Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

The PROCARE consortium: toward an improved allocation strategy for kidney allografts.

Kidney transplantation is the best treatment option for patients with end-stage renal failure. At present, approximately 800 Dutch patients are registered on the active waiting list of Eurotransplant. The waiting time in the Netherlands for a kidney from a deceased donor is on average between 3 and 4 years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality among patients on the waiting list is high. In order to increase the number of kidney donors, several initiatives have been undertaken by the Dutch Kidney Foundation including national calls for donor registration and providing information on organ donation and kidney transplantation. The aim of the national PROCARE consortium is to develop improved matching algorithms that will lead to a prolonged survival of transplanted donor kidneys and a reduced HLA immunization. The latter will positively affect the waiting time for a retransplantation. The present algorithm for allocation is among others based on matching for HLA antigens, which were originally defined by antibodies using serological typing techniques. However, several studies suggest that this algorithm needs adaptation and that other immune parameters which are currently not included may assist in improving graft survival rates. We will employ a multicenter-based evaluation on 5429 patients transplanted between 1995 and 2005 in the Netherlands. The association between key clinical endpoints and selected laboratory defined parameters will be examined, including Luminex-defined HLA antibody specificities, T and B cell epitopes recognized on the mismatched HLA antigens, non-HLA antibodies, and also polymorphisms in complement and Fc receptors functionally associated with effector functions of anti-graft antibodies. From these data, key parameters determining the success of kidney transplantation will be identified which will lead to the identification of additional parameters to be included in future matching algorithms aiming to extend survival of transplanted kidneys and to diminish HLA immunization. Computer simulation studies will reveal the number of patients having a direct benefit from improved matching, the effect on shortening of the waiting list, and the decrease in waiting time.

Predictive impact of allele-matching and EBMT risk score for outcome after T-cell depleted unrelated donor transplantation in poor-risk acute leukemia and myelodysplasia.

Many parameters predict for outcome after unrelated donor (URD) allogeneic hematopoietic stem cell transplantation (alloSCT). High-resolution HLA-matching significantly impacts outcome and also the European Group of Blood and Marrow Transplantation (EBMT) risk score, based on patient age, disease stage, donor type, time from diagnosis to SCT and gender combination, may predict for non-relapse mortality and overall survival (OS). We evaluated the individual and combined effects of allele-matching and the EBMT risk score in 327 patients with poor-risk acute leukemia or myelodysplasia, who received a T-cell depleted URD alloSCT. Matching for HLA-A, -B, -C and -DRB1 alleles (8/8 match) was associated with a 5-year OS of 40% compared with 30% for mismatched (≤7/8) pairs (P=0.02). Patients with EBMT risk scores of 1-2, 3, 4 and 5-7 had 5-year OS estimates of 53, 43, 30 and 20%, respectively (P<0.001). The favorable prognostic impact of an 8/8 donor was most pronounced if the EBMT risk score was low (1-2). Five-year OS was 74±8% vs 39±11% for fully matched patients with a low-risk EBMT score as compared with EBMT low-risk patients with ≤7/8 donors. These data underscore the importance of incorporating both the EBMT risk score and the degree of high-resolution HLA-matching in the risk assessment prior to URD alloSCT.

Adult metachromatic leukodystrophy treated by allo-SCT and a review of the literature.

Five patients with adult-onset metachromatic leukodystrophy (MLD) underwent allo-SCT. Conditioning was reduced in intensity and grafts were obtained from voluntary unrelated donors. All but one graft were depleted of T-lymphocytes. Patient age at transplantation varied from 18 to 29 (median, 27) years. Two patients rejected their graft and MLD progressed. The recipient of the unmanipulated graft converted to complete donor chimerism with normalization of arylsulphatase A (ARSA) levels. Despite ARSA normalization, he deteriorated. Another patient was a mixed chimera. Following escalated doses of donor lymphocyte infusions he converted to complete donor chimerism. His levels of ARSA correlated positively with the percentage of donor cells and MLD was not progressive. The fifth patient died after 35 days from complications associated with GVHD. We conclude that results of allo-SCT in symptomatic MLD patients are poor. However, allo-SCT may stop progression of MLD in selected patients.

Complete genomic sequence of a novel HLA-B allele, B*4456N.

A novel allele, HLA-B*4456N, was identified upon cloning and sequencing of genomic DNA.

Addition of ATG to the conditioning regimen is a major determinant for outcome after transplantation with partially lymphocyte-depleted grafts from voluntary unrelated donors.

We retrospectively analysed the outcome of voluntary unrelated donor (VUD)-SCT in 56 patients after conditioning without or with ATG. All received partially lymphocyte-depleted grafts. Four of 17 patients (24%) who were not given ATG rejected their grafts, as did one of 33 (3%) conditioned with ATG (P=0.02). The incidences of acute graft-versus-host disease grade III/IV were 29 and 6%, respectively (P=0.02), and probabilities of 1-year transplant-related mortality were 64% (95% CI, 44-84%) and 27% (95% CI, 12-42%), respectively (P=0.004). Projected at 3 years, probability of survival was 18% (95% CI, 2-34%) after conditioning without ATG and 60% (95% CI, 43-70%) after conditioning with ATG (P=0.002). Probabilities of disease-free survival (DFS) were 18% (95% CI, 2-34%) and 45% (95% CI, 27-63%), respectively (P=0.005). Patients who did not receive ATG had a probability of current DFS of 18% (95% CI, 3-34%) and this was 60% (95% CI, 43-77%) for the patients conditioned with ATG (P<0.001). We conclude that the addition of ATG to the conditioning regimen is associated with a significantly more favourable outcome in recipients of partially T-cell-depleted grafts from VUDs.

Pseudothrombocytopenia: a report of a new method to count platelets in a patient with EDTA- and temperature-independent antibodies of the IgM type.

Pseudothrombocytopenia is usually associated with blood specimens anticoagulated with ethylenediamine tetraacetic acid (EDTA) or other anticoagulants. It may be caused by temperature-independent, EDTA-dependent antibodies of the immunoglobulin-M (IgM) type. Here a patient with EDTA-independent and temperature-independent pseudothrombocytopenia mediated by IgM or IgM-containing immune complex is reported, and a reliable method is described for a proper counting of platelets in such cases.

HLA-C mismatches induce strong cytotoxic T-cell reactivity in the presence of an additional DRB/DQB mismatch and affect NK cell-mediated alloreactivity.

BACKGROUND: The functional relevance of HLA-C mismatches in an alloresponse is still much debated, putting into doubt the relevance of matching for this antigen in selection of an allogeneic bone marrow donor. In addition to presenting peptides to T cells, HLA-C also functions as a ligand for killing inhibitory receptors (KIRs) on natural killer (NK) cells. In the current study we provide an in vitro basis to address the question of whether mismatches for this antigen are a risk factor for acute graft-versus-host disease (GVHD). METHODS AND RESULTS: By analysis of cytotoxic and helper T-lymphocyte precursor frequency (CTLp-f and HTLp-f) in 153 pairs, we are able to show that isolated HLA-C mismatches appear less immunogenic than do isolated HLA-A mismatches. Strikingly, the presence of an HLA-C mismatch next to a HLA-DRB or HLA-DQB mismatch leads to a synergistic increase in CTLp-f outcome. Moreover, we are the first to show that absence of a single inhibitory epitope as a result of an HLA-C mismatch can be sufficient to induce NK mediated alloreactivity, that is, kill and proliferate. CONCLUSIONS: We conclude that, in most cases, isolated HLA-C mismatches may be acceptable with respect to T-cell-mediated alloreactivity; however, the presence of a strong helper epitope (DR/DQ mismatch) appears sufficient to overcome the low immunogenicity of HLA-C. HLA-C mismatches that affect KIR epitopes, can induce NK mediated alloreactivity. This suggests that, in HLA-A-, -B-, -DR-, and -DQ-matched patients, NK cells may play a role in the induction and development of acute GVHD.

Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool for distinguishing permissible from non-permissible class I mismatches in T-cell-depleted allogeneic bone marrow transplantation.

Matching for HLA has been the gold standard in bone marrow donor selection. But, with the ever increasing number of identified HLA alleles, it is becoming more difficult to find a fully HLA-identical donor other than a sibling. Retrospective analysis revealed that HLA mismatches do not necessarily give rise to acute graft-versus-host-disease (GVHD). However, we have no means of defining these 'permissible' mismatches before bone marrow transplantation (BMT). Thus, we set out to establish whether functional matching by means of helper and cytotoxic T-lymphocyte precursor frequency analysis (HTLp-f and CTLp-f respectively) can be applied to this end. Fifty-five recipient-donor pairs other than HLA-identical siblings, the recipient of which received a T-cell-depleted graft, were analysed by high-resolution HLA typing and/or HTLp-f/CTLp-f analysis. The predictive value of the CTLp-f assay for development of acute GVHD was confirmed. More importantly, our data indicate that the CTLp-f assay was able to discriminate permissible from non-permissible HLA-A, -B or -Cw mismatches, but not for DRB/DQB mismatches. The absolute number of alloreactive CTLs present in the graft correlated with the risk of acute GVHD. Although HTLp-f and CTLp-f together had a high negative predictive value, HTLp-f outcome by itself was not correlated with acute GVHD. As we have no evidence yet that HTLp-f or CTLp-f can identify permissible DRB/DQB mismatches, high-resolution matching for these antigens remains the best option. The combination of high-resolution DRB/DQB typing and the CTLp-f assay would enable the accurate prediction of the risk of acute GVHD while extending the pool of potential donors. Furthermore, it would enable adjustment of the number of T- cells in the graft accordingly to improve clinical outcome.

DPB1*7601, a novel DPB1 variant in the Caucasoid population.

A new DPB1 allele has been identified in a Caucasoid individual, DPB1*7601. The sequence of the complete second exon has been confirmed by cloning and subsequent sequencing. This allele differs by one amino acid, at codon 36, from DPB1*1401, as indicated by SBT and PCR-SSP analysis. The amino-acid motif introduced by the change is shared by DPB1*0401 and some rare alleles. It remains unclear whether the change is due to interallelic microgen conversion or a single point mutation.

Helper and cytotoxic T cell precursor frequencies are not predictive for development of acute graft-versus-host disease after partially T cell-depleted HLA-identical sibling BMT.

Despite the use of partially T cell-depleted grafts, 20% of the recipients of an HLA-identical sibling marrow graft develop aGVHD > or = II. This indicates that the current method for selecting a sibling donor, ie serological typing for HLA-A, B and DR, and a mixed lymphocyte culture (MLC) or molecular typing for HLA-DRB/DQB, is not predictive for aGVHD. In order to optimise our selection procedure, we retrospectively analysed patients who developed aGVHD > or = II by means of sequencing based typing for HLA-DPB and frequency analysis of alloreactive helper and cytotoxic T lymphocyte precursors (HTLp-f and CTLp-f). Patients who did not develop aGVHD or developed aGVHD grade I served as controls. Retrospective typing for HLA-DPB revealed only a single disparity in the group with aGVHD > or = II, indicating that mismatches for antigens other than HLA are the major cause of aGVHD in these patients. Furthermore, in our patient group, neither HTLp-f nor CTLp-f were predictive for development of aGVHD indicating that these assays in their current set-up are insufficiently sensitive to predict aGVHD in BMT with a partially T cell-depleted graft. We conclude, that HLA-identical siblings can be identified by means of serological typing for HLA-A and B and intermediate resolution molecular typing for DRB and DQB, but that for the prediction of aGVHD cellular tests with higher sensitivity and specificity as compared to the currently used HTLp-f and CTLp-f assays need to be developed.

A single [3H]thymidine-based limiting dilution analysis to determine HTLp and CTLp frequencies for bone marrow donor selection.

Histocompatibility between recipient and donor is a critical factor in allogeneic BMT which, to a large extent, determines the incidence of GVHD after BMT. Functional histocompatibility assays, such as the helper T lymphocyte precursor frequency assay (HTLp) and the cytotoxic T lymphocyte precursor frequency assay (CTLp), have proved to be helpful tools in facilitating donor selection procedures. However, a major drawback of these assays is that they are laborious and require large numbers of cells. We therefore adapted a [3H]thymidine-based assay, the 'JAM' test, as a read-out for CTLp frequencies, to replace the more cumbersome 51Cr-release assay. Furthermore, we applied an experimental setup that enables the assessment of HTLp and CTLp frequencies from a single limiting dilution assay to reduce the number of cells needed. The newly developed assay is relatively easy to perform and has the advantage that different subsets of T cells can be quantified in a single ongoing alloreaction. When the combined assay was applied in unrelated donor selection it proved to be a sensitive method that enables differentiation in suitability of distinct donors for a single patient. Therefore, the combined HTLp/CTLp assay appears to be a practical and sensitive method for identifying functional histocompatibility in related and unrelated donor/recipient combinations.

Specificity and Ig class of preformed alloantibodies causing a positive crossmatch in renal transplantation. The implications for graft survival.

Sixty-five kidney transplantations performed across a non-current alloantibody-positive T cell crossmatch or an alloantibody-positive B cell crossmatch were studied retrospectively. The DTT crossmatch was used to discriminate between IgM and IgG donor-reactive antibodies. Subsequently the HLA specificity of donor-reactive IgG antibodies was determined in the MAILA assay. The first transplantations performed across a non-current positive T cell DTT crossmatch (IgG) were associated with poor graft survival, as only 5 of 11 (45%) transplants were functioning at 1 year. The HLA specificity of donor T cell reactive IgG antibodies appeared to determine the fate of the graft: only 2 of 7 (29%) patients with donor HLA class I-reactive antibodies had functioning grafts at 1 year, whereas all 3 patients with donor T cell-reactive antibodies, lacking HLA specificity, had functioning grafts. In 17 first transplantations, 15 grafts (88%) transplanted across an IgM-positive B cell crossmatch were functioning at 1 year. In 9 re-transplantations we found 6 grafts (67%) functioning at 1 year. B cell-reactive IgG antibodies, however, were associated with poor graft survival. In 7 first transplantations 2 grafts (29%) were functioning at 1 year, and in 17 re-transplantations 8 grafts (47%) were functioning at 1 year. For 19 patients the HLA specificity of donor B cell-reactive IgG antibodies was determined. Thirteen patients had HLA class II (-DR and/or -DQ)--specific antibodies; of these, 4 (31%) had a functioning graft at 1 year. Two of 3 (67%) patients with weak HLA class I--reactive antibodies and 2 of 3 (67%) patients with B cell--reactive IgG antibodies without HLA specificity had a functioning graft at 1 year. Although the number of cases analyzed is small, the following conclusions can be drawn: First, in general, the presence of donor HLA class I-, HLA-DR-, and HLA-DQ-reactive IgG antibodies is a contraindication to transplantation. However, under certain so-far-unknown conditions, transplantation across donor-reactive HLA specific IgG alloantibodies might be possible. Second, renal transplantation can be safely performed across B cell-reactive IgM antibodies. Third, donor-reactive IgG antibodies that do not recognize HLA do not seem to be harmful.

Immunotherapy with monoclonal anti-idiotypic antibodies: tumour reduction and lymphokine production.

A patient with a B-cell chronic lymphocytic leukaemia was treated with a murine IgG1 monoclonal anti-idiotypic antibody (MoAb anti-id). After a total of 773.2 mg MoAb anti-id had been administered with a maximum daily dose of 83.2 mg, 90% tumour reduction was established within 2 weeks. Although in vitro the tumour cells were stimulated by MoAb anti-id no signs of tumour cell activation were observed in vivo during therapy with MoAb anti-id. The rapid tumour reduction suggests that the proliferation-enhancing property of anti-id is not a contra-indication for immunotherapy. The FcR II receptors on the patients monocytes could interact with the IgG1 monoclonal used. MoAb anti-id administration induced strongly decreased platelet counts, dependent on the amount of serum idiotype that had to be cleared. Antibody administration activated the macrophage/monocyte system, reflected in the neopterin profile, resulting in TNF alpha production and simultaneously strong reductions of circulating tumour cells. The partial remission lasted 3 months, then the tumour reappeared. These data show that a straightforward therapy with MoAb anti-id, in itself, has a strong potential, but is not sufficient to eradicate the tumour permanently. Further study will be needed to improve the clinical results with this kind of therapy.

Phenotypic and functional changes of tumour cells from patients treated with monoclonal anti-idiotypic antibodies.

In this paper data are presented indicating that immunotherapy with monoclonal anti-idiotypic antibodies (MoAb anti-id) can provoke different responses in the B-cell tumour concerned. With respect to the course of disease during and after immunotherapy, the in vitro findings may very well explain the in vivo observations in the two patients (D.E.F., B.O.R.) with B-cell chronic lymphocytic leukaemia (B-CLL) who were treated with MoAb anti-id. After initial tumour reduction, there was a recurrence of tumour cells with altered functional and phenotypic properties. In both cases the recurring tumour cells still expressed the same idiotype. In one patient (D.E.F.) the phenotypic changes (a surface Ig change from IgM, IgG, IgA, and IgD to weakly positive IgM and IgD) and functional changes (a 10-fold increase in [3H]thymidine uptake and a decreased idiotype secretion in vitro), together with the in vivo findings with respect to the course of disease--at relapse an impressive tumour regrowth rate with constant serum idiotype level--suggest that immunoselection might have taken place favouring the survival and relapse of a less mature, more aggressive tumour cell population with a lower idiotype expression. In the second patient (B.O.R.), the phenotypic changes (an isotype change from IgM and IgD to IgM with the loss of IgD, and a gradual decrease in expression of CD19 and CD24) and functional changes (a 10-fold increase of idiotype secretion in vitro), together with the in vivo finding that the serum idiotype level had increased 25-fold compared with the preimmunotherapy serum level with comparable tumour load, strongly suggest an immunotherapy-induced differentiation of the malignant B cell. We also describe an increased expression of CD74, detected by MoAb BoM22, on the recurring tumour cells of patient B.O.R., whereas the expression of HLA-DP, -DQ and -DR did not change. The significance of this finding is unclear.

Thrombocytopenia due to aurothioglucose, sulphasalazine, and hydroxychloroquine.

A 56 year old woman with rheumatoid arthritis developed relapsing thrombocytopenia during successive treatments with aurothioglucose, sulphasalazine, and hydroxychloroquine. The presence of IgM or IgG antibodies or immune complexes reactive with autologous platelets could not be shown. Relapsing thrombocytopenia may indicate a genetically determined HLA-DR3 and B8 aberrant immunological response to stimuli such as certain second line drugs.

The development of non-responsiveness to immunotherapy with monoclonal anti-idiotypic antibodies in a patient with B-CLL.

A patient having a B cell chronic lymphocytic leukaemia was treated with a monoclonal anti-idiotypic antibody (MoAb anti-id). Up to 24.5 g of MoAb anti-id has been administered to the patient over a period of 1 year without serious side effects. Despite a substantial amount of serum idiotype (id = 100 micrograms/ml) and a low expression of id on the tumour cells (+/- 6000 molecules per cell) clearance of serum id and a marked tumour reduction was obtained. Therapy resistance developed and coincided with a decreased clearance rate of circulating id-anti-id immune complexes and an increased modulation of cellular id expression, in vivo. This suggests that a decreased clearance rate of anti-id coated tumour cells provided more time for id modulation in vivo, resulting in therapy resistance. Therefore, the overall capacity of the natural effector system may have an important influence on the ultimate therapeutic effect of immunotherapy with MoAb anti-id. Although the partial remission obtained was not long-lasting, this study shows that MoAb anti-id therapy can be effective even when id expression on the tumour cells is low and a substantial amount of serum id is present.