Frans Claas

Association between specific HLA combinations and probability of kidney allograft loss: the taboo concept

BACKGROUNDnHLA matching improves the outcome of cadaveric renal transplantation. However, many allografts function well even in the presence of one or more HLA mismatches, which raises the question of whether some mismatches are better recognised by the recipients immune system than others. We aimed to identify mismatched HLA donor-recipient combinations that were associated with increased graft loss.nnnMETHODSnWe selected 2877 first, unrelated renal transplants with a single HLA A, B, or DR mismatch, undertaken between 1982 and 1992, from the Eurotransplant database. To enhance statistical power the analysis was restricted to mismatches of an HLA antigen that occurred in 100 or more donors. 1342 transplants met this criterion and were grouped into a definition set (n = 873) and a validation set (n = 469). In the definition set, we studied further only those recipient HLA antigens that occurred in at least 30 cases within each donor antigen mismatch subset. By a Cox proportional hazards model, donor-recipient combinations that led to significantly higher graft loss than in the whole group were defined. Such combinations were classified as taboo; the remaining combinations were classified as indifferent.nnnFINDINGSn106 individual recipient antigens were found at least 30 times with a corresponding donor mismatch in the definition set; 11 of the 106 had a significant effect on graft survival. Seven combinations were classified as taboo. Taboo combinations, confirmed as such in the validation set, were associated with graft survival of 81% at one year and 50% at 5 years, significantly lower than the rates in the group with indifferent combinations (89% and 69%; p = 0.04) or among 1190 recipients with no mismatches (89% and 72%; p = 0.03). The findings were substantiated by a multivariate analysis that included the effect of patient immunisation, cold ischaemia time, age, and sex.nnnINTERPRETATIONnMismatched donor antigens are differentially recognised depending on the HLA phenotype of the recipient. The findings may have important clinical consequences for graft survival after transplantation.

Fundamental Role for HO‐1 in the Self‐Protection of Renal Allografts

Tissue attenuates to injury by the effects of heme oxygenase (HO)‐1. The induction of HO‐1 expression is modulated by a (GT)n dinucleotide polymorphism in the promoter of the gene, of which increased activity is associated with short (S) (≤27) repeats. We investigated the influence of this HO‐1 gene polymorphism on renal transplant survival.

Detection of panel-reactive anti-HLA class I antibodies by enzyme-linked immunosorbent assay or lymphocytotoxicity: Results of a blinded, controlled multicenter study

A soluble HLA ELISA for the detection of anti-HLA class I IgG antibodies was developed and compared to complement-dependent microlymphocytotoxicity. ELISA plates were coated with a panel of sHLA class I antigens isolated from the culture supernatants of 46 different EBV-transformed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Absorbance was read using an ELISA plate reader and assay results were analyzed by computer. Antibody specificities were determined by Fishers exact test tail analysis. The reproducibility of ELISA assay results was evaluated in a blinded, controlled multicenter study. A total of 102 serum specimens from patients on waiting lists to receive kidney transplants were tested five times by ELISA in five different laboratories. The correlation coefficients (r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and the average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint titration of several serum specimens demonstrated equivalent sensitivity of ELISA and microlymphocytotoxicity (using the anti-globulin antibody protocol). Most of the antibody specificities determined by ELISA were in agreement with specificities determined by microlymphocytotoxicity. To evaluate the correlation of ELISA and microlymphocytotoxicity (CDC) assay results the same 102 specimens were tested six times by CDC in five different laboratories. The interlaboratory correlation coefficient (r) of %PRA values determined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the average agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus microlymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This demonstrates that the detection of anti-HLA class I antibodies by soluble HLA ELISA is a reliable alternative to microlymphocytotoxicity testing.

Immunogenic human leukocyte antigen class II antigens on human cardiac valves induce specific alloantibodies

BACKGROUNDnThe kinetics of panel reactive antibodies (PRA) and incidence of antibodies directed against human leukocyte antigen (HLA) class II were studied in patients who received a cryopreserved cardiac valve allograft.nnnMETHODSnA complement-dependent microlymphocytotoxicity test was used to determine the percentage of panel reactive antibodies. Anti-HLA class II antibodies were measured by two-color fluorescence assays.nnnRESULTSnThe panel reactive antibodies became positive in 25 (78%) of 32 recipients between 1 and 16 months after implantation. Antibodies against HLA class II antigens were detected in 11 (37%) of 30 patients. In 9 (82%) of 11 cases these antibodies were donor specific. The induction of antibodies against donor HLA class II antigens suggests that intact HLA class II antigens are expressed by viable cells within the graft. Dithiothreitol analysis showed that the antibodies were of the immunoglobulin G type. Apparently, the HLA class II antigens are expressed in an immunogenic way, as activation of specific T-helper cells is essential for the switch from immunoglobulin M to immunoglobulin G antibodies.nnnCONCLUSIONSnAllogeneic valve transplantation is associated with the production of donor-specific anti-HLA class I and II antibodies that could contribute to graft failure. This possibly detrimental effect might be prevented by cross matching in sensitized patients.

Influence of non-inherited maternal HLA antigens on occurrence of rheumatoid arthritis

Many HLA-associated diseases occur in patients not carrying the putative predisposing antigen. The suggestion that this might be due to disease heterogeneity is not sufficiently supported by available data. We hypothesise that HLA-DR4-associated genetic susceptibility to rheumatoid arthritis is due to an effect of DR4 on T-cell receptor repertoire expression and that the presence of antigen in the mother is capable of producing this effect in her children, even when DR4 is not inherited by them. To investigate this possibility we HLA typed 94 rheumatoid arthritis patients and their parents and 86 control families. An increased frequency, compared with controls, of non-inherited maternal HLA-DR4 was found predominantly in the mothers of DR4-negative patients. Unexpectedly, we also found an increased frequency of non-inherited maternal HLA-DR6 and a decreased frequency of non-inherited maternal HLA-DR3 in the mothers of DR4-positive patients. The results of our analyses are consistent with our hypothesis.

Alloreactive lymphoid infiltrates in human heart transplants: Loss of class II-directed cytotoxicity more than 3 months after transplantation

From 535 endomyocardial biopsies (87 heart transplant recipients) 283 cell cultures could be generated. All cultures tested contained T lymphocytes and in most cases CD4 was the predominant phenotype at any time posttransplant. A significantly higher proportion of CD8-dominated cultures was found among cultures from biopsies without myocytolysis. In the first 3 months post transplant 57% of cultures showed cytotoxicity against both class I and class II mismatched donor major histocompatibility complex (MHC) antigens, changing to an incidence of 33% at greater than 90 days. This proved to be due to a significant decrease in the number of cultures with human leukocyte antigen class II-directed cytotoxicity. This study shows that early after transplantation a heart transplant is infiltrated with activated donor-specific cytotoxic T cells which recognize a broad spectrum of mismatched donor MHC antigens, and that in time this spectrum becomes more restricted.

The relative radioresistance of interleukin-2 production by human peripheral blood lymphocytes: consequences for the development of a new limiting dilution assay for the enumeration of helper T lymphocyte precursor frequencies

We describe a limiting dilution assay for the enumeration of alloreactive helper T lymphocyte precursor frequencies in human peripheral blood. The proliferation rate of the murine indicator cell line, cytotoxic T lymphoblastic line 2 (CTLL-2) induced by interleukin-2 (IL-2) culture supernatants was determined by staining with the fluorescent DNA dye propidium-iodide. Lymphocytes from healthy individuals as well as from patients with end stage kidney disease and no previous allosensitization exhibited a relative radioresistance of their IL-2 production up to gamma irradiation doses of 40-60 Gy. This differs from previous findings in the literature, showing a total inhibition of the IL-2 production in unsensitized individuals using a gamma irradiation dose of 20 Gy. The consequences of this relative radioresistance are that for a reliable stimulator cell inactivation in assays for the enumeration of helper T lymphocyte precursors gamma irradiation doses of at least 50 (-60) Gy are needed. Increasing the gamma irradiation dose for the inactivation of the stimulator cells can result in a decrease of the antigen presenting capacity of these cells.

Drug-induced agranulocytosis: review of possible mechanisms, and prospects for clozapine studies

Although toxicity and inborn errors of metabolism may also be involved, immunological reactions play an important role in the induction of drug-induced agranulocytosis. Drug-induced antibodies may lead to agranulocytosis by at least three different immunological mechanisms. Immune complexes may selectively adhere to granulocytes or their immature precursor cells, the drug may bind to the granulocytes as carriers of the immunogenic drug and finally the drug may induce antibodies directed to granulocyte-specific structures. The use and the interpretation of in vitro assays to detect drug-dependent antibodies against granulocytes or myeloid precursor cells are discussed. These assays will be used to detect a possible immunological mechanism involved in clozapine-induced agranulocytosis. Further studies will concern the identification of possible genetic risk factors associated with clozapine-induced agranulocytosis.

Transfusion-associated graft-versus-host disease in immunocompetent patients: a self-protective mechanism

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare but hazardous complication caused by transfusion of leucocyte-containing blood. It is not clear why some patients are at risk for TA-GVHD, but others are not. We studied TA-GVHD in immunocompetent individuals by looking at donor-anti-host reactivity after transfusion of leucocyte-containing blood. We tested reactivity in 62 donor-recipient combinations in vitro before and at different times after blood transfusion with mixed lymphocyte culture. One patient was studied in more detail. Reactivity of blood donors against hosts gradually decreased after blood transfusion. This decrease significantly correlated with time (p < 0.001). Studies in a single patient showed that absence of donor-host reactivity was due to host T cells that inhibited the response. These data indicate that an active mechanism exists in immunocompetent individuals which inhibits the graft-versus-host reaction of the donor against the host. This mechanism might be exploited in organ transplantation by pre-transplant blood transfer.

Neonatal tolerance revisited

Of the many thousand patients who are waiting for a kidney transplant an increasing percentage cannot be transplanted because they have formed broadly reactive leucocyte antibodies. Broadly reactive leucocyte antibodies are here defined as antibodies that react with more than 85°70 of a panel of at least 50 unrelated donors. At present the percentage of such patients in Eurotransplant is over 10070. In the rest of Europe it is nearly 15070 [1]. These patients are very difficult to transplant because the crossmatch with potential donors is nearly always positive. Only if donor and recipient are HLA-A and -B identical or compatible is there a reasonable chance for a negative crossmatch and a successful transplant. However, even with more than six thousand patients on the waiting list of Eurotransplant we are not able to provide an HLA identical graft for more than 5-8070, and this is only possible if the HLAo phenotype of the patient is relatively frequent. It is therefore important to determine whether those patients who often react not only to 85070 of the panel but to almost all panel cells (a so-called 100070 panel reactivity) have formed antibodies against all HLA antigens or whether there remain some antigens towards which they have not formed antibodies. There are two protocols with which one can identify so-called permissible mismatches for these highly immunized patients. We call such anti-