (3). 4. These two models were then compared with observed antibody specificity frequencies. The antigen mismatch model consistently underestimated observed frequencies. On the other hand, the epitope mismatch model seems to be a much better representation of observed frequencies. 5. In conclusion, we bring attention to the occurrence of higher-than-expected frequencies of antibodies against HLA, especially rare antigens like A43 and B76, and we show that mismatched epitopes might provide a plausible explanation for these observed high frequencies.