Association of anti-human leukocyte antigen and anti-angiotensin II type 1 receptor antibodies with liver allograft fibrosis after immunosuppression withdrawal.

BACKGROUND: Many pediatric patients who receive a living-donor liver transplant undergo withdrawal of immunosuppression (IS). For them, the high incidence of long-term progressive graft fibrosis is of particular concern. METHODS: We conducted a cross-sectional study including 81 pediatric patients who underwent IS withdrawal after living-donor liver transplant at Kyoto University Hospital and whose serum samples and pathological data could be obtained during the analysis period. We examined the association of donor-specific anti-human leukocyte antigen (HLA) antibody (DSA) and angiotensin II type 1 receptor antibody (anti-AT1R Ab) with posttransplant graft fibrosis. Normalized mean fluorescence intensity (MFI) 5,000 or higher and anti-AT1R Ab concentrations 17 U/mL or higher were both considered high level. The patients were classified into an advanced fibrosis group (AFG) (Ishak score ≥ 3) and a control group (CG) (Ishak score ≤ 2). RESULTS: Only one patient demonstrated DSA class I. Among those who demonstrated DSA class II, more AFG patients than CG patients demonstrated high-level mean fluorescence intensity, although the difference was not significant (64% vs. 39%; P=0.053). The incidence of high-level DSA-DRB1, however, was significantly higher in the AFG than that in the CG (40% vs. 4%; P<0.001), but there was no significant difference in DSA-DQB1 or DSA-DRB345. High-level anti-AT1R Ab was significantly more frequent in the AFG than in the CG (65% vs. 36%; P=0.02). All patients with both high-level DSA-DRB1 and high-level anti-AT1R Ab were found to have advanced fibrosis (P<0.001). CONCLUSION: Anti-AT1R Ab and DSA-DRB1 may be candidates as biomarkers of graft fibrosis; both HLA and non-HLA immunity may be involved in graft fibrosis after IS withdrawal.

Different mechanisms causing loss of mismatched human leukocyte antigens in relapsing t(6;11)(q27;q23) acute myeloid leukemia after haploidentical transplantation.

Mismatched human leukocyte antigens (HLAs) on leukemic cells can be targeted by donor T cells in HLA-mismatched/haploidentical stem cell transplantation. In two cases of acute myeloid leukemia with t(6;11)(q27;q23) abnormality presented here, flow cytometry analysis showed a lack of HLA-A unshared between recipients and donors in relapsing leukemic cells after HLA-haploidentical transplantation. However, high-resolution HLA genotyping showed that one case lacked a corresponding HLA haplotype, whereas the other preserved it. These cases suggest that leukemic cells, which lacked mismatched HLA expression, might have an advantage in selective expansion under donor T-cell immune surveillance after HLA-haploidentical transplantation. Most importantly, down-regulation of unshared HLA expression potentially occurs by genetic alterations other than loss of HLA alleles.

ACPA-negative RA consists of two genetically distinct subsets based on RF positivity in Japanese.

HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10(-6) and 0.0011, OR: 1.57 (1.28-1.91) and 1.37 (1.13-1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21-1.89) and 3.08 (1.68-5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote.

A large-scale association study identified multiple HLA-DRB1 alleles associated with ACPA-negative rheumatoid arthritis in Japanese subjects.

BACKGROUND: HLA-DRB1 is associated with rheumatoid arthritis (RA). However, it has recently been suggested that HLA-DRB1 is only associated with patients with RA who have anticitrullinated peptide/protein antibodies (ACPA), which are specific to RA. OBJECTIVE: To elucidate whether specific HLA-DR alleles are associated with ACPA-negative RA development. METHODS: HLA-DRB1 typing was carried out in 368 Japanese ACPA-negative patients with RA and 1508 healthy volunteers as the first set, followed by HLA-DRB1 typing of 501 cases and 500 controls as the second set. The HLA-DRB1 allele frequency and diplotype frequency were compared in each group, and the results of the two studies were combined to detect HLA-DRB1 alleles or diplotypes associated with ACPA-negative RA. RESULTS: HLA-DRB1*12:01 was identified as a novel susceptibility allele for ACPA-negative RA (p=0.000088, OR=1.72, 95% CI 1.31 to 2.26). HLA-DRB1*04:05 and *14:03 showed moderate associations with ACPA-negative RA (p=0.0063, OR=1.26, 95% CI 1.07 to 1.49 and p=0.0043, OR=1.81, 95% CI 1.20 to 2.73, respectively). The shared epitope was weakly associated with ACPA-negative RA, but no dosage effect was detected (p=0.016, OR=1.17, 95% CI 1.03 to 1.34). A combination of HLA-DRB1*12:01 and DRB1*09:01 showed a strong association with susceptibility to ACPA-negative RA (p=0.00013, OR=3.62, 95% CI 1.79 to 7.30). Homozygosity for HLA-DR8 was significantly associated with ACPA-negative RA (p=0.0070, OR=2.16, 95% CI 1.22 to 3.82). It was also found that HLA-DRB1*15:02 and *13:02 were protective against ACPA-negative RA (p=0.00010, OR=0.68, 95% CI 0.56 to 0.83 and p=0.00059, OR=0.66, 95% CI 0.52 to 0.84, respectively). CONCLUSIONS: In this large-scale association study multiple alleles and diplotypes were found to be associated with susceptibility to, or protection against, ACPA-negative RA.

Significant association between chronic antibody-mediated rejection and donor-specific antibodies against HLA-DRB rather than DQB in renal transplantation.

De novo production of antidonor HLA antibody has been reported to be associated with chronic antibody-mediated rejection (CAMR). However, some donor-specific antibodies (DSA) do not seem to cause graft injury. Identification of the DSA responsible for CAMR and establishment of effective screening method for early detection of CAMR are therefore essential. All sera from 586 maintenance renal transplant recipients were examined for HLA antibody using ELISA and Luminex-based assay. Positive sera were divided into high (>20% of positive control), moderate (10-20%), and low (2-10%). Donor specificities were analyzed using single antigen beads. ELISA detected only about half of high HLA antibodies (class I: n = 19, class II: n = 46) measured by Luminex-based assay. DSA against class I and class II were identified in 42% and 87% of high antibodies, respectively, including 78% against DQB and 44% against DRB. Renal dysfunction due to CAMR was closely related to high/moderate DRB DSA (n = 11), but not low DRB DSA (n = 9) nor high/moderate/low DQB DSA alone (n = 20). It was speculated that DRB DSA would be more detrimental to the graft, while DQB DSA were readily detectable in blood circulation. Further study, including detailed pathologic analysis of graft biopsy and long-term follow-up, is necessary.

Successful cord blood transplantation using a reduced-intensity conditioning regimen for advanced childhood-onset cerebral adrenoleukodystrophy.

The CCALD, which is caused by a mutation of the ABCD1 gene that encodes a peroxisomal membrane protein, progresses to a stage where the patient is in a vegetative state and can cause death within 3-5 yr after the appearance of neurological symptoms. Although HSCT is the only means of preventing the progression of this disease, HSCT is currently recommended only for cases diagnosed in the early stages. Previous reports on HSCT in advanced CCALD have indicated that the complications of the HSCT procedure seem to outweigh its benefits with respect to survival and neurological outcome. In this case, we successfully treated advanced CCALD with CBT using a reduced-intensity conditioning regimen to reduce regimen-related toxicity and transplant-associated morbidity and mortality. Neither neurological deterioration nor deterioration of MRI abnormalities were observed during the clinical course. We report that CBT using the reduced-intensity conditioning regimen was well tolerated, stopped disease progression and contributed to a good neuropsychological outcome in this case of advanced CCALD.

Anti-citrullinated peptide antibody-negative RA is a genetically distinct subset: a definitive study using only bone-erosive ACPA-negative rheumatoid arthritis.

OBJECTIVES: ACPA is a highly specific marker for RA. It was recently reported that ACPA can be used to classify RA into two disease subsets, ACPA-positive and ACPA-negative RA. ACPA-positive RA was found to be associated with the HLA-DR shared epitope (SE), but ACPA negative was not. However, the suspicion remained that this result was caused by the ACPA-negative RA subset containing patients with non-RA diseases. We examined whether this is the case even when possible non-RA ACPA-negative RA patients were excluded by selecting only patients with bone erosion. METHODS: We genotyped HLA-DRB1 alleles for 574 ACPA-positive RA, 185 ACPA-negative RA (including 97 erosive RA) and 1508 healthy donors. We also tested whether HLA-DR SE is associated with RF-negative or ANA-negative RA. RESULTS: ACPA-negative RA with apparent bone erosion was not associated with SE, supporting the idea that ACPA-negative RA is genetically distinct from ACPA-positive RA. We also tested whether these subsets are based on autoantibody-producing activity. In accordance with the ACPA-negative RA subset, the RF-negative RA subset showed a clearly distinct pattern of association with SE from the RF-positive RA. In contrast, ANA-negative as well as ANA-positive RA was similarly associated with SE, suggesting that the subsets distinguished by ACPA are not based simply on differences in autoantibody production. CONCLUSIONS: ACPA-negative erosive RA is genetically distinct from ACPA-positive RA.

[Early relapse of hemophagocytic syndrome after reduced-intensity cord blood transplantation for relapsed acute lymphoblastic leukemia].

We report a 4-year-old girl who presented with acute onset of hemophagocytic syndrome (HPS) after induction therapy and HPS relapsed immediately after reduced-intensity cord blood transplantation (RI-CBT) for relapse of acute lymphoblastic leukemia. The patient underwent CBT from 2 locus-mismatched donor, after reduced-intensity conditioning therapy consisting of fludarabine, melphalan, and total body irradiation 4Gy. Prednisolone and cyclosporine were administered for prophylaxis against graft-versus-host disease. Bone marrow examination on day 20 revealed activated macrophages displaying hemophagocytosis. The origin of macrophages was 2(nd) donor derived. After administration of steroids, intravenous immunoglobulin and VP-16, the patient exhibited complete chimerism and remained in complete remission for over one year.

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.

MBL2 single nucleotide polymorphism diversity among four ethnic groups as revealed by a bead-based liquid array profiling.

In the present work we established a rapid, cost-effective and high-throughput method for genotyping using a multiplexed microsphere-based suspension array platform - Luminex xMAP which enabled us to analyze 3 SNPs in the MBL2 gene promoter and 5' UTR, and 3 coding SNPs exon 1 haplotypes, associated with different levels of MBL2 expression. Using this system MBL2 diversity in four different ethnic groups, namely, Asian (Japanese), Caucasian, Hispanic and African-American-assessed. Results showed significant variability in terms of allele, genotype, and haplotype distribution. Characteristic MBL haplotype patterns were defined for each ethnic group. A prevalence of haplotypes coding functional proteins capable of complement activation and pathogen opsonization was observed. Regardless of the significant diversity of individual haplotypes, a high, almost similar (25-28%) proportion of haplotypes associated with MBL deficiency was found in the four ethic groups. The proportion of individuals homozygous for the haplotypes resulting in complete MBL2 deficiency was also significant (2-10%). Considering the role of MBL2 in innate immunity and as a clinically relevant marker, the genotyping approach developed and the knowledge of the genetic variation in different ethnic groups will be relevant to future medical genetic studies.

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.

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.

Phenotype frequencies of autosomal minor histocompatibility antigens display significant differences among populations.

Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen-matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen-matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated.

Effects of HLA allele and killer immunoglobulin-like receptor ligand matching on clinical outcome in leukemia patients undergoing transplantation with T-cell-replete marrow from an unrelated donor.

The responsible human leukocyte antigen (HLA) locus and the role of killer immunoglobulin-like receptor (KIR) ligand matching on transplantation outcome were simultaneously identified by multivariate analysis in 1790 patients with leukemia who underwent transplantation with T-cell-replete marrow from an unrelated donor (UR-BMT) through the Japan Marrow Donor Program. The graft-versus-leukemia (GVL) effect depended on leukemia cell type. HLA-C mismatch reduced the relapse rate in acute lymphoblastic leukemia (ALL) (hazard ratio [HR] = 0.47; P = .003), and HLA-DPB1 mismatch reduced it in chronic myeloid leukemia (CML) (HR = 0.35; P < .001). In contrast, KIR2DL ligand mismatch in the graft-versus-host (GVH) direction (KIR-L-MM-G) increased in ALL (HR = 2.55; P = .017). An increased rejection rate was observed in KIR2DL ligand mismatch in the host-versus-graft direction (HR = 4.39; P = .012). Acute GVH disease (GVHD) was increased not only in the mismatch of HLA-A, -B, -C, and -DPB1, but also in KIR-L-MM-G. As a whole, the mismatch of HLA-A, -B, and -DQB1 locus and KIR-L-MM-G resulted in increased mortality. In conclusion, not only the mismatch of HLA-C and -DPB1, but also KIR-L-MM-G affected leukemia relapse, which should be considered based on leukemia cell type. Furthermore, KIR-L-MM induced adverse effects on acute GVHD (aGVHD) and rejection, and brought no survival benefits to patients with T-cell-replete UR-BMT.

Fetal-maternal microchimerism: impact on hematopoietic stem cell transplantation.

Reciprocal cell traffic between mother and fetus during pregnancy gives rise to postpartum fetal-maternal lymphohematopoietic microchimerism, which is frequently detected in blood or tissue from healthy individuals. Although such microchimerism has been implicated in the pathogenesis of autoimmune diseases and tissue repair, recent clinical experiences have suggested the association of microchimerism with acquired immunologic hyporesponsiveness to non-inherited maternal HLA antigens (NIMAs) or inherited paternal HLA antigens (IPAs); T cell-replete HLA-haploidentical hematopoietic stem cell transplantation from a microchimeric IPA/NIMA-mismatched donor confers relatively lower incidence of severe graft-versus-host disease. The underlying mechanisms by which fetal-maternal microchimerism contributes to IPA/NIMA-specific tolerance are still elusive, although emerging experimental evidence suggests an involvement of the central deletion of IPA/NIMA-reactive T cells, the induction of peripheral regulatory T cells, and affinity-dependent modulation of NIMA-reactive B cells.

Human leukocyte antigens in Japanese patients with biliary atresia: retrospective analysis of patients who underwent living donor liver transplantation.

Biliary atresia (BA) is a neonatal obstructive cholangiopathy characterized by a fibrosclerosing obliteration of the extrahepatic bile duct. The aim of this study was to investigate the relationship between human leukocyte antigens (HLA) and susceptibility to BA. We retrospectively analyzed 392 Japanese patients with BA and without extrahepatic anomalies who underwent living donor liver transplantations at our institute. Healthy Japanese volunteers (n = 828) served as normal controls. A significant positive association was observed between BA and HLA-DR2 (39.0% of patients vs. 30.4% of controls, odds ratio = 1.46, p = 0.029). Two-locus analyses disclosed that DR2 was not independently associated with BA, but the increased frequency of HLA-A24 and -B52 reflected the linkage disequilibrium between -A24, -B52, and -DR2. Moreover, the frequency of the haplotype HLA-A24-B52-DR2 was significantly higher in patients with BA than in the general Japanese populations described in the literature (odds ratio = 2.20, p = 0.00124). These results indicate that the gene for BA susceptibility is in close linkage disequilibrium with the HLA-A24-B52-DR2 haplotype observed in the Japanese population. We speculate that a gene at the locus close to HLA plays an important role in the pathogenesis of BA.

Unmanipulated HLA-haploidentical bone marrow transplantation for the treatment of fatal, nonmalignant diseases in children and adolescents.

Fetomaternal microchimerism has been demonstrated, and immunologic tolerance to unshared HLA antigens between mother and offspring may be suggested. We used T-cell-repleted bone marrow transplantation (BMT) from their HLA-haploidentical mothers to treat 6 patients with fatal nonmalignant diseases. The number of mismatched HLA loci in the graft-versus-host disease (GVHD) direction was 3 in 4 patients and 2 in 2 patients. The number in the host-versus-graft direction was 3 in 4 patients, 2 in 1 patient, and 1 in 1 patient. Microchimerism of inherited paternal antigens was demonstrated in 5 donors, and microchimerism of noninherited maternal antigens was detected in 3 recipients. GVHD prophylaxis consisted of short-course methotrexate, tacrolimus, and mycophenolate mofetil (3 patients) or short-course methotrexate, tacrolimus, and methylprednisolone (1 patient). Engraftment was achieved in 5 patients who had received preconditioning, and T-cell engraftment was confirmed in 1 patient with severe combined immunodeficiency. Acute GVHD developed in 3 patients: grade 1 in 2 patients and grade 2 in 1 patient. Chronic GVHD was observed in 5 patients: localized type in 3 patients and extended type in 2 patients. Five patients were alive 11 to 30 months after BMT and 1 patient died of chronic GVHD. Unmanipulated haploidentical BMT from a maternal donor may be the treatment of choice of poor-prognosis nonmalignant diseases.