Gottfried F. Fischer

Maternal alloimmunization against fetal platelet antigens: a prospective study.

Summary. Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies to fetal platelet antigens. This prospective study was carried out to evaluate the incidence of anti‐platelet antibodies in 933 mother‐child pairs where the mother and child were typed for the human platelet antigens (HPA)‐l, ‐2,‐3,‐5. Sera from mismatched mother‐child pairs were screened for anti‐platelet antibodies, anti‐HLA class I and blood group ABO IgG antibodies. Platelet‐specific antibodies were anti‐HPA‐3a in one and anti‐HP A‐5b in 17 neonates, respectively. All these neonates had normal platelet counts. One woman had autoreactive antibodies. Anti‐HLA class I and anti‐blood group A IgG antibodies were detected in five and four neonates, respectively, born with a platelet count <150×109/l. None of the 11 homozygous HP A‐lb mothers became immunized against their heterozygous offspring. The maternal HLA‐allotypes HLA‐DR52 and ‐DR6, typically found in individuals immunized against HPA‐la and ‐5b, respectively, were found in three of 11 HPA‐b/b non‐responders and eight of the anti‐HPA‐5b responders. The results indicate that a risk for NAIT due to HPA‐2 and ‐3 alloimmunization is low. The HLA allotypes do not predict the risk for NAIT due to HPA‐1 or ‐5 alloimmunization. Maternal anti‐HPA‐5b antibodies do not correlate with the platelet count in the neonate.

Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level.

Due to IgE cross‐reactivity, birch pollen‐allergic individuals frequently develop type I hypersensitivity reactions to celery tuber. We evaluated the T cell response to the major allergen in celeriac, Api g 1, and the cellular cross‐reactivity with its homologous major allergen in birch pollen, Bet v 1. Api g 1‐specific T cell lines (TCL) and clones (TCC) were established from peripheralblood mononuclear cells of allergic patients. Epitope mapping of Api g 1 with overlapping Api g 1‐derived peptides revealed one dominant T cell‐activating region, Api g 1109–126. TCL and TCC generated with Api g 1 cross‐reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet v 1 were stronger than to Api g 1. Epitopemapping with Bet v 1‐derived peptides revealed that T cells specific for several distinct epitopes distributed over the complete Bet v 1 molecule could be activated by Api g 1. Bet v 1109–126 was identified as the most important T cell epitope for cross‐reactivity with Api g 1. This epitope shares 72% amino acid sequence similarity with the major T cell‐activating region of the food allergen, Api g 1109–126. Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross‐reactivity with the major birch pollen allergen. The activation of Bet v 1‐specific Th2 cells by Api g 1, in particular outside the pollen season, may have consequences for birch pollen‐allergic individuals.

Regeneration of erythropoiesis after related- and unrelated-donor BMT or peripheral blood HPC transplantation: a major ABO mismatch means problems.

BACKGROUND: Blood group incompatibility in allogeneic BMT is common but does not appear to affect the outcome in terms of incidence of graft rejection or delayed engraftment. However, major ABO incompatibility may be associated with prolonged erythroid aplasia.

The T Cell Response to Art v 1, the Major Mugwort Pollen Allergen, Is Dominated by One Epitope

Mugwort (Artemisia vulgaris) pollen allergens represent the main cause of pollinosis in late summer in Europe. At least 95% of sera from mugwort pollen-allergic patients contain IgE against a highly glycosylated 24- to 28-kDa glycoprotein. Recently, this major allergen, termed Art v 1, was characterized, cloned in Escherichia coli, and produced in recombinant form. In the present study we characterized and compared the T cell responses to natural (nArt v 1) and recombinant Art v 1 (rArt v 1). In vitro T cell responses to nArt v 1 and rArt v 1 were studied in PBMC, T cell lines (TCL), and T cell clones (TCC) established from PBMC of mugwort-allergic patients. Stimulation of PBMC or allergen-specific TCL with either nArt v 1 or rArt v 1 resulted in comparable proliferative T cell responses. Eighty-five percent of the TCC reactive with rArt v 1 cross-reacted with the natural protein. The majority of the CD4+CD8−TCR αβ+ Art v 1-specific TCC, obtained from 10 different donors, belonged to the Th2 phenotype. Epitope mapping of TCL and TCC using overlapping peptides revealed a single immunodominant T cell epitope recognized by 81% of the patients. Inhibition experiments demonstrated that the presentation of this peptide is restricted by HLA-DR molecules. In conclusion, the T cell response to Art v 1 is characterized by one strong immunodominant epitope and evidently differs from the T cell responses to other common pollen allergens known to contain multiple T cell epitopes. Therefore, mugwort allergy may be an ideal candidate for a peptide-based immunotherapy approach.

Severe immune hemolysis after minor ABO‐mismatched allogeneic peripheral blood progenitor cell transplantation occurs more frequently after nonmyeloablative than myeloablative conditioning

BACKGROUND : Hemolysis as a result of donor‐recipient minor ABO mismatching is a complication of allogeneic peripheral blood progenitor cell (PBPC) transplantation (PBPCT). The increased B‐lymphocyte content of PBPC grafts and immunosuppressive regimens without methotrexate (MTX) may increase incidence and severity of this event.

Characterization of T Cell Responses to Hev b 3, an Allergen Associated with Latex Allergy in Spina Bifida Patients

The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure such as health care workers and patients with spina bifida (SB). Due to a birth defect of the spinal canal and the resulting neurological and orthopedic defects, these patients require multiple surgeries during childhood. SB patients display a unique pattern of sensitization: IgE-reactivity is preferentially directed against Hev b 3 and Hev b 1, two latex allergens with high sequence similarity. In this study, we analyzed the T cell response to Hev b 3 in latex-allergic SB patients using poly-, oligo-, and monoclonal T lymphocyte cultures. All T cell clones (TCC) were CD3/CD4-positive and expressed the αβ TCR. According to their cytokine production pattern (IL-4 vs IFN-γ), 12 of 21 TCC were classified as Th2-like, 2 of 21 were Th1-like, and 7 of 21 belonged to a Th0-like subset. Using 11 T cell lines and 21 TCC, nine T cell stimulating fragments were determined out of 52 overlapping 12-mer peptides representing the complete amino acid sequence of Hev b 3. Ag presentation of one dominant T cell epitope could be associated with a four-amino acid binding motif (YSTS, position 11–13) in the β1 chain of HLA-DR molecules expressed by the respective patients. No reactivity was observed when Hev b 3-reactive T cell lines or TCC were incubated with peptides representing homologous parts of the Hev b 1 molecule, i.e., no cross-reactivity between Hev b 3 and Hev b 1 at the T cell level was evident.

Allogeneic disparities in immunoglobulin-like transcript 5 induce potent antibody responses in hematopoietic stem cell transplant recipients

In hematopoietic stem cell transplant (HSCT) recipients, the recognition of polymorphic antigens by the donor-derived immune system is an important mechanism underlying both graft-versus-host disease and graft-versus-leukemia (GVL) effect. Here we show that a subset of HSCT recipients (13.9%, n = 108) have antibodies directed to surface molecules of dendritic cells. We have used one such serum in conjunction with retroviral expression cloning to identify the highly polymorphic surface molecule immunoglobulin-like transcript 5 (ILT5) as one of the targets of dendritic cell-reactive antibodies. ILT5 reactive antibodies were found in 5.4% of HSCT patients but not in solid organ transplantation recipients, patients with collagen diseases, multiparous women, or polytransfused or healthy persons. We show that ILT5-specific antibodies can mediate killing of ILT5-bearing cells and furthermore demonstrate ILT5 expression in some leukemic cells, indicating that it might be a target for GVL effects. Thus, our results represent the first description of potent allogeneic antibody responses to a non-major histocompatibility complex cell surface molecule in hematopoietic stem cell transplanted patients and warrant further studies to elucidate the role of antibodies to polymorphic cell surface molecules in GVL and graft-versus-host responses.

Long-term follow-up of patients after related- and unrelated-donor bone marrow transplantation for chronic myelogenous leukemia

Abstract Between January 1983 and December 1997, 88 patients (36 female, 52 male, median age 37 years, range 19–57) with chronic myelogenous leukemia (CML) underwent allogeneic bone marrow transplantation (BMT) at the University Hospital of Vienna. Sixty patients were in chronic phase, 18 in accelerated phase, and ten in blast crisis. Marrow donors were HLA-identical siblings for 64 patients (BM 58, PBSC 6), 2-antigen-mismatched related donors (RD) for two, HLA-identical unrelated donors (URD) for 17, and 1-antigen-mismatched URD for five. The median time from diagnosis to BMT was 22 months (range 2–91), and 63 patients had received prior interferon (IFN)-alpha therapy, 46 (73%) for more than 6 months. Conditioning therapy consisted of cyclophosphamide (CY) and total body irradiation (TBI) in 71 patients and CY and busulfan (BU) in 16. One patient received etoposide and TBI. For graft-versus-host disease (GVHD) prophylaxis methotrexate (MTX) was given to 12 patients, MTX and cyclosporin A (CSA) to 67, CSA alone to four, and CSA and methylprednisolone to five. Durable engraftment was documented in 80 of 82 patients (98%). As of December 31, 1997, 52 patients (59%) were alive, 38 (58%) after sibling transplantation with a median observation time of 73 months and 14 (64%) after URD transplantation with a median observation time of 12 months. Probability of overall survival is 59%, for patients undergoing transplantation in chronic phase and 44% for patients undergoing transplantation in advanced stage CML. Probability of disease-free survival (DFS) after sibling and URD BMT is 55% and 59%, respectively. Ten patients (12%) experienced relapse of CML. Transplant-related mortality was 32% both after RD and after URD transplantation. Acute GVHD occurred in 53 of 80 evaluable patients (66%), consisting of grade III or IV in 14 patients (18%). Chronic GVHD developed in 40 of 63 eligible patients (63%), including extensive disease in 26 patients (41%). Thus, sibling and URD BMT offer high cure rates with acceptable toxicity to patients with CML.

A Combination of Two Distinct in vitro Amplification Procedures for DNA Typing of HLA-DRB and -DQB 1 Alleles

The differential hybridisation of oligonucleotide probes to polymerase chain reaction (PCR)‐amplified DNA has become a standard procedure for tissue typing. We describe a typing method in which differential ligation replaces differential hybridisation, which is a significant simplification of this strategy. After amplification by the PCR two labelled, sequence‐specific oligonucleotides hybridise, in the fluid phase, to one strand of heat‐denatured amplification product in juxtaposition. In the case of perfectly complementary sequences surrounding the gap, a thermostable ligase catalyses the ligation of the two oligonucleotides, otherwise they stay separated. The use of heat‐resistant ligase enables easy repetition of the denaturation‐annealing‐ligation cycle in a thermocycler. The ligation products are detected by an enzyme linked immunosorbent assay. We tested this typing approach in a model system, the characterisation of three functional alleles of HLA‐DRB 3 using three probe pairs. No discrepancies were observed in typing 100 individuals of known genotypes. A total of 33 probe pairs combined with generic and group‐specific amplification allowed the typing of alleles of HLA‐DRB and ‐DQB1 loci at low resolution. We confirmed ligation‐based typing results of 259 individuals with sequence‐based HLA‐DRB1 typing and HLA‐DQB1 typing using PCR with sequence‐specific primers (SSPs). In addition, more than 1,500 ligation‐based HLA‐DRB1 typings were concordant with SSP typing. Excellent signal‐to‐noise ratios in the enzyme‐linked immunosorbent assay make ligation‐based typing remarkably robust. The time requirement of 2.5 h post‐PCR enables practicable typing of putative organ donors. The whole procedure is more easily amenable to automation than methods based on differential hybridisation requiring additional incubators and extra handling for hybridisation and washing.

16th IHIW: Population Global Distribution of Killer Immunoglobulin-like Receptor (KIR) and Ligands

In the last fifteen years, published reports have described KIR gene‐content frequency distributions in more than 120 populations worldwide. However, there have been limited studies examining these data in aggregate to detect overall patterns of variation at regional and global levels. Here, we present a summary of the collection of KIR gene‐content data for 105 worldwide populations collected as part of the 15th and 16th International Histocompatibility and Immunogenetics Workshops, and preliminary results for data analysis.