Donor HLA-specific Abs: to BMT or not to BMT?

The engraftment failure associated with Abs to donor-specific HLA (DSA) limits options for sensitized BMT candidates. Fourteen of fifteen patients with no other viable donor options were desensitized and transplanted using a regimen of plasmapheresis and low-dose i.v. Ig modified to accommodate pre-BMT conditioning. DSA levels were assessed by solid-phase immunoassays and cell-based crossmatch tests. DSA levels were monitored throughout desensitization and on day -1 to determine if there was any DSA rebound that would require additional treatment. A mean reduction in DSA level of 64.4% was achieved at the end of desensitization, with a subsequent reduction of 85.5% after transplantation. DSA in 11 patients was reduced to levels considered negative post-BMT, whereas DSA in three patients remained at low levels. All 14 patients achieved donor engraftment by day +60; however, seven patients suffered disease relapses. Four patients experienced mild, grade 1 GVHD. Factors influencing the response to desensitization include initial DSA strength, number, specificity, DSA rebound and a mismatch repeated from a prior transplant. While desensitization should be reserved for patients with limited donor options, careful DSA assessment and monitoring can facilitate successful engraftment after BMT.

Dynamic challenges inhibiting optimal adoption of kidney paired donation: findings of a consensus conference.

While kidney paired donation (KPD) enables the utilization of living donor kidneys from healthy and willing donors incompatible with their intended recipients, the strategy poses complex challenges that have limited its adoption in United States and Canada. A consensus conference was convened March 29-30, 2012 to address the dynamic challenges and complexities of KPD that inhibit optimal implementation. Stakeholders considered donor evaluation and care, histocompatibility testing, allocation algorithms, financing, geographic challenges and implementation strategies with the goal to safely maximize KPD at every transplant center. Best practices, knowledge gaps and research goals were identified and summarized in this document.

Calculated PRA: initial results show benefits for sensitized patients and a reduction in positive crossmatches.

The calculated panel reactive antibody (CPRA), which is based upon unacceptable HLA antigens listed on the waitlist form for renal transplant candidates, replaced PRA as the measure of sensitization among US renal transplant candidates on October 1, 2009. An analysis of the impact of this change 6 months after its implementation shows an 83% reduction in the number of kidney offers declined nationwide because of a positive crossmatch. The increasing acceptance and utilization of unacceptable HLA antigens to avoid offers of predictably crossmatch-positive donor kidneys has increased the efficiency of kidney allocation, resulting in a significant increase in the percentage of transplants to broadly sensitized (80+% PRA/CPRA) patients from 7.3% during the period 07/01/2001-6/30/2002 to 15.8% of transplants between 10/1/09-3/31/10. The transplant rates per 1000 active patient-years on the waitlist also increased significantly for broadly sensitized patients after October 1, 2009. These preliminary results suggest that 'virtual' positive crossmatch prediction based on contemporary tools for identifying antibodies directed against HLA antigens is effective, increases allocation efficiency and improves access to transplants for sensitized patients awaiting kidney transplantation.

Incidence of humoral sensitization in HLA partially mismatched hematopoietic stem cell transplantation.

As part of the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), seven centers participated in a collaborative project to determine whether any significant humoral sensitization occurred post-transplant among recipients of HLA partially mismatched hematopoietic cell transplants (HCTs). A total of 140 donor/recipient pairs were enrolled with a total of 367 pre-and post-transplant sera analyzed. The majority of the samples (69.1%) were obtained within 30-90 days post-HCT. HLA-specific antibodies were defined using single antigen bead assays on a Luminex platform with a positive cutoff value of 1000 normalized median fluorescence intensity (MFI). There was an overall incidence of post-HCT sensitization toward donor HLA mismatches of 5.7%; however, all cases were among recipients of one HLA haplotype-mismatched grafts under nonmyeloablative, pre-transplant conditioning. Among the one haplotype-mismatched recipients, 15.7% (8/51) developed donor HLA-specific antibodies and 29.4% also had antibodies directed toward third party HLA antigens. Among the donor-specific antibodies, 9.8% were directed toward HLA class I antigens; 7.8% were against class II antigens; and 2.0% had both class I and II specificity. The relative strength of post-transplant antibodies was low with no significant difference in the mean maximum MFI values between third party and donor-specific antibodies. Because only a small number (10.2%) of the post-transplant samples were obtained 180 days or more post-HCT, longer term study is needed to evaluate any clinical relevance of these low-to-moderate levels of donor-specific antibody in one haplotype-mismatched recipients, as well as to determine whether any other antibodies occur at later times.

Proinflammatory events are associated with significant increases in breadth and strength of HLA-specific antibody.

Identification of factors responsible for an increase in the breadth or strength of HLA-specific antibody (HSA) is critical to the continued successful management and transplantation of sensitized patients. A retrospective review of our HLA registry identified 107 patients with known HSA and sufficient information in their electronic patient record to determine the presence or absence of a proinflammatory event. The patients were stratified according to transplant status [sensitized and on the transplant waitlist (n = 65); immunosuppressed recipients of a positive crossmatch (+XM) transplant (n = 42)]. Eighty-three percent of waitlist candidates and 55% of sensitized kidney transplant recipients with a documented proinflammatory event had an associated increase in HSA. Interestingly, among patients with a culture-proven infection, 97% of the waitlist patients and 54.8% of +XM recipients had an associated rise in HSA. Overall, proinflammatory events were associated with a greater increase among waitlist patients than +XM recipients, 5.3-fold [IRR 5.25, (95% CI 4.03-6.85), p < 0.001] versus 2.5-fold [IRR 2.54, (95% CI 1.64-3.95), p < 0.001] increase in HSA. Therefore, sensitized patients known to have an infection or undergoing surgery should be monitored for expansion of HSA.

Reduced intensity HLA-haploidentical BMT with post transplantation cyclophosphamide in nonmalignant hematologic diseases.

Allogeneic blood or marrow transplantation (BMT) is potentially curative for a variety of life-threatening nonmalignant hematologic diseases such as paroxysmal nocturnal hemoglobinuria (PNH) and hemoglobinopathies. The application of BMT to treat these disorders is limited by the lack of suitable donors and often end-organ damage from the underlying disease. We treated three patients with thrombotic PNH, one of whom also had sickle cell disease, with a nonmyeloablative, HLA-haploidentical BMT with post-transplant CY. Rapid engraftment without GVHD occurred in two of the patients, including the patient with sickle cell disease. Both patients are disease free with full donor chimerism and require no immunosuppressive therapy, with follow-up of 1 and 4 years, respectively. Nonmyeloablative, HLA-haploidentical BMT with post-transplant CY is a promising approach for patients with life-threatening nonmalignant hematologic disease who lack an HLA-matched sibling donor.

Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using posttransplantation cyclophosphamide.

Cyclophosphamide (Cy) is a potent immunosuppressive agent that is selectively toxic to lymphocytes proliferating in response to recent antigen stimulation. In animal models, both graft rejection and GVHD after histoincompatible BMT can be inhibited by the posttransplantation administration of high-dose Cy. Therefore, a phase I clinical trial was undertaken to determine the minimal conditioning, including posttransplantation Cy, that permits the stable engraftment of partially HLA-mismatched marrow (up to 3 HLA antigens) from first-degree relatives. Thirteen patients (median age, 53 years) with high-risk hematologic malignancies received conditioning with fludarabine, 30 mg/m2 per day from days -6 to -2, and TBI, 2 Gy on day -1. All patients received Cy, 50 mg/kg on day 3, mycophenolate mofetil from day 4 to day 35, and tacrolimus from day 4 to day > or = 50. Three patients in cohort 1 received no additional conditioning, and 2 experienced graft rejection. Ten patients in cohort 2 received identical conditioning with the addition of Cy 14.5 mg/kg on days -6 and -5. Sustained donor cell engraftment occurred in 8 of these patients, with a median time to absolute neutrophil count > 500/microL of 15 days (range, 13-16 days) and to unsupported platelet count > 20,000/microL of 14 days (range, 0-26 days). All patients with engraftment achieved > or = 95% donor chimerism within 60 days of transplantation. Two patients with myelodysplastic syndrome rejected their grafts but experienced autologous neutrophil recovery at 24 and 44 days. Histologic acute GVHD developed in 6 patients (grade II in 3 patients, grade III in 3 patients) at a median of 99 days (range, 38-143 days) after transplantation and was fatal in 1 patient. At a median follow-up of 191 days (range, 124-423 days), 6 of 10 patients in cohort 2 were alive, and 5 were in complete remission of their disease, including both patients with graft rejection. These data demonstrate that partially HLA-mismatched bone marrow can engraft rapidly and stably after nonmyeloablative conditioning that includes posttransplantation Cy. Clinically significant antitumor responses occur, even among patients who reject their donor grafts.

Identification of a novel DR4 allele, DRB1*0442.

A new DRB1 allele encoding DR4, DRB1*0442, was identified in three Caucasian siblings by reverse in-line hybridization and defined by sequencing based typing. The DRB1*0442 allele differs from DRB1*0404 by a single nucleotide at position 227 (T-->A) of codon 47 in exon 2. At the amino acid level, this substitution results in a change from tyrosine to phenylalanine. Serologically, the new allele appears to retain the DR4 antigenicity; however, this substitution may affect peptide-binding specificity.

Antigen, allele, and haplotype frequencies report of the ASHI minority antigens workshops: part 1, African-Americans.

HLA typing was performed on 977 African Americans residing throughout most of the United States. Class I and class II antigens and class II alleles were defined for all individuals and class I alleles were determined for a subset of individuals. The occurrence of 854 of the individuals in family groups permitted direct counting of allele and haplotype frequencies. The data were analyzed for antigen, allele, and haplotype frequencies; recombination frequencies; segregation distortion; distribution of haplotype frequencies; linkage disequilibria; and geographic distribution of DR antigens. Tables of the antigen, allele, the most common two and three point haplotypes, and 88 extended haplotypes that include class I and class II alleles are presented. Notable findings include a lower than expected frequency of recombination between the B and DR loci (theta= 0.0013), lower than expected frequency of inheritance (44.5% vs 54.5%) of the DRB1*1503; DQB1*0602 haplotype, lower than anticipated linkage disequilibrium values for DR; DQ haplotypes, and a skewed geographic distribution of DR antigens.

Effect of Bw4 and Bw6 epitope mismatches on antibody production, acute and chronic rejection, and graft survival in renal allografts.

BACKGROUND: Highly sensitized patients often have antibodies directed against the HLA Bw4 and Bw6 epitopes. Because of the high frequency of these epitopes, when present, these antibodies result in a high incidence of positive cross-matches. We sought to determine whether antibodies specific for Bw4 or Bw6 affected renal allograft outcome. METHODS: The effect of mismatches for the HLA class I public epitopes, Bw4 and Bw6, was examined in 72 recipients of one haplotype matched recipients of living, related donor renal allografts selected to control for degree of HLA mismatch. Analysis of the production of HLA-specific antibody was performed for 180 recipients of failed cadaveric allografts by complement-dependent cytotoxicity tests and by an enzyme-linked immunoadsorbent assay (ELISA). RESULTS: No significant difference was observed in the incidence of acute rejection, number of rejection episodes or 1-year allograft survival among Bw4/6 matched versus mismatched recipients of one haplotype matched allografts. Additionally, no significant difference in the development of chronic allograft nephropathy was noted among 56 recipients followed long-term (> or =3 years). In the recipients of failed cadaveric transplants, Bw4/6 mismatching was associated with the frequency and magnitude of production of HLA-specific antibody. However, the panel reactive antibodies correlated with the number of HLA-A and -B mismatches, and there was no additional impact of Bw4/6 mismatching. IgG, HLA-specific antibodies were found to be significantly increased among patients homozygous for Bw4 or Bw6, whether or not there was a Bw4/6 mismatch. CONCLUSIONS: Mismatching for Bw4 or Bw6 does not confer any independent, increased risk for humoral sensitization or renal allograft failure.

Sequence-based typing of HLA class I alleles in Alaskan Yupik Eskimo.

In comparison to South America, native North Americans tend to be less diverse in their repertoire of HLA class I alleles. Based upon this observation, we hypothesized that the Yupik Eskimo would exhibit a limited number of previously identified class I HLA alleles. To test this hypothesis, sequence-based typing was performed at the HLA-A, -B and -C loci for 99 Central Yupik individuals from southwestern Alaska. Two new class I alleles, A*2423 and Cw*0806, were identified. While A*2423 was observed in only one sample, Cw*0806 was present in 26 of the 99 individuals and all of the Cw*0806 samples contained B*4801. Allele Cw*0806 differs from Cw*0803 by a single nucleotide substitution such that Cw*0803 may be the progenitor of Cw*0806. Allele Cw*0803 was originally characterized as unique to South America, but detection of Cw*0803 in the Yupik indicates that Cw*0803 was a founding allele of the Americas. The presence of new alleles and previously unrecognized founding alleles in the Yupik population show that natives of North America are more diverse than previously envisioned.

Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: I. Evaluation of the GTI QuikID assay and analysis of antibody patterns.

The development of solid phase immunoassays using solubilized HLA molecules as targets has provided a means of detecting HLA-specific antibodies that overcomes many of the shortcomings of lymphocyte based assays. We have evaluated a commercially available assay, the GTI QuikID (QID), that uses solubilized class I molecules from 40 subjects selected for their HLA phenotype, to characterize HLA-specific antibodies. We tested 595 sera from 319 subjects and compared the results obtained with QID to those obtained with cytotoxicity (CYT) and with GTI QuikScreen (QS) as well as to historic data. The correlation of QID with CYT (r = 0.54) was comparable to that between QID and QS (r = 0.60). The majority of disparities between QS and QID were apparent false negatives with QID that could be overcome by analyzing QID data at lower cutoff values. In contrast, most of the disparities between QID and CYT were false negatives in CYT due to the relatively low sensitivity of that assay. As expected, the ELISA was more sensitive (97%) than CYT (78%) but had a somewhat lower specificity (87% vs. 92%) due, most likely, to selection of sera that excluded most sera that were known to be nonspecific by CYT. Determination of antibody specificity could be achieved quickly by manual analysis of the QID data because of the way the data are presented by the manufacturer's software. Interestingly, the frequencies of different antibodies detected by ELISA differed from those detected by CYT with ELISA identifying more sera containing antibodies to both A and B locus antigens.

Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: II. Clinical relevance.

Until recently, the nature of humoral sensitization to HLA has been characterized by data from lymphocyte-based assays, predominantly cytotoxicity tests. We have examined the characteristics, determined by enzyme-linked immunosorbent assay (ELISA), of sera from 191 subjects known to have produced HLA-specific antibody. We found that ELISA detected higher frequencies compared with cytotoxicity of many (74.5%), but not all, HLA-specific antibodies; in many cases (42.6%) the frequencies of these antibodies were higher than predicted from population frequencies whereas some antibodies (23.4%) occurred with lower than expected frequencies. Some of the increase in frequencies can be accounted for by crossreactivity, i.e., sensitization to epitopes shared among two or more allelic products. The presence of epitopes shared between a recipient's antigen and a mismatched antigen in a donor also tended to narrow the specificity of antibody produced. However the data also indicate differences in immunogenicity among different antigens suggesting that crossreactive group matching would be beneficial in some but not all cases. Finally, we present case studies to illustrate the value of ELISA in predicting humoral rejection episodes and in monitoring the efficacy of rejection therapies.

Elimination of alloantibodies by immunoablative high-dose cyclophosphamide.

BACKGROUND: Alloimmunization is a major problem for patients being considered for solid organ transplantation and in patients who require blood transfusion support. We previously demonstrated that high-dose cyclophosphamide (200 mg/kg) without hematopoietic stem cell transplantation leads to durable complete remissions in aplastic anemia and other autoimmune disorders. We now examine the ability of high-dose cyclophosphamide to eliminate alloreactivity. METHODS: IgG-specific antibodies to HLA class I were assayed using enzyme-linked immunosorbent assays in 18 consecutive patients with severe aplastic anemia before and after treatment with high-dose cyclophosphamide. RESULTS: Anti-HLA antibodies were detected before or shortly after therapy in 5 of the 18 patients studied. Complete remission of aplastic anemia was achieved in four of these five patients. High-dose cyclophosphamide markedly reduced anti-HLA antibody titers in these four patients; they were completely eradicated in three patients. Only one patient did not achieve significant reduction in the alloantibody titer after high-dose cyclophosphamide. CONCLUSIONS: High-dose cyclophosphamide without stem cell transplantation can eradicate HLA-specific alloantibody.

Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients.

BACKGROUND: Hyperacute rejection (HAR) and acute humoral rejection (AHR) remain recalcitrant conditions without effective treatments, and usually result in graft loss. Plasmapheresis (PP) has been shown to remove HLA- specific antibody (Ab) in many different clinical settings. Intravenous gamma globulin (IVIG) has been used to suppress alloantibody and modulate immune responses. Our hypothesis was that a combination of PP and IVIG could effectively and durably remove donor-specific, anti-HLA antibody (Ab), rescuing patients with established AHR and preemptively desensitizing recipients who had positive crossmatches with a potential live donor. METHODS: The study patients consisted of seven live donor kidney transplant recipients who experienced AHR and had donor-specific Ab (DSA) for one or more mismatched donor HLA antigens. The patients segregated into two groups: three patients were treated for established AHR (rescue group) and four cross-match-positive patients received therapy before transplantation (preemptive group). RESULTS: Using PP/IVIG we have successfully reversed established AHR in three patients. Four patients who were cross-match-positive (3 by flow cytometry and 1 by cytotoxic assay) and had DSA before treatment underwent successful renal transplantation utilizing their live donor. The overall mean creatinine for both treatment groups is 1.4+/-0.8 with a mean follow up of 58+/-40 weeks (range 17-116 weeks). CONCLUSIONS: In this study, we present seven patients for whom the combined therapies of PP/IVIG were successful in reversing AHR mediated by Ab specific for donor HLA antigens. Furthermore, this protocol shows promise for eliminating DSA preemptively among patients with low-titer positive antihuman globulin-enhanced, complement-dependent cytotoxicity (AHG-CDC) cross-matches, allowing the successful transplantation of these patients using a live donor without any cases of HAR.