Wil A. Allebes

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.

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.

Pre-kidney-transplant blood transfusions do not improve transplantation outcome: a Dutch national study

BACKGROUND Female renal transplant candidates are prone to be sensitized by prior pregnancies, and undetected historical sensitization might decrease transplantation outcome. Hypothesis of our study was that pre-transplant blood transfusions (PTFs) can elucidate historical sensitization and that the avoidance of the associated antigens can improve transplantation outcome. METHODS Data from all female non-immunized renal transplant candidates who received a random PTF (rPTF) (n = 620), matched PTF (mPTF) (one HLA-A and B and one HLA-DR match) (n = 86) or donor-specific blood transfusion (DST) (n = 100) between 1996 and 2006 were collected. Complement-dependent cytoxicity was used to detect anti-HLA antibodies. Sensitization and transplantation outcomes after a PTF were analyzed. Non-immunized female renal transplant recipients who did not receive a PTF were used as the control group. RESULTS In 165 patients, anti-HLA antibodies (IgG) were detected after the PTF. Both historical and primary sensitizations were found. A DST induced donor-specific anti-HLA antibodies in 25% of the DST recipients. Our policy did not improve transplantation outcome in recipients of a kidney from a deceased donor (n = 368) or in recipients of a living donor [DST (n = 49) and mPTF (n = 66)]. CONCLUSIONS A PTF did elucidate historical sensitization but induce primary sensitization as well. No beneficial effect of PTFs on transplantation outcome was found, and PTFs with the intention to detect historical sensitization are therefore not suggested.

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 ⩾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 ⩾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 ⩾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.

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.

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 μg/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.

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

Summary: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.

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.

Allostimulatory capacity of conditionally immortalized proximal tubule cell lines for bioartificial kidney application

Novel renal replacement therapies, such as a bioartificial kidney (BAK), are needed to improve current hemodialysis treatment of end-stage renal disease (ESRD) patients. As BAK applications may reveal safety concerns, we assessed the alloimmunization and related safety aspects of readily available conditionally immortalized human proximal tubule epithelial cell (ciPTEC) lines to be used in BAK. Two ciPTEC lines, originally derived from urine and kidney tissue, were characterized for the expression and secretion of relevant molecules involved in alloimmunization and inflammatory responses, such as HLA class-I, HLA-DR, CD40, CD80, CD86, as wells as soluble HLA class I and proinflammatory cytokines (IL-6, IL-8 and TNF-α). A lack of direct immunogenic effect of ciPTEC was shown in co-culture experiments with peripheral blood mononuclear cells (PBMC), after appropriate stimulation of ciPTEC. Tight epithelial cell monolayer formation on polyethersulfone flat membranes was confirmed by zonula occludens-1 (ZO-1) expression in the ciPTEC tight junctions, and by restricted inulin-FITC diffusion. Co-culture with (activated) PBMC did not jeopardize the transepithelial barrier function of ciPTEC. In conclusion, the absence of allostimulatory effects and the stability of ciPTEC monolayers show that these unique cells could represent a safe option for BAK engineering application.