Incidence and specificity of HLA antibodies in multitransfused patients with acquired aplastic anemia.

BACKGROUND: This study aimed to establish the prevalence and characteristics of anti-HLA in antibody acquired aplastic anemia patients following cessation of antithymocyte globulin therapy and to characterize antibody in terms of epitope specificity. STUDY DESIGN AND METHODS: One hundred and fifty multitransfused, untransplanted patients from eight European centers were investigated by serologic methods. RESULTS: Sixty-two percent were antibody positive. Eighteen HLA-Class-I-specific antibodies (15 IgG, 3 IgM) were identified in 13 patients; 13 antibodies were specific for HLA-A epitopes and 5 for HLA-B. Epitope analysis identified significant correlation between serum reactivity and amino acid substitutions associated with HLA-Class-I epitopes. An excess of antibodies to HLA-A1-associated cross-reactive groups was identified. There was no significant difference in antibody frequency in patients taking cyclosporine compared to those who were not. CONCLUSION: Data suggested a contribution from B cell memory of alloantigens introduced during pregnancy. In some cases, antibody production continued many years after the last transfusion, and although the target varied between individual patients, the antibody to HLA was focused on a few specific Class I epitopes, the majority of which mapped to the HLA-A molecule.

Quantitation of cyclosporine-sensitive and -resistant allospecific cytotoxic cells at birth.

In the absence of clinically relevant models of acute rejection we have attempted to develop an assay to measure cyclosporine-resistant allospecific cytotoxic cells in vitro, beginning at birth. The principle of limiting dilution analysis was applied to investigate umbilical cord bloods as responders. Responders were incubated for 1 h in different concentrations of cyclosporine and irradiated HLA mismatched stimulator cells from healthy adults added, followed by recombinant IL-2. After 7 days, responders were tested against three europium-labelled PHA blasts: stimulator, responder and third party. A significant number of cyclosporine-resistant allospecific cytotoxic cell precursors were found in cord blood indicating prior activation. They may have been primed in utero against non-inherited maternal HLA antigens. Cyclosporine-resistant allospecific cytotoxic cell precursors were demonstrated in human umbilical cord blood using a quantitative assay. These cells may influence the reaction to subsequent transplants.

A novel polymorphism in the human interleukin-13 (IL-13) promoter.

Using PCR-SSCP and automated nucleotide sequencing we have identified a novel single nucleotide C --> T polymorphism in the human IL-13 promoter, at position -1055 relative to the transcription start site. Allele frequency analysis in a population of normal cord blood donors indicated frequencies of 0.833 (C) and 0. 167 (T).

Modified cytotoxic T lymphocyte precursor frequency assay by measuring released europium in a time resolved fluorometer.

The frequency of cytotoxic T lymphocyte precursors (CTLpf) can be quantified by using the principle of limiting dilution analysis (LDA). Chromium 51 (51Cr) and europium (Eu) release assays are based on the measurement of marker release after lysis of targets by the effector cells. Although, 51Cr release has been widely used to quantify cell lysis since its introduction, it has several disadvantages such as handling and disposal of radioisotopes as well as health risk to personnel involved performing the assay. This situation has led us to adopt a non-radioactive cytotoxicity assay. After 7 days culture the PHA-stimulated targets are labeled with europium DTPA chelate. Lysis of labeled targets by effectors releases the Eu-DTPA complex in culture medium--a highly fluorescent substance. The amount of fluorescence can be measured in a time resolved fluorometer. We describe here some modifications of the original protocol which include optimising IL-2 requirements, reduction of incubation times, addition of an extra spin before 37 degrees C incubation, readjustment of target cells per volume of labeling buffer and other crucial parameters increasing the specificity and sensitivity of CTLpf assay. We are in agreement with others that the Eu-release assay is specific and reproducible. It can be used for the CTLpf estimation as well as other T cell and non-T cell cytotoxicity assays.

The predictive value of epitope analysis in highly sensitized patients awaiting renal transplantation.

The accumulation of highly sensitized patients (HSP) on renal transplant waiting lists is a problem faced by all transplant registries. We have studied the HLA class I serological reactivity of 20 random HSP and have related antibody specificity to primary amino acid sequence. In six patients we identified significant correlations (chi 2 test, r > or = 0.93) between panel reactivity and specific amino acid substitutions characteristic of HLA-A, -B, and -C public epitopes. Antibody reactivity was associated with up to three public epitopes in each patient. The 12 separate antibody specificities identified were associated with 10 residues. Seven correlated with HLA-A locus substitutions (Glu-62/Gly-65, Lys-66, Arg-114, His-114/Tyr-116/Lys-127, Thr-142/His-145 [x2], and Thr-149), two with HLA-B locus substitutions (Thr-24, Ser-24) and three with interlocus antibodies associated with either HLA-A and B (Leu-82/Arg-83 [x2]) or with HLA-B and -C substitutions (Leu-163). This information allowed us to predict HLA class I allelic products of known primary sequence that would react negatively with each HSP serum. Windows of acceptable mismatches (WAMMs) can thus be delineated with a view to crossmatch negative transplantation without the need for exhaustive serological analysis. Surprisingly we found that WAMMs for these patients included up to 80% of the 10 commonest HLA class I haplotypes in the British population with four patients being crossmatch compatible with A1,3; B7,8. These observations lead us to propose a more intelligent approach to transplanting HSP based on epitope analysis and definition of WAMMs.

Current status of unrelated-donor bone marrow transplantation. The International Marrow Unrelated Search and Transplant (IMUST) Study.

1. The International Marrow Unrelated Search and Transplant (IMUST) Study 1 provides novel prognostic data on outcome of unrelated-donor (UD) searches for patients with well-defined clinical characteristics. Case-types analyzed by multifactorial methods reveal the importance of HLA phenotype, ethnic mismatching, and stage of disease at search request, in predicting search outcome. White patients, with common HLA types and early disease, were least likely to suffer search failure. In contrast, searches for non-White patients with unusual HLA phenotypes and advanced disease were most likely to fail. Of importance, 70% of patients had HLA phenotypes defined as uncommon. 2. Overall donor yield at the 2 UK registries between 1989 and 1991 was 7%, significantly below expectations. Reasons for this shortfall are that theoretical predictions did not consider ethnic mismatch and logistical delays incurred by outdated UD search routines and most importantly HLA-typing inaccuracies. 3. IMUST Study 2 is a prospective multicenter-controlled cohort study comparing HLA-identical sibling donor (ID) and UD-bone marrow transplantation (BMT) for factors affecting BMT outcome. Generous support was provided by 83 BMT centers worldwide. An interim analysis of 165 UD- and 368 ID-BMT, with at least 6 months follow-up after BMT, is described. Unifactorial analysis showed a probability of engraftment at day 100 of 89% after UD- compared with 98% after ID-BMT (p < 0.001). Probability of Grades II-IV acute graft-versus-host disease (AGvHD) at 100 days was 52% after UD- compared with 42% after ID-BMT (p < 0.01). Probability of overall survival at day 400 was 42% after UD- compared with 63% after ID-BMT (p < 0.001). Survival on day 400 of those patients receiving UD-BMT for early disease was encouraging at 52%. 4. Multifactorial analysis was performed on combined data from UD- and ID-BMT cohorts to identify various factors predicting engraftment, AGvHD, and overall survival. Survival after UD-BMT was increased by center experience of UD-BMT and the use of additional pretransplant immunosuppression. Survival was decreased in UD- compared with ID-BMT, by female donor to male recipient and poor-risk disease. Engraftment was improved in ID- compared with UD-BMT, and after UD-BMT at centers experienced in UD-BMT. Engraftment worsened with the use of ex-vivo T-cell depletion for GvHD prophylaxis, in chronic myeloid leukemia, and in male recipients of female marrow.(ABSTRACT TRUNCATED AT 400 WORDS)

A rapid solid-phase rosette-inhibition assay for the detection of Fc receptor (FcRII)-blocking alloantibodies [corrected].

A procedure for the detection of FcR-blocking alloantibodies is described which uses human B lymphocytes immobilised on plastic by poly-L-lysine. Antibodies which inhibited rosette formation between B lymphocytes or Daudi cells and ox erythrocytes coated with rabbit antibodies (EA) were detected in 10 out of 10 sera containing anti-HLA A2 antibodies and 3 out of 3 sera containing anti-HLA class II antibodies. Inhibition of rosette formation (EAI activity) was mediated by protein G-separated IgG. Analysis of rosette formation using these 13 sera and lymphocytes from 39 donors revealed that the degree of inhibition was bimodal; most sera were either clearly inhibitory or non-inhibitory in the assay. However, there was no correlation between inhibition of rosette formation (EAI activity) and lymphocytotoxicity. Four pairs of sera showed similar patterns of reactivity (r greater than 0.6, p less than 0.01; 2 x 2 chi-square test), and cells from 2 donors showed antithetical reactions with 12 of 13 sera (r = -0.86, p less than 0.001). These data suggest that the solid-phase rosette inhibition assay is a rapid and reproducible means of detecting antibodies reactive with non-HLA class I or class II antigens on human B cells.

Applications of automated simultaneous double fluorescence (SDF): I. Unbiased, quantitative HLA-A,B phenotyping.

An automated HLA typing method has been developed and standardized. The procedure involves simultaneous excitation and reading of two fluorochromes, carboxy-fluorescein diacetate and propidium iodide, by a purpose-built Terasaki tray scanner known as the Astroscan 2100. Data obtained from 95 donors typed by this method and by the classical NIH dye exclusion method prove this system to be suitable for routine HLA-A,-B typing (similarity coefficient 0.9861). This comparison has highlighted significant observer bias, whereby the observer tends to downgrade very weakly positive reactions to negative and upgrade moderately positive reactions to strong positive. Elimination of this bias reduces the chances of any clinically disastrous consequence.

Applications of automated simultaneous double fluorescence (SDF). II. HLA class I phenotyping using immunomagnetically separated T lymphocytes.

HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant. Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4-coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test. These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.

Production of monoclonal human antibody to HLA-DR5 (DRw11) by mouse/human heterohybridomas.

We describe here the production of a human monoclonal antibody to the HLA-DR5 antigen. A human B-cell line secreting cytotoxic antibody that reacted preferentially with DR5-positive targets was fused to the mouse myeloma P3X63Ag8.653 and the resulting heterohybridomas cloned twice. The clones secreted human IgM (lambda light chain), which showed specificity for the DR5 antigen in cytotoxicity assays and reacted with DRw11-positive but not DRw12-positive targets. These results demonstrate the potential of this approach to the production of human monoclonal antibodies to transplantation antigens.