Activated human T lymphocytes express MHC class I heavy chains not associated with beta 2-microglobulin.

We present here the molecular characterization of a new activation-induced surface structure on human T lymphocytes, termed LA45, with high homology (93% at protein level) to MHC class I molecules. Antigen modulation and sequential immunoprecipitation experiments revealed that LA45 and HLA class I proteins do not crossreact with the corresponding antibodies. Furthermore, LA45 is not associated with beta 2-m. On the other hand, we could show that the separation of HLA-A,B,C and beta 2m molecules, induced by SDS-denaturation, leads to a conformational change in the heavy chain in such a way that it becomes reactive with LA45. The 90/45 kD LA45 proteins thus appear to be non-beta 2m-associated MHC class I alpha chains that are selectively expressed by activated but not by resting human T lymphocytes.

A novel HLA-C allele, Cw*0617.

Sequencing analysis of exons 1-3 of the human leukocyte antigen (HLA)-C gene showed a novel allele, HLA-Cw*0617. While the amino acid sequence is identical with the HLA-Cw*060201 allele, the leader peptide differs in three amino acids.

Haematopoietic stem cell donor registries: World Marrow Donor Association recommendations for evaluation of donor health.

The ability to identify unrelated haematopoietic stem cell donors in one country for recipients in another country requires cooperation and standardization in many areas. The donor assessment and testing are very important issues affecting quality and safety of donation. This special report details the World Marrow Donor Association's recommended procedures regarding the medical evaluation of donors, with the intent to protect the volunteer from the risk to damage his health and to offer the recipient the appropriate quality of stem cells. This document describes criteria for permanent or temporary deferral, guidelines for risk evaluation of infectious disease, examples of conditions requiring assessment and questionnaires designed to elicit relevant information about a donor's medical history and general health.

Impact of HLA class I high-resolution mismatches on chronic graft-versus-host disease and survival of patients given hematopoietic stem cell grafts from unrelated donors.

There is consensus that matching of unrelated donors (URD) and patients for HLA class II alleles improves the outcome of hematopoietic stem cell transplantation (HSCT). However, the significance of HLA class I allelic mismatches for transplant outcome is under discussion and reports on long-term effects like chronic graft-versus-host disease (GVHD) are rare. Thus, we investigated the association of human leukocyte antigen (HLA) class I allele mismatches and outcome in 144 patients given HSCT from URD who were matched for HLA-DRB1, DRB3/4/5, and DQB1 alleles. The risk of chronic GVHD was significantly increased in patients with class I mismatched donors, the mismatch either detected by low- or high-resolution typing. A single HLA class I allele mismatch significantly increased the risk of chronic GVHD in multivariate analysis. Overall survival was significantly reduced in patient/donor pairs with more than one-allele class I mismatch. Thus, selection of unrelated donors for transplantation should be based on high-resolution HLA class I typing.

Lack of association between HLA antigens and anorexia nervosa.

Studies of the human leukocyte antigen (HLA) system were performed in 37 patients with anorexia nervosa to confirm an alleged association with HLA-B16 and haplotype HLA-A26,B38. No correlation between anorexia nervosa and HLA, including HLA-DR, could be found.

Evidence that sensitivity to cyclosporine is influenced by the HLA-DR phenotype of kidney graft recipients.

Clinical as well as experimental studies have shown a great interindividual variability in the immunosuppressive efficacy of CsA. Evaluating previous in vitro findings of a correlation between sensitivity of alloresponsiveness to CsA and the HLA-DR phenotype CsA levels were compared in kidney transplant recipients with and without rejections during the early posttransplant period and tested for a possible relationship to the HLA-DR phenotype of the recipient. In patients treated with CsA and prednisolone only, rejection frequency was significantly higher in HLA-DRw6 positive than in DRw6 negative graft recipients (77% vs. 53%, P = 0.045). In the DRw6 positive group incidence of rejection was independent of CsA blood levels, whereas in DRw6 negative patients frequency of rejection episodes decreased as a function of increasing CsA levels. Therefore the relative risk in developing graft rejection continuously increased in HLA-DRw6 positive patients. In HLA-DR2 positive graft recipients, however, a decrease in the relative risk could be observed with increasing CsA levels. Within patients with bioptically verified rejection episodes HLA-DR2 positive recipients had significantly lower CsA levels than DR2 negative patients (P = 0.01). In other HLA-DR phenotypes no association with CsA blood levels could be assessed. Also no statistically significant difference could be found in nonrejecting patients. These clinical findings demonstrate an association of sensitivity to immunosuppressive treatment and the HLA-DR phenotype of the graft recipient. Our results would indicate a very low CsA sensitivity of HLA-DRw6 positive graft recipients and thus might offer an explanation for previous findings about an increase in the incidence of rejection reported on those patients.

Influence of HLA phenotypes on the inhibition of in vitro alloreactivity by cyclosporine.

Interindividual variations in the immunosuppressive effect of Cyclosporine have been observed in clinical organ transplantation. Searching for an in vitro correlate we investigated a possible relation between inhibition of alloresponsiveness by CsA and the HLA phenotypes of the responder or stimulator in mixed lymphocyte reactions. Peripheral blood mononuclear cells from 28 healthy volunteers were used as responder or stimulator cells (gamma-irradiated) and the inhibitory effect of graded amounts of CsA was determined in 130 criss-cross combinations. Sensitivity of alloresponsiveness to the drug was expressed as the dose causing 50% inhibition (ED50) and was read from the inhibition curves generated after four-parameter logistic curve fitting. ED50 ranged from 0.35 ng/ml to 33.4 ng/ml and correlated only weakly with the magnitude of the response (r = 0.12). In MLC with HLA DR4-positive responder cells, ED50 was significantly lower (Pc = 0.0035, Kruskal Wallis) when compared with MLC with responder cells of other DR haplotypes. For HLA DR5-positive responder cells ED50 was significantly higher (Pc = 0.042) when compared with DR5-negative responder cells. No significant correlation between ED50 and any particular haplotype of the stimulator cells could be observed. Sensitivity to CSA did not differ in MLC with 1 or 2 mismatches in the HLA-DR locus. In summary, we found that sensitivity of in vitro alloreactivity was different for particular HLA DR phenotypes, which may have important implications for the immunosuppressive therapy of transplanted patients with cyclosporine.

HLA-C locus antigens seen by one-dimensional isoelectric focusing: definition of the so far known HLA-C specificities and of two subtypes.

A panel of homozygous typing cells was employed for biochemical definition of all known HLA-C locus antigens from Cw1 through Cw11. Each HLA-C antigen yielded, after precipitation with one of the monoclonal antibodies W6/32, 4E, or HC10, a specific banding pattern on one-dimensional isoelectric focusing gels. In addition, two biochemical subtypes of Cw1 and Cw7 could be identified. These biochemical HLA-C "splits" were characterized not only by different isoelectric points but also by strong linkage disequilibriums with certain HLA-B antigens.

Informed consent--suggested procedures for informed consent for unrelated haematopoietic stem cell donors at various stages of recruitment, donor evaluation, and donor workup.

The Ethics Working Group of the World Marrow Donor Association (WMDA) was established to address the increasing and complex number of ethical issues surrounding unrelated haematopoietic stem cell donation where the selected donor and recipient reside in different countries. This paper considers the topic of informed donor consent, but recognises that the recommendations contained within the paper may be subject to cultural variances in interpretation, and to adjustment to meet the legal requirements of individual countries. Nevertheless, the extent of international cooperation establishes sufficient common denominators for the recommendations to be widely adhered to in the interests of best practice.

Excellent long-term survival after allogeneic marrow transplantation in patients with severe aplastic anemia.

Between 1982 and 1996, 20 patients (10 male, 10 female) with severe aplastic anemia (SAA) with a median age of 25 years (17-37 years), received grafts from an HLA-identical sibling (n = 17), HLA-identical unrelated donor (n = 2) or identical twin (n = 1). The median time from diagnosis to marrow transplantation (BMT) was 15 months (range 1-96 months). More than half of the patients had received more than 10 units of red blood cells or platelet transfusions prior to BMT. Pretransplant immunosuppression consisted of cyclophosphamide (CY) alone (n = 10), CY in combination with total body irradiation (n = 8), and CY and antithymocyte globulin (n = 2). For graft-versus-host disease (GVHD) prophylaxis methotrexate (MTX) alone (n = 9) or MTX with cyclosporin A (n = 10) were given. One patient died on day 18 after marrow grafting due to infection; all other patients had complete and sustained engraftment (95%). Eight patients developed acute GVHD (42%), nine patients chronic GVHD (53%) including four with extensive disease manifestation. One patient experienced a secondary malignancy 11 years after BMT. Eighteen patients followed for a median of 9.45 years (0.42-14.7 years) have sustained hematological reconstitution and are alive and well with a Karnofsky performance score of at least 90%. Thus, excellent long-term survival and low morbidity make allogeneic or syngeneic BMT the treatment of choice for younger patients with severe aplastic anemia.

Matched unrelated donor marrow transplantation in patients with advanced acute leukemia.

Patients with advanced acute leukemia (AL) have a poor prognosis with death due to disease or complications of therapy. High-dose chemoradiotherapy followed by allogeneic marrow transplantation (BMT) has been used to overcome resistance of the leukemic clone resulting in long-term survival of up to 20%. Due to lack of suitable related donors BMT from an HLA-compatible unrelated donor (MUD) has been increasingly applied in these patients during the last years. Between January 1991 and August 1997 twenty five patients with advanced acute myeloid (n=19) or lymphoid (n=6) leukemia, 11 males and 14 females, age 22 to 41 (median 32) years received MUD (n=22) or 1-antigen mismatched unrelated donor (n=3) grafts. In four patients an autologous BMT had been performed previously. For conditioning all patients were given total body irradiation containing regimens. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (CSA) and methotrexate (n=24) or CSA and methylprednisone (n=1). In 23 patients (92%) class II region compatibility was assessed by DRB1, DRB3, DRB5, and DQB1 allele typing by hybridization of amplified DNA with ligation based typing. In 2 patients HLA-DR typing was performed by two colour fluorescence cytotoxicity test and mixed lymphocyte cultures. As of November 1997 10/25 patients (40%) are surviving leukemia-free after a median observation time of 17 (range, 3 to 38) months. Transplant-related mortality was an overall of 36% and 28% in patients receiving their first BMT. In 6/25 patients (24%) relapse occurred 2 to 26 months after BMT. Incidence of acute GVHD grade I to IV was 85%. The probability of relapse projected at 3 years was 35%. High-dose chemoradiotherapy followed by MUD marrow infusion has a curative potential for patients with advanced acute leukemia and offers the chance of long-term leukemia-free survival. Currently, up to 80% of patients with acute myelogenous leukemia (AML) and acute lymphoid leukemia (ALL) under the age of 50 years achieve complete hematological remission (CR) with conventional dose chemotherapy. However, in patients who are refractory to induction chemotherapy or relapse prognosis is still poor. There, high-dose chemoradiotherapy followed by allogeneic marrow infusion has been used to overcome resistance of the refractory leukemic clone and has resulted in long-term survival. For selected patients lacking a human leukocyte antigen (HLA) compatible family donor marrow transplantation (BMT) with a suitable unrelated marrow donor (MUD) has become a feasible and effective treatment. Here, we report our experience in patients with advanced acute leukemia given marrow grafts from unrelated donors.

Low transplant-related mortality in patients receiving unrelated donor marrow grafts for leukemia.

Transplantation with unrelated donor (UD) marrow has been shown to potentially cure patients with leukemia. Between January 1991 and April 1998, 54 patients with leukemia have received an UD BMT at our institution. Five patients received their UD BMT as a second transplant after a preceding autologous or syngeneic BMT and were excluded from further analysis. Forty-nine patients with leukemia (acute leukemia n = 26; CML n = 23) and a median age of 36 years (range 19-51) were analyzed. For conditioning, all patients received a combination of fractionated TBI and CY. GVHD prophylaxis consisted of MTX and CsA in all patients. As of 30 April 1998, 27 of 49 (55%) patients survive after a median observation time of 18 months. The probability of overall survival for standard risk and high risk patients is 54% and 31% (P = 0.05). Probability of transplant-related mortality (TRM) is 27%, 24% in standard risk and 31% in high risk patients (P = 0.44). Patients younger than 40 years (n = 33) had a similar TRM as patients 40 years and older (n = 16). The probability of relapse is 41% for the whole group, 29% for standard risk and 55% for high risk pts (P<0.05). Our data confirm that UD BMT is an effective treatment for patients with leukemia. TRM is almost similar to related sibling BMT, most probably due to improvements in HLA typing technology, conditioning regimen and supportive patient care.

Long-term outcome and quality of life of patients who are alive and in complete remission more than two years after allogeneic and syngeneic stem cell transplantation.

We assessed long-term outcome in 155 patients who had undergone an allogeneic/syngeneic stem cell transplant (SCT) and were in complete remission for more than 2 years after transplant. Probability of late transplant-related mortality was 6%, and affected only patients with chronic graft-versus-host disease (cGVHD). Thirteen percent of patients experienced relapse. Overall survival projected at 10 and 15 years was 83% and 76%, respectively. Secondary malignancies occurred in two patients, 7.5 and 11 years after SCT. Three female and four male patients parented children 19 to 84 months after SCT. Quality of life (QoL) was assessed in a cross-sectional study by the means of a 30-item questionnaire (QLQ-C30) of the EORTC. The questionnaire was sent to 127 patients remaining alive and answered by 106 patients. Seventy-three percent reported a good to very good QoL within 5 years after SCT and 78% after this time point. However, patients with cGVHD had significant impairment of physical, role and social functioning and only 60% of them were fit for work. These results from long-term survivors show that high cure rates with good to very good QoL can be achieved by allogeneic or syngeneic SCT.

[Genetic control of mixed lymphocyte culture]. / Untersuchungen zur genetischen Steuerung der gemischten Lymphozytenkultur.

Eighteen healthy families were HLA typed with highly specific cytotoxic antisera; 10 of them were also HLA-DR typed. All of them were tested in the mixed lymphocyte culture. Two different methods of mixed lymphocyte culture-with respect to the number of cells used-were compared. The influence of D and DR antigens on mixed lymphocyte culture is discussed. A rather rare finding was that two unrelated people were homozygous for DR, but not for D and, hence, different with respect to their D and DR antigens. Seven individuals, among them two pairs of identical siblings, were found to be homozygous for HLA-D and HLA-DR. The possibility of using them as typing cells for the alleles of the HLA-D locus was discussed.

[Reactions of primed lymphocytes (PLT) and their genetic determination (author's transl)]. / Reaktionen geprägter Lymphozyten (PLT) und deren genetische Steuerung.

Lymphocytes of one family member, if cultured in a primary culture with HLA haploidentical lymphocytes of another family member are primed by the foreign HLA haplotype. Primed lymphocytes can recognize cells bearing the priming haplotype in a secondary culture. The kinetics of this reaction (primed lymphocyte typing, PLT) and its genetic regulation were investigated in one family with a HLA-DR homozygous, HLA-D heterozygous member, one family with two HLA-D homozygous probands, one family with a recombination within the HLA system and in three normal families.

[Mixed lymphocyte culture as a compatibility test for bone marrow transplantation]. / Die gemischte Lymphozytenkultur als Verträglichkeitstest für die Knochenmarktransplantation.

The reactions in mixed lymphocyte culture (MLC) between family members from 35 healthy families and 80 families of potential bone marrow recipients are shown. From the low MLC reactions seen in 128 pairs of HLA identical siblings, the limit of "Negative Reaction in Mixed Lymphocyte Culture" valid for our own laboratory can now be defined. The importance of mixed lymphocyte culture as a histocompatibility test for bone marrow transplantation is discussed.

[Computer-assisted histocompatibility assessment in mixed lymphocyte culture (MLC)]. / Computergestützte Verträglichkeitsbeurteilung in der gemischten Lymphozytenkultur (MLC).

Analysis of the results of mixed lymphocyte culture (MLC) for compatibility testing preceding transplantation of bone marrow and other organs has so far required a vast input, both in terms of laboratory staff and work hours. We have developed a computer programme which performs this work rapidly. Graphics of the reaction patterns can be obtained, moreover, and these can prove a helpful tool in interpretation of the results.

[The significance of mixed lymphocyte culture and mitogen-stimulated culture for kidney transplantation (author's transl)]. / Die Bedeutung der gemischten Lymphozytenkultur und der Mitogenstimulation für die Nierentransplantation.

In order to detect HLA-D identity or incompatibility, MLC tests were performed in 21 recipient-donor combinations before kidney transplantation (20 cadaver donors, one living related donor). In 20 cases, the MLC reaction was positive (R.R. greater than 10%). 13 of these patients rejected the transplant during the first three months after transplantation. One case was observed in which the MLC reaction between recipient and donor was negative. In this case, the graft has been functioning for more than a year. The lymphocytes of 7 kidney graft recipients were investigated in mitogen-stimulated lymphocyte cultures. In some cases, rejection crises were recognized in the test on the basis of a significantly enhanced reactivity of mitogen-stimulated lymphocytes. When no increase of proliferation was detected in the cultures, a rejection crisis became most improbable. One crisis failed to be detected and 6 false positive reactions occurred.

[Serologic diagnosis and therapeutic possibilities in habitual abortion]. / Serologische Diagnostik und Therapiemöglichkeit bei Abortus habitualis.

Immunological laboratory investigations and success in the therapy of habitual abortion have led to a new view of the disease. Though we are currently unable to fully understand the underlying mechanisms, it seems to be generally accepted that insufficient production of HLA antibodies at an early stage of gestation is pathogenetically involved. Employing sensitive methods (IPI = immune phagocytosis inhibition assay) antibodies directed against fetal (paternal) histocompatibility antigens can be detected in the sera of healthy women already in the fifth week of gestation. The lack of such antibodies in the sera of aborting women is therefore an important diagnostic criterion. Strikingly good histocompatibility between the partners in couples with habitual abortion is occasionally encountered, providing an explanation for the lack of immune response in these cases. Patients with a classical clinical picture can be treated by immunization with the partner's leucocytes. The rate of success for subsequent pregnancies is about 80%. A positive effect was also observed in patients with primary infertility (about 20%).

[Lymphocyte stimulation in the primed lymphocyte typing test by as yet unknown HLA-linked antigens (WP-alleles)]. / Lymphozytenstimulation im PLT-Test durch bisher unbekannte HLA-gekoppelte Antigene (WP-Allele).

Lymphocytes from HLA-identical unrelated donors often show positive stimulation in the mixed lymphocyte culture, possibly caused by alleles of HLA-linked, but not yet defined gene loci. In order to investigate such determinants lymphocytes from HLA-DR identical individuals were primed and tested in the PLT system. Family and population data indicate that such antigens exist and can be detected in vitro.