Jürgen Enczmann

Analyses of HLA-C–specific KIR repertoires in donors with group A and B haplotypes suggest a ligand-instructed model of NK cell receptor acquisition

To determine the influence of KIR and HLA class I polymorphism on human NK cell repertoires, 32 different clonotypes representing all possible combinations of 4 inhibitory KIR and NKG2A were analyzed by multicolor flow cytometry. In donors homozygous for the common group A KIR haplotype, a significant influence of HLA-C ligands was seen: KIR repertoires were dominated by clonotypes expressing a single KIR for the respective cognate ligand, either the C1-specific KIR2DL3 or C2-specific KIR2DL1. In contrast, in donors possessing the polymorphic group B haplotypes, a similar adaptation to cognate HLA-C was lacking. We suggest that this discrepancy is largely the result of a suppressive effect of the group B-specific KIR2DL2 on the frequency of KIR2DL1(+) NK cells. In functional assays, KIR2DL2 not only recognized C1 but also C2 ligands, showing overlapping specificity with KIR2DL1. Moreover, using an NK cell differentiation assay we show sequential acquisition of KIR2DL2 before KIR2DL1 on developing NK cells. Together, these observations are compatible with a ligand-instructed model of NK cell education, in which recognition of HLA class I by an inhibitory receptor (KIR2DL2) suppresses subsequent expression of a second receptor (KIR2DL1) of related specificity. Importantly, the ligand-instructed model fits to the observed KIR repertoires in both broad KIR haplotype groups.

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.

Prospective, Comprehensive, and Effective Viral Monitoring in Children Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Major advances in the monitoring and treatment of viral infections after hematopoietic stem cell transplantation (HSCT) have been achieved over the last decade. The appropriate extent of viral monitoring and antiviral therapy remains controversial, and reports in pediatric patients receiving allogeneic unmanipulated hematopoietic stem cells (HSCs) are sparse. A total of 40 pediatric patients who underwent HSCT with either peripheral blood stem cells (PBSCs, n = 30) or bone marrow (BM; n = 10) were prospectively monitored every week for viral DNAemia (VDNA) by simultaneous detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), human adenovirus (ADV), and polyoma BK virus (BKV) using real-time TaqMan polymerase chain reaction (PCR). All patients received prophylactic acyclovir and preemptive ganciclovir (GCV) when 500 copies/microg DNA (EBV/HHV6) or >1 copy/microg DNA (CMV) were detected on 2 consecutive measurements. VDNA occurred in 25 of 40 recipients (CMV, 11/40 patients [28%]; EBV, 19/40 [48%]; HHV6, 2/40 [5%]; ADV/BKV, 1/40) and was found exclusively after neutrophil engraftment and in most cases up to day +100. Recurrent VDNA (P = .028) and (readily treatable) viral disease (P = .003) were observed predominantly in patients suffering from nonmalignant diseases, a cohort characterized by delayed lymphocyte engraftment. VDNA occurred more frequently in HLA-mismatched HSCT and in the 24 of 40 patients receiving antithymocyte globulin (ATG). The incidence of EBV, but not that of CMV, was increased in the ATG group. Yet, in these patients, viral loads of both EBV and CMV were higher, but with prompt initiation of preemptive GCV, no posttransplantation lymphoproliferative disorder or other life-threatening morbidities occurred. HHV6 was typically detected at low viral loads (<10(2) copies/microg DNA), with only 5% of HSC recipients fulfilling our HHV6 criteria for triggering GCV treatment. In multivariate analysis, ATG treatment, HLA mismatch, recipient CMV seropositivity, and stem cell source, but not severe acute graft-versus-host disease were identified as independent risk factors for VDNA. This comprehensive viral monitoring program with defined thresholds for initiation of preemptive GCV effectively prevents the development of critical viral disease, even in high-risk patients receiving ATG.

In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway

BACKGROUND Pluripotent unrestricted somatic stem cells (USSC) from UC blood can differentiate into hepatic cells in the in utero sheep model, resulting in 20% human albumin-producing parenchymal hepatic cells without cell fusion or tumor-formation events. Additionally, we have shown in vitro differentiation of USSC by hepatocyte growth factor and oncostatin M induction, causing changes in the gene expression towards the endodermal lineage. Positive glycogen synthase expression and a positive periodic acid-schiff reaction demonstrated a functional production of polysaccharides in the cells. METHODS We describe the in vitro differentiation of USSC towards an endodermal pathway using different matrices, growth factors and organic substances. Also, co-cultures of USSC with primary cells of endodermal tissue were prepared to mimic the biologic niche. We investigated the effect of direct co-culture of USSC with primary rat hepatocytes or with sheep tissue of endodermal origin. Direct co-cultures were set up to ensure cell-cell contacts. For co-cultures without cell-cell contacts, transwell inlays with 1-microm membranes were used to separate the cells. Furthermore, the effect of endodermally conditioned medium was investigated. Changes in the gene expression patterns were analyzed by RT-PCR. RESULTS We have shown that USSC can differentiate in vitro into an endodermal-like cell with a phenotype similar to hepatic cells. Differentiation of USSC with growth factors, retinoic acid, matrigel matrix and different co-cultures led to an increased expression of albumin and also to the detection of GSC, SOX 17, Cyp2B6, Cyp3A4, Gys2, HNF4a, ISL-1 and Nkx6.1. In addition, functional albumin secretion was observed. DISCUSSION Although the differentiation assays demonstrated here produce only an immature hepatocyte-like cell, endodermaly differentiated USSC might be a useful alternative for cell replacement in the future.

Matching of the minor histocompatibility antigen HLA-A1/H-Y may improve prognosis in corneal transplantation.

Background. Minor histocompatibility (H) antigens are peptides of allelic intracellular proteins that play an important role in human leukocyte antigen (HLA) matched transplantations. In an animal model of keratoplasty, minor H antigens have even been reported to exceed the immunogenicity of major H antigens (MHC). This investigation is to assess any benefit of matching the broadly expressed gender (H-Y) and HA-3 antigens in HLA-A1 donor positive human keratoplasty. Methods. A total of 229 HLA-A1 donor positive keratoplasties were analyzed. A Cox proportional hazards model and Kaplan-Meier analysis were applied to estimate the effect of H-Y or HA-3 mismatches on rejection-free graft survival. Results. Eighty-one cases were mismatched for H-Y (male donor to female recipient). A mean follow up of two years showed graft survival as high as 88% in the H-Y compatible group compared to only 77% in the H-Y mismatched group (P=0.02). Eight out of 62 cases were mismatched for HA-3. No statistically significant influence of HA-3 matching on rejection-free graft survival was observed (85% vs. 73%, P=0.52). Conclusion. HLA-A1/H-Y matching and matching for other broadly expressed minor H antigens may further improve prognosis in keratoplasty.

Beneficial effect of matching at the HLA-A and -B amino-acid triplet level on rejection-free clear graft survival in penetrating keratoplasty.

Objective. The beneficial effect of human leukocyte antigen (HLA) matching on long-term prognosis in penetrating keratoplasty is now unequivocal but has to be weighed against the additional waiting period on an individual basis. HLAMatchmaker is a molecularly based algorithm for histocompatibility determination that can identify immunologically acceptable mismatches and thus potentially reduce time on the waiting list dramatically without negatively affecting prognosis. Methods. The HLAMatchmaker algorithm (triplet-string matching) was applied on each of 545 normal-risk keratoplasties for which complete HLA type was known at split-level resolution. Two homogenous groups were defined. Group I consisted of the 147 penetrating keratoplasties with up to 13 triplet-string mismatches (the typical upper limit of foreign in case of a single HLA-A or HLA-B allele mismatch) and was compared to the remaining 398 patients with more triplet mismatches (group II) using the Kaplan-Meier method and log-rank statistics. Analysis of clear graft survival on the basis of conventional HLA-A and HLA-B matching was performed as well. Reduction of time on the waiting list as compared to conventional HLA-A and HLA-B matching was predicted individually. Results. Triplet-string matching yielded 85% rejection-free clear graft survival 3 years after penetrating keratoplasty in group I but only 76% in group II (P <0.05), whereas conventional HLA-A and HLA-B matching did not result in any statistically significant reduction of immune reactions because of lack of statistical power (P =0.08). Triplet-string matching (13 mismatches accepted) reduces median time on the waiting list by 80%. Conclusions. Triplet-string matching seems to improve mid- to long-term prognosis in penetrating keratoplasties while simultaneously reducing time on the waiting list in most cases. It should thus be considered for histocompatibility determination in penetrating keratoplasty.

Recipient cytokine genotypes for TNF-α and IL-10 and the minor histocompatibility antigens HY and CD31 codon 125 are not associated with occurrence or severity of acute GvHD in unrelated cord blood transplantation: A retrospective analysis

Background. In HLA-identical sibling bone marrow transplantation, certain recipient cytokine gene polymorphism genotypes and minor histocompatibility differences influence the occurrence and severity of acute graft-versus-host disease (aGvHD). The present study investigated the role of cytokine tumor necrosis factor (TNF)-&agr; and interleukin (IL)-10 gene polymorphisms HY, HA-1, and CD31 minor histocompatibility antigen (mHag) mismatch in the development of aGvHD after unrelated cord blood (CB) transplant (CBT). Methods. DNA samples of 115 CB recipients and their unrelated CB grafts were analyzed for genotype associated with TNF-&agr; (TNFd3/d3) and IL-10 (IL-10−1064, 11–16) and for disparities in major and three minor histocompatibility antigens, HY, HA-1, and CD31 codon 125. Results were correlated with the incidence of aGvHD grades II to IV. Results. Neither the donor nor the recipient GvHD risk alleles TNFd3/d3 and IL-10−1064 (11–16) were associated with the development of aGvHD grades II to IV and I to IV. Because of the heterogeneity of CBTs, the data were reanalyzed separately for patients with malignancies (n=83) or with inborn errors (n=24). No significant association was observed between the severity of aGvHD and the possession of either TNFd3/d3 or IL-10 (11–16) genotypes. Mismatches for the mHags HY, HA-1, and CD31 exon 125 between donor and recipient did not associate with aGvHD grades II to IV. Conclusions. In contrast to HLA-identical sibling bone marrow transplantation, in mismatched unrelated CBT, neither the cytokine genotypes TNFd3/d3 alone or in combination with IL-10−1064 alleles nor the minor histocompatibility antigens HY, HA-1, and CD31 exon 125 were associated with aGvHD grades II to IV. Further determination of the cytokine gene polymorphism genotypes in CBTs compared with bone marrow transplants may identify those polymorphisms that could be potential predictive markers for the occurrence of aGvHD.

[Long-term results of homologous penetrating limbokeratoplasty in total limbal stem cell insufficiency after chemical/thermal burns].

BACKGROUND Since 1991 homologous penetrating limbokeratoplasty has been performed in 32 patients with severe limbal stem cell insufficiency following chemical/thermal burns. The long-term results considering the effects of HLA matching are presented for the first time. PATIENTS All patients received systemic cyclosporin A and/or mycophenolate mofetil in the postoperative course. In 9 patients grafts with 0-1 HLA mismatches in the HLA A, B and DR loci, in 6 patients grafts with 2-6 mismatches and in 17 patients untyped grafts were used. Long-term clear graft survival was estimated according to Kaplan and Meier. RESULTS Five years postoperatively, 50% of the grafts with 0-1 mismatches, 32% of the grafts with 2-6 mismatches and 18% of the untyped grafts were centrally clear (log-rank-test, p>0.05). CONCLUSIONS Although statistically not significant, HLA matched grafts seem to deliver better results than untyped grafts in penetrating limbokeratoplasty. Improvement of matching strategies and immunosuppression may possibly further improve current results.

Novel mutations of the von Hippel-Lindau tumor-suppressor gene and rare DNA hypermethylation in renal-cell carcinoma cell lines of the clear-cell type

Renal‐cell carcinoma (RCC) is the most common neoplasm of the kidney, accounting for about 3% of all adult malignancies. Histopathologically, 80% of all cases can be classified as clear‐cell RCC. Of these, approximately 55% to 70% are associated with mutations in the von Hippel‐Lindau (VHL) tumor‐suppressor gene. Here, new mutations of the VHL gene were defined by the use of temperature gradient gel electrophoresis and subsequent sequencing. In addition, DNA hypermethylation, an alternative mechanism of VHL gene silencing, was evaluated by methylation‐specific PCR. Twenty‐six clear‐cell, 3 chromophilic, and 2 chromophobic RCC cell lines were analyzed. Among the clear‐cell RCC cell lines tested, 12 (47%) contained 13 mutations overall: 8 (62%) in exon 1, 3 (23%) in exon 2, and 2 (15%) in exon 3. Ten of these mutations have thus far not been described. All single base pair changes were transversions. Six mutations led to alteration of a single amino acid. Seven mutations generated a frameshift or a stop codon. One cell line contained a complex duplication of 36 bp. All cell lines with mutations showed loss of heterozygosity in the VHL gene. No mutations could be detected in the chromophilic or chromophobic RCC samples. Significant hypermethylation was not observed in any of the cell lines. These data provide further evidence that distinct mutations in the VHL gene are a characteristic feature of clear‐cell RCC. In contrast, hypermethylation of the gene is probably a rare event. The high frequency of transversion mutations suggests a role for exogenous carcinogens in the etiology of clear‐cell RCCs. Int. J. Cancer 87:650–653, 2000.

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German Caucasoid patients and their corresponding 821 potential unrelated stem cell transplants

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.