Beth W. Colombe

A 2-step approach to myeloablative haploidentical stem cell transplantation: a phase 1/2 trial performed with optimized T-cell dosing.

Studies of haploidentical hematopoietic stem cell transplantation (HSCT) have identified threshold doses of T cells below which severe GVHD is usually absent. However, little is known regarding optimal T-cell dosing as it relates to engraftment, immune reconstitution, and relapse. To begin to address this question, we developed a 2-step myeloablative approach to haploidentical HSCT in which 27 patients conditioned with total body irradiation (TBI) were given a fixed dose of donor T cells (HSCT step 1), followed by cyclophosphamide (CY) for T-cell tolerization. A CD34-selected HSC product (HSCT step 2) was infused after CY. A dose of 2 × 10(8)/kg of T cells resulted in consistent engraftment, immune reconstitution, and acceptable rates of GVHD. Cumulative incidences of grade III-IV GVHD, nonrelapse mortality (NRM), and relapse-related mortality were 7.4%, 22.2%, and 29.6%, respectively. With a follow-up of 28-56 months, the 3-year probability of overall survival for the whole cohort is 48% and 75% in patients without disease at HSCT. In the context of CY tolerization, a high, fixed dose of haploidentical T cells was associated with encouraging outcomes, especially in good-risk patients, and can serve as the basis for further exploration and optimization of this 2-step approach. This study is registered at www.clinicaltrials.gov as NCT00429143.

The effect of hla class I (A and B) and class II (DR) compatibility on liver transplantation outcomes : An analysis of the OPTN database

The purpose of this study was to explore the relationship between human leukocyte antigen (HLA) compatibility and liver transplantation outcomes by analyzing the effect of HLA compatibility on 5‐year graft survival. We analyzed first liver transplants between 1987 and 2002 in the Organ Procurement and Transplantation Network (OPTN) database, where A, B, or DR loci data were available. Graft failure was defined as retransplantation or death from transplant‐related cause. We evaluated associations between total and locus‐specific match levels and 5‐year graft survival. Multivariable Cox proportional‐hazard models were used to evaluate statistical interactions and adjust for the effect of potential confounders. Among 29,675 first‐time transplants, the overall degree of HLA match had no effect on 5‐year graft survival, even after controlling for potential confounders. Univariate and multivariable analyses showed that the 0 HLA antigen mismatch cohort of patients had higher 5‐year graft failure rates than the other 6 antigen mismatch groups. However, this occurred in a small group with a disproportionately large number of live donors and split‐liver recipients. When these recipients were excluded from the analysis, the effect was no longer seen. Finally, multivariable, locus‐specific analyses showed no association between 5‐year graft survival and degree of match/mismatch and the A, B, or DR loci. In conclusion, this careful examination of the OPTN database, with respect to HLA match or mismatch and liver graft survival, reaffirms that HLA matching has no clinically significant impact on this outcome. Liver Transpl 12:652–658, 2006.

HERITABLE FACTORS DISTINGUISH TWO TYPES OF ALOPECIA AREATA

Alopecia areata (AA) has been shown to be associated with the inheritance of HLA class II alleles. HLA-DQ3 appears to be the general susceptibility allele for AA. Patients with long-standing disease patterns, namely, longterm patchy AA and long-term alopecia totalis and alopecia universalis (AT/AU), can be differentiated by their particular HLA associations. Long-standing AT/AU patients have unique and highly significant associations with HLA antigens DR4, DR11, and DQ7. A complex of early onset, disease severity, family incidence, and associations with HLA DR antigens are characteristic of long-standing AT/AU as distinct from long-standing patchy AA.

Acute Humoral Rejection in a Zero Mismatch Deceased Donor Renal Transplant Due to an Antibody to an HLA-DP α

We present a case of a highly sensitized 54-year-old African American male with three prior failed transplants who received a zero mismatch deceased donor kidney and had antibody-mediated rejection directed against a human leukocyte antigen (HLA)-DP donor-specific antigen. The donor was a 21-year-old man with normal renal function. The recipient’s calculated panel reactive antibody was 99%. The standard anti-human globulin-enhanced T-cell and National Institute of Health (NIH) B-cell cytotoxicity crossmatches were negative. Flow cytometric Band T-cell crossmatches were weakly positive by a few channels over the established cutoffs. Intraoperatively, 1 mg/kg antithymocyte globulin, 250 mg methylprednisolone, and 1 g/kg intravenous immunoglobulin (IVIG) were given. Postoperatively, an additional four daily doses of 1 mg/kg antithymocyte globulin, a tapering dose of steroids, and two doses of 500 mg/kg of IVIG were given. The recipient did not have delayed graft function but presented 2 weeks later with increased creatinine and oliguria. Biopsy revealed both borderline acute cellular and acute antibody-mediatedrejection with diffuse C4d positivity in the peritubular capillaries on immunofluorescence. High-resolution HLA typing of the donor was pursued, and a mismatch at the DPA1 locus was identified. Singleantigen bead screening of pretransplant and posttransplant sera revealed a strong complement-fixing antidonor antibody (Luminex C1q antibody fixing assay, reference laboratory: Stanford University Blood Center Histocompatibility Laboratory) against DPA1*0103 with a titer by doubling dilution of 1/1024. The patient received high-dose steroids, multiple rounds of alternate day plasmapheresis, each followed by 100 mg/kg IVIG and 1 g rituximab. His antibody titers decreased to 1/64 and his creatinine nadired at 1.4 mg/ dL. Six months later, he had another 1a cellular and chronic antibody-mediated rejection with a creatinine of 2.1 mg/dL. The United Network for Organ Sharing allocates zero-mismatched kidneys based on six antigens derived from the paired loci A, B, and DR encoded by the major histocompatibility complex located on chromosome 6. The rationale for this policy is improved graft survival in the setting of a more favorable immunologic environment. High-resolution HLA typing is not routinely performed for renal transplantation, and thus, DP status is usually unknown. DPA1 and DPB1 are similarlypolymorphicasA,B,andDR,andasof January 2010, they have 28 and 138 known alleles, respectively (1). DP antigens were discovered in 1980 in restimulation experiments of previously primed T cells. They belong to HLA class II major histocompatibility complex and are composed of polymorphic and chains of similar molecular weight (32 kDa). They are characterized by weak primary allogeneic proliferative response but strong secondary cellmediated cytotoxicity (2). HLA DP mismatches do not influence first kidney transplants but significantly impact outcomes for retransplants (3–5). It has been shown that donor-recipient pairs matched at A, B, and DR have a greater than 80% probability of being mismatched at DP loci (6, 8). We pursued DP typing for the donor, and our pair was matched at A, B, Cw, DR, and DQ alleles as determined by high-resolution (allele level) DNA sequence-specific priming typing (Table 1). The pretransplant sera of recipient contained a strong antidonor antibody against the donor’s DPA1*0103 allele, with a mean fluorescence intensity in the range of 8000 to 14,000. An additional mismatch at DPB1*0601 was revealed and studied, but the antibodies against this allele were found to have mean fluorescence intensity less than 1000. Clinically significant antidonor antibodies to DPB1 have been associated with rejection in both variably matched and zero-mismatched renal allografts (7–9). In addition, antibodies to DPB1 have also been implicated in chronic humoral rejection in retransplants and have been found to share epitope with other HLA molecules (10). Further epitope classification of DPB1 has been pursued, which has helped in the identification of antibodies directed against them (11). It is known that failed renal allografts can elicit an antibody response to DP (12). However, rejection related to antibodies to DPA1 has not been described previously, and our case is unique as it is the first one to report antibody-mediated rejection because of a high-strength antibody to DPA1. It is logical to think that incorporating appropriate donor and recipient HLA-Cw and DP antigens/alleles and antibody information using luminex solid phase assays into UNet would improve concordance between the virtual crossmatching being used for kidney allocation and the actual flow crossmatches. For zero-mismatched grafts, Cw concordance is likely, whereas DP is not. We think that the data forming the basis for virtual crossmatch TABLE 1. Our donor and recipients’ typing by high-resolution DNA sequence-specific priming method

A Two-Step Approach to Myeloablative Haploidentical Transplantation: Low Nonrelapse Mortality and High Survival Confirmed in Patients with Earlier Stage Disease

Haploidentical hematopoietic stem cell transplantation (HSCT) is an attractive alternative donor option based on the rapid availability of an acceptable donor for most patients and decreased cost compared with costs of other alternative donor strategies. The safety of haploidentical HSCT has increased in recent years, making it ethically feasible to offer to patients with earlier stage disease. We developed a 2-step approach to haploidentical HSCT that separates the lymphoid and myeloid portions of the graft, allowing fixed T cell dosing to improve consistency in outcome comparisons. In the initial 2-step trial, the subset of patients without morphologic disease at HSCT had high rates of disease-free survival. To confirm these results, 28 additional patients without evidence of their disease were treated and are now 15 to 45 (median, 31) months past HSCT. To date, the 2-year cumulative incidence of nonrelapse mortality is 3.6%, with only 1 patient dying of nonrelapse causes, confirming the safety of this approach. Based on low regimen toxicity, the probabilities of disease-free and overall survival at 2 years are 74% and 77%, respectively, consistent with the findings in the initial trial and supporting the use of this approach in earlier stage patients lacking a matched related donor.

Characterization of rejection episodes in patients following positive crossmatch and ABO-incompatible live donor renal transplantation.

For kidney transplant recipients with donor‐specific antibody (DSA) to HLA‐ (+XM) or ABO‐antigens (ABOI), there is a need to improve detection and treatment of antibody‐mediated rejection (AMR). The methods included a retrospective review of consecutive patients that received plasmapheresis and immune globulin (PPIVIg) to abrogate +XM or ABOI. Twelve patients were transplanted after PPIVIg (+XM = 9, ABOI = 2, +XM/ABOI = 1). No hyperacute rejections occurred. Rejection occurred in seven patients [four AMR, three acute cellular rejection (ACR)]. In four +XM patients, DSA was detected during graft dysfunction despite lack of histologic and C4d features of AMR. In one patient, DSA preceded the histologic and immunofluorescent features of AMR. In another patient with borderline changes and DSA, graft function improved after PPIVIg, despite lack of histologic or immunofluorescent evidence of AMR. One patient with Banff IIA ACR and DSA treated with antithymocyte antibody but not PPIVIg had recurrent rejections and poor graft function. In +XM and ABOI recipients with graft dysfunction: (i) DSA may represent AMR in the absence of C4d or histologic features of AMR; (ii) DSA can precede C4d or light microscopic features of AMR; (iii) A poor outcome may result if DSA or continued allograft dysfunction is present and not treated despite a negative biopsy.

Two patterns of sensitization demonstrated by recipients of donor-specific transfusion. Limitations to control by Imuran.

Characteristics of the sensitization response to donorspecific transfusion (DST) have been studied in the context of the pretransfusion panel reactive antibody (PRA) status of the recipient. Two distinct patterns of response to DST and Imuran treatment have been found. In patients with one-haplotype-matched donors, the panel nonreactive patient (PRA <10%) has a 19% incidence of DST sensitization that is further reduced by Imuran treatment to 6%; antibodies are both anti-T cell and anti-B cell, are transient, and are specific to the mismatched HLA antigens of the blood donor. Panel-reactive patients (PRA >10%) have a 56% incidence of DST sensitization; the antibodies appear within 2 weeks of the first transfusion, are anti-T cell, and are generally of broad specificity and persistent duration consistent with amplification of a previous antigenic exposure; Imuran seems to have little or no effect in reducing the incidence of sensitization in these panel-reactive patients. However, panel reactive patients whose PRA levels spontaneously fall to panel-nonreactive levels immediately prior to DST therapy have an exceedingly low (0–8%) incidence of sensitization with or without Imuran coverage.

Human leukocyte antigen and adult living‐donor liver transplantation outcomes: An analysis of the organ procurement and transplantation network database

Human leukocyte antigen (HLA) compatibility has no clinically significant impact in cadaveric liver transplantation. Less is known regarding living‐donor liver transplantation (LDLT). Our prior analysis of the Organ Procurement and Transplantation Network (OPTN) database suggested a higher graft failure rate in patients who underwent LDLT from donors with close HLA match. We further investigated the effect of HLA‐A, ‐B, and ‐DR matching on 5‐yr graft survival in adult LDLT by analyzing OPTN data regarding adult LDLT performed between 1998 and 2005. We evaluated associations between 5‐yr graft survival and total, locus‐specific, and haplotype match levels. Separate analyses were conducted for recipients with autoimmune (fulminant autoimmune hepatitis, cirrhosis secondary to autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis) or nonautoimmune liver disease. Multivariable Cox proportional hazard models were used to evaluate interactions and adjust for potential confounders. Among 631 patients with available donor/recipient HLA data, the degree of HLA match had no significant effect on 5‐yr graft survival, even when analyzed separately in recipients with autoimmune vs. nonautoimmune liver disease. To be able to include all 1,838 adult LDLTs, we considered a first‐degree related donor as substitute for a close HLA match. We found no difference in graft survival in related vs. unrelated pairs. In conclusion, our results show no detrimental impact of close HLA matching on graft survival in adult LDLT, including in recipients with underlying autoimmune liver disease. Liver Transpl 13:1405–1413, 2007.

Sensitization trends after renal allograft failure: the role of DQ eplet mismatches in becoming highly sensitized.

Sensitization following renal allograft failure (AF) is highly variable. Some patients remain non‐sensitized (NS), while others become highly sensitized (HS). We studied 66 NS patients who experienced AF after initial kidney transplantation. Post‐failure, two main groups of NS panel reactive antibody (PRA) class I and II <10% and HS patients (PRA class I or II ≥80%) were identified. The impact of acute rejection (AR), immunosuppression withdrawal (ISW) at AF, allograft nephrectomy, graft intolerance syndrome (GIS), and both standard serologic and eplet‐based mismatches (MM) in inducing HS status after failure was examined. Late PRA testing post‐failure revealed 18 patients remained NS and 34 patients became HS. African American recipients, ISW at AF, DQB1 eplet MM, and presence of GIS were associated with becoming HS. Presence of total zero eplet MM, zero DQA1/B1 eplet MM, continuation of immunosuppression after failure, and a hyporesponsive immune status characterized by recurrent infections were features of NS patients. DQ eplet MM represents a significant risk for becoming HS after AF. Studies comparing ISW vs. continuation in re‐transplant candidates with high baseline DQ eplet MM burden should be performed. This may provide insights if sensitization post‐AF can be lessened.

Successful living donor renal transplantation despite ABO incompatibility and a positive crossmatch.

Abstract:  Potential live kidney donors have been rejected when the prospective recipients are blood type or crossmatch incompatible. By utilizing plasmapheresis combined with intravenous immune globulin (PP/IVIg) prior to surgery, donor‐specific antibodies against blood group or human leukocyte antigens (HLA) have been removed, thereby allowing successful renal transplantation. A 26‐yr‐old male with a panel reactive antibody level of 100% and repeated positive crossmatches against deceased donor kidney offers, including zero HLA mismatched donors, successfully underwent ABO‐incompatible kidney transplantation from his HLA‐identical but nevertheless crossmatch‐incompatible sister. The initial anti‐A blood group isoagglutinin titers were 128, 256, and 1024 at room temperature, 37°C, and 37°C anti‐IgG enhanced, respectively. With an individualized PP/IVIg regimen based on donor‐specific antibody titer, however, the relevant antibodies were adequately reduced and hyperacute rejection avoided. Subsequent antibody‐mediated rejection, likely directed against a minor histocompatibility antigen, was diagnosed on postoperative day 7 and successfully treated. Neither ABO, or crossmatch incompatibility, or both in combination prohibit kidney transplantation.