Miyuki Ozawa

Predicting Kidney Graft Failure by HLA Antibodies: a Prospective Trial

HLA antibodies have been shown to be associated with late graft loss of organ transplants in prior studies. Recently they were even shown to appear years BEFORE rejection. ( 1) An international cooperative study of 4763 patients from 36 centers was undertaken to determine the frequency of HLA antibodies in patients with functional transplants. The overall frequency of HLA antibodies among kidney transplant recipients was 20.9%; 19.3% in the liver, 22.8% in the heart, and 14.2% in the lung. Patients treated with CsA‐MMF had significantly lower antibodies (9.8%) than those treated with CsA‐Aza (18.1%) (0.00008). ( 2) Second, a prospective trial was performed in 23 kidney transplant centers to determine whether HLA antibodies could predict failures within 1 year. Among the 2278 patients followed up, 91 grafts failed and 34 patients died. Of 500 patients who had HLA antibodies, 6.6% failed compared with 3.3% among 1778 patients without antibodies (p = 0.0007). Among 244 patients who made de novo antibodies, 8.6% failed compared with 3.0% failures among 1421 patients who did not make antibodies (p = 0.00003). Death occurred in 1.5% of patients and was not associated with antibodies. Thus, after 1 year in this prospective trial, patients with HLA antibodies had graft failure at a significantly higher rate than those without antibodies.

Incidence and impact of de novo donor-specific alloantibody in primary renal allografts.

Background To date, limited information is available describing the incidence and impact of de novo donor-specific anti–human leukocyte antigen (HLA) antibodies (dnDSA) in the primary renal transplant patient. This report details the dnDSA incidence and actual 3-year post-dnDSA graft outcomes. Methods The study includes 189 consecutive nonsensitized, non-HLA-identical patients who received a primary kidney transplant between March 1999 and March 2006. Protocol testing for DSA via LABScreen single antigen beads (One Lambda) was done before transplantation and at 1, 3, 6, 9, and 12 months after transplantation then annually and when clinically indicated. Results Of 189 patients, 47 (25%) developed dnDSA within 10 years. The 5-year posttransplantation cumulative incidence was 20%, with the largest proportion of patients developing dnDSA in the first posttransplantation year (11%). Young patients (18–35 years old at transplantation), deceased-donor transplant recipients, pretransplantation HLA (non-DSA)–positive patients, and patients with a DQ mismatch were the most likely to develop dnDSA. From DSA appearance, 9% of patients lost their graft at 1 year. Actual 3-year death-censored post-dnDSA graft loss was 24%. Conclusion We conclude that 11% of the patients without detectable DSA at transplantation will have detectable DSA at 1 year, and over the next 4 years, the incidence of dnDSA will increase to 20%. After dnDSA development, 24% of the patients will fail within 3 years. Given these findings, future trials are warranted to determine if treatment of dnDSA-positive patients can prevent allograft failure.

Serial Ten-Year Follow-Up of HLA and MICA Antibody Production Prior to Kidney Graft Failure

The role of HLA antibodies in chronic allograft rejection was examined utilizing a unique resource of sera collected annually and stored over a 12‐year period from patients with rejected or retained grafts. In patients selected for not having preformed HLA antibodies, 679 postoperative serial serum samples from 39 patients who rejected their grafts and 26 with functioning grafts were tested for HLA Class I and Class II antibodies by flow cytometry and for MICA antibodies by cytotoxicity on recombinant cell lines. HLA antibodies were found in 72% of patients who rejected grafts, compared to 46% with functioning transplants (p < 0.05). In addition, the incidence of IgG HLA plus MICA antibodies was higher (77%) among those with failed transplants than those with functioning transplants (42%) (p < 0.01). Finally, if patients with IgM anti‐HLA antibodies were included, 95% of the 39 patients who rejected their grafts had HLA or MICA antibodies, compared to 58% with functioning grafts (p < 0.01). Patients who rejected transplants had HLA and MICA antibodies more frequently than those with functioning grafts. These antibodies found in the peripheral circulation, were not necessarily donor‐specific, but their association with failure is consistent with a causality hypothesis.

Predictive value of HLA antibodies and serum creatinine in chronic rejection: results of a 2-year prospective trial.

In this large collaborative study, 2,231 transplanted patients with functioning kidneys were tested for HLA antibodies, then examined 2 years later for graft survival. Among 478 patients with antibodies, 15.1% failed in 2 years, compared to 6.8% failure in 1,753 patients without antibodies (P=0.00000002). Cytotoxicity testing correlated better with outcome than flow cytometry or ELISA testing on HLA coated beads, possibly because it detected non-HLA antigens. When the patients were further broken down into those with serum creatinine at the time of testing of 0.5–1.9, 4.4% of antibody patients failed at 2 years, compared to 4.3% of patients without antibodies. This 0.1% difference increased among patients with serum creatinine values of 2.0–2.9 to 17.9%, and among those with 3.0–3.9 to 16.3%. We conclude that HLA antibodies posttransplantation is predictive of subsequent graft failure, and its predictive value can be enhanced among patients with higher serum creatinine values.

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.

The Importance of Anti‐HLA‐Specific Antibody Strength in Monitoring Kidney Transplant Patients

Approximately 25% of patients who undergo kidney transplantation develop HLA‐specific antibodies, the strength of which has not been previously correlated with graft failure. The strength of these de novo antibodies is investigated in this study. Serial dilution of strong HLA‐specific allo‐antibodies (up to 1:25,600) and testing with HLA‐antigen‐coated beads showed that the titer of the reaction to different HLA antigens is directly correlated to maximum fluorescence values and the molecules of equivalent soluble fluorochrome (MESF) values obtained by Luminex machines. Thus, the strength of antibodies can be measured utilizing maximum fluorescence and MESF. The strength of antibodies in the sera from 39 patients who subsequently had graft failure were markedly higher than those in the sera of 26 patients who continued to have good graft function (p = 0.0084). A clear increase in the strength of antibodies was identified in nine patients with a subsequent increase in serum creatinine levels. If analyzed for donor specificity, a strong association was noted for donor‐specific MESF and failure (p = 0.00000027). Our results suggest that it is important to monitor the strength of antibodies when evaluating patient sera posttransplant.

The role of IgA anti-HLA class I antibodies in kidney transplant survival

The unusually high 88% one-year cadaver kidney graft survival rate in patients with IgA nephropathy (IgAN) prompted us to investigate the influence of IgA anti-HLA class I antibodies on subsequent graft survival. We found that patients with various original diseases with IgA antibodies to the HLA molecule had high 91% one-year graft survival compared with 58% one-year survival for those who did not have preformed IgA antibodies against the HLA molecule prior to transplantation (P<0.0005). The IgA antibodies were detected by reaction with class I HLA molecules isolated by capture with monoclonal antibody and detected with an enzyme-linked immunosorbent assay. In contrast, IgG antibodies to the HLA molecule resulted in a lower one-year graft survival rate (74%) than in those patients without IgG antibodies (87%) (p=0.08). IgA antibodies to the HLA molecule, when present, tended to react at a high frequency on a random lymphocyte panel. These findings suggest that sensitization resulting in IgA anti-HLA antibodies may counteract the deleterious effect of an IgG antibody response in clinical kidney transplantation.

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.

No Occurrence of De Novo HLA Antibodies in Patients With Early Corticosteroid Withdrawal in a 5-year Prospective Randomized Study

The purpose of this study was to determine the effect of early corticosteroid cessation on the occurrence of de novo human leukocyte antigen (HLA) antibody posttransplant. Renal transplant recipients (n=37) were randomized to early corticosteroid withdrawal at day 7 posttransplant (n=21 patients), or to chronic steroids (n=16), all in combination with thymoglobulin as induction agent, tacrolimus and mycophenolic acid as maintenance therapy. To establish the time course of HLA antibody appearance, sera collected pretransplant and for up to 5 years posttransplant were screened for the appearance of HLA antibodies. In this 5-year longitudinal study, only one patient in the control group developed a de novo donor-specific HLA antibody. We conclude that renal transplant recipients on steroid withdrawal by the end of week 1 are not at higher risk for developing HLA antibodies compared with a standard steroid regimen up to 5 years posttransplant.

Predicting operational tolerance in pediatric living-donor liver transplantation by absence of HLA antibodies.

Background The role of anti–human leukocyte antigen (HLA) antibodies in operational tolerance (OT) after pediatric living-donor liver transplantation (LDLT) remains inconclusive. We investigated whether the presence of HLA antibodies impeded the development of OT. Methods We retrospectively examined the prevalence of anti-HLA antibodies in pediatric LDLT recipients before transplantation and at 3 weeks after transplantation and analyzed the significance of those antibodies in relation to later OT. Forty pediatric LDLTs were performed between April 1996 and December 2000 and followed up through July 2011, with sera available for measurement of HLA antibodies. Seventeen patients achieved OT (mean follow-up, 4571.9±544.7 days) and 23 patients did not achieve OT (mean follow-up, 4532.0±425.4 days). Protocol liver biopsy was done for 14 OT patients and 16 non-OT patients. Their sera were tested for anti-HLA class I and II antibodies using the LABScreen single antigen beads test, in which a 1000 mean fluorescence value was considered positive. Results The prevalence of antibodies after transplantation in non-OT patients was higher than in OT patients (95.2% vs. 73.3%; P<0.001). The highest mean fluorescence intensity of antibodies was significantly higher in non-OT patients than in OT patients. The prevalence of HLA-B, HLA-C, HLA-DQ, and HLA-DR antibodies was significantly higher in non-OT patients than in OT patients. The highest mean fluorescence intensity of HLA-A, HLA-B, and HLA-DQ observed in non-OT patients was significantly higher than those in OT patients. Conclusions In our study, posttransplantation HLA antibodies were associated with the future absence of OT. A prospective study with more patients is necessary to confirm the predictive value of HLA antibodies for OT.