Antibody-epitope conjugates deliver immunogenic T-cell epitopes more efficiently when close to cell surfaces.

Antibody-mediated delivery of immunogenic viral CD8+ T-cell epitopes to redirect virus-specific T cells toward cancer cells is a promising new therapeutic avenue to increase the immunogenicity of tumors. Multiple strategies for viral epitope delivery have been shown to be effective. So far, most of these have relied on a free C-terminus of the immunogenic epitope for extracellular delivery. Here, we demonstrate that antibody-epitope conjugates (AECs) with genetically fused epitopes to the N-terminus of the antibody can also sensitize tumors for attack by virus-specific CD8+ T cells. AECs carrying epitopes genetically fused at the N-terminus of the light chains of cetuximab and trastuzumab demonstrate an even more efficient delivery of the T-cell epitopes compared to AECs with the epitope fused to the C-terminus of the heavy chain. We demonstrate that this increased efficiency is not caused by the shift in location of the cleavage site from the N- to the C-terminus, but by its increased proximity to the cell surface. We hypothesize that this facilitates more efficient epitope delivery. These findings not only provide additional insights into the mechanism of action of AECs but also broaden the possibilities for genetically fused AECs as an avenue for the redirection of multiple virus-specific T cells toward tumors.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections.

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide’s and Fluspirilene’s efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Immediate impact of COVID-19 on transplant activity in the Netherlands.

The rapid emergence of the COVID-19 pandemic is unprecedented and poses an unparalleled obstacle in the sixty-five year history of organ transplantation. Worldwide, the delivery of transplant care is severely challenged by matters concerning - but not limited to - organ procurement, risk of SARS-CoV-2 transmission, screening strategies of donors and recipients, decisions to postpone or proceed with transplantation, the attributable risk of immunosuppression for COVID-19 and entrenched health care resources and capacity. The transplant community is faced with choosing a lesser of two evils: initiating immunosuppression and potentially accepting detrimental outcome when transplant recipients develop COVID-19 versus postponing transplantation and accepting associated waitlist mortality. Notably, prioritization of health care services for COVID-19 care raises concerns about allocation of resources to deliver care for transplant patients who might otherwise have excellent 1-year and 10-year survival rates. Children and young adults with end-stage organ disease in particular seem more disadvantaged by withholding transplantation because of capacity issues than from medical consequences of SARS-CoV-2. This report details the nationwide response of the Dutch transplant community to these issues and the immediate consequences for transplant activity. Worrisome, there was a significant decrease in organ donation numbers affecting all organ transplant services. In addition, there was a detrimental effect on transplantation numbers in children with end-organ failure. Ongoing efforts focus on mitigation of not only primary but also secondary harm of the pandemic and to find right definitions and momentum to restore the transplant programs.

DCD donor hemodynamics as predictor of outcome after kidney transplantation.

Insufficient hemodynamics during agonal phase-ie, the period between withdrawal of life-sustaining treatment and circulatory arrest-in Maastricht category III circulatory-death donors (DCD) potentially exacerbate ischemia/reperfusion injury. We included 409 Dutch adult recipients of DCD donor kidneys transplanted between 2006 and 2014. Peripheral oxygen saturation (SpO2-with pulse oximetry at the fingertip) and systolic blood pressure (SBP-with arterial catheter) were measured during agonal phase, and were dichotomized into minutes of SpO2 > 60% or SpO2 < 60%, and minutes of SBP > 80 mmHg or SBP < 80 mmHg. Outcome measures were and primary non-function (PNF), delayed graft function (DGF), and three-year graft survival. Primary non-function (PNF) rate was 6.6%, delayed graft function (DGF) rate was 67%, and graft survival at three years was 76%. Longer periods of agonal phase (median 16 min [IQR 11-23]) contributed significantly to an increased risk of DGF (P = .012), but not to PNF (P = .071) and graft failure (P = .528). Multiple logistic regression analysis showed that an increase from 7 to 20 minutes in period of SBP < 80 mmHg was associated with 2.19 times the odds (95% CI 1.08-4.46, P = .030) for DGF. In conclusion, duration of agonal phase is associated with early transplant outcome. SBP < 80 mmHg during agonal phase shows a better discrimination for transplant outcome than SpO2 < 60% does.

[Waiting list registration for kidney transplants must improve]. / Wachtlijstregistratie niertransplantatie moet beter.

OBJECTIVE: To investigate how the composition of the waiting list for postmortem kidney transplant has developed, and whether the waiting list reflects actual demand. DESIGN: Retrospective research and cohort study. METHOD: We used data from the period 2000-2014 from the Dutch Transplant Foundation, 'RENINE' and Eurotransplant. This concerned data on postmortem kidney donation, live donor transplants, the waiting list and kidney transplantation. RESULTS: The postmortem kidney transplant waiting list included transplantable (T) and non-transplantable (NT) patients. The number of T-patients declined from 1271 in 2000 to 650 in 2014, and the median waiting time between the start of dialysis and postmortem kidney transplant decreased from 4.1 years in 2006 to 3.1 years in 2014. The total number of patients on the waiting list, however, increased from 2263 in 2000 to 2560 in 2014 and in the same period the number of new patient registrations increased from 772 to 1212. In about 80% of the NT-patients the reason for their NT status was not registered. A cohort analysis showed that NT-patients have a 2-times lower chance of a postmortem kidney transplant and a 2-times higher chance of leaving the waiting list without transplantation or of live-donor transplantation. CONCLUSION: The demand for donor kidneys remains high. The increased number of transplants resulted in a declining waiting list for T-patients while the total waiting list is getting longer. Waiting list registration and maintenance need to be improved, to give better insight into the real demand.

T helper 2 response in allergic bronchopulmonary aspergillosis is not driven by specific Aspergillus antigens.

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by an allergic immunological response to Aspergillus fumigatus. In this study, we investigated whether certain Aspergillus antigens are more allergenic than others, as was postulated previously. We stimulated peripheral blood mononuclear cells from patients with ABPA with the classically described A. fumigatus allergens Aspf1, Aspf2, Aspf3, and Aspf4, as well as two other Aspergillus antigens, Crf1 and Catalase1. Activated CD4+ T cells displayed a T helper 2 phenotype with the production of IL-4 in response to stimulation with several of these different antigens. Immune responses were not limited to the classically described A. fumigatus allergens. In healthy individuals, we demonstrated a similar recognition profile to the different antigens, but in contrast the activated CD4+ T cells exerted a T helper 1 phenotype and mainly produced IFN-γ after stimulation with A. fumigatus antigens. In conclusion, irrespective of the A. fumigatus antigen, the T-cell immune response in patients with ABPA is skewed to a T helper 2 cytokine secretion profile.

Cognate CD4 T-cell licensing of dendritic cells heralds anti-cytomegalovirus CD8 T-cell immunity after human allogeneic umbilical cord blood transplantation.

UNLABELLED: Reactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+ T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+ T-cell licensing of dendritic cells (DCs) is required to generate effective CD8+ T-cell responses. For humans, this was not fully understood. We here show that CD4+ T cells are essential for licensing of human DCs to generate effector and memory CD8+ T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65-specific CD4+ T cells precedes the rise in CMV-pp65-specific CD8+ T cells. Second, the elicitation of CMV-pp65-specific CD8+ T cells from rare naive precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+ T cells. Finally, also CD8+ T-cell memory responses require CD4+ T-cell-mediated licensing of DCs in our system, by secretion of gamma interferon (IFN-γ) by pp65-specific CD4+ T cells. Together, these data show that human DCs require licensing by cognate antigen-specific CD4+ T cells to elicit effective CD8+ T-cell-mediated immunity and fight off viral reactivation in CBT patients. IMPORTANCE: Survival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) that they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T cells, called helper T cells, is required.

Risk associations between HLA-DPB1 T-cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent of HLA-DPA1.

HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.

In vitro generation of mature, naive antigen-specific CD8(+) T cells with a single T-cell receptor by agonist selection.

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8(+) T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4(+)CD8(+) double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8(+) T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8(+) T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

Multi-cistronic vector encoding optimized safety switch for adoptive therapy with T-cell receptor-modified T cells.

T-cell receptor (TCR) gene transfer is an attractive strategy to equip T cells with defined antigen-specific TCRs using short-term in vitro procedures to target both hematological malignancies and solid tumors. TCR gene transfer poses different safety issues that might warrant the inclusion of a suicide gene. High affinity TCRs may result in on-target toxicity, and off-target reactivity directed against healthy tissue can be observed due to mixed TCR dimers. Inclusion of a suicide gene as a safety switch may abrogate these unwanted toxicities. Human CD20 has been proposed as a nonimmunogenic suicide gene targeted by widely used clinical-grade anti-CD20 antibodies that can additionally function as a selection marker. However, transduction of T cells with a multi-cistronic vector encoding both TCR and CD20 resulted in poor coexpression. In this study, we demonstrated that codon optimization of TCR and CD20 resulted in profound coexpression of both the preferentially expressed antigen in melanoma (PRAME)-TCR and CD20, allowing selective as well as efficient elimination of these engineered T cells in vitro. These results demonstrate the great potential of codon optimized CD20 to be broadly used in clinical trials as a safety switch.

Notch induces human T-cell receptor γδ+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway.

In wild-type mice, T-cell receptor (TCR) γδ(+) cells differentiate along a CD4 CD8 double-negative (DN) pathway whereas TCRαß(+) cells differentiate along the double-positive (DP) pathway. In the human postnatal thymus (PNT), DN, DP and single-positive (SP) TCRγδ(+) populations are present. Here, the precursor-progeny relationship of the various PNT TCRγδ(+) populations was studied and the role of the DP TCRγδ(+) population during T-cell differentiation was elucidated. We demonstrate that human TCRγδ(+) cells differentiate along two pathways downstream from an immature CD1(+) DN TCRγδ(+) precursor: a Notch-independent DN pathway generating mature DN and CD8αα SP TCRγδ(+) cells, and a Notch-dependent, highly proliferative DP pathway generating immature CD4 SP and subsequently DP TCRγδ(+) populations. DP TCRγδ(+) cells are actively rearranging the TCRα locus, and differentiate to TCR(-) DP cells, to CD8αß SP TCRγδ(+) cells and to TCRαß(+) cells. Finally, we show that the γδ subset of T-cell acute lymphoblastic leukemias (T-ALL) consists mainly of CD4 SP or DP phenotypes carrying significantly more activating Notch mutations than DN T-ALL. The latter suggests that activating Notch mutations in TCRγδ(+) thymocytes induce proliferation and differentiation along the DP pathway in vivo.

Unrelated donor marrow transplantation in children: transplant policy and outcome in Leiden Paediatrics SCT-Centre.

The transplant policy for unrelated donor (UD) BMT at Leiden Paediatrics' SCT-Centre consisted of the use of (1) fully HLA-matched donors or, if not available, HLA-class I matched and/or cytotoxic T-lymphocyte precursor (CTLp)-negative donors and (2) protective isolation of the recipient and antimicrobial suppression of his/her gut microflora to prevent infections and acute GVHD. Engraftment, GVHD, relapse in the case of malignancy and survival were studied retrospectively in 126 evaluable children, transplanted between 1988 and 2005. In addition to the effect of HLA-matching, that of other transplant-relevant variables on the outcome was also studied. Actuarial OS was 65% and the EFS was 59%, 13% graft failures occurred and 7.5% > or =grade II acute GVHD. HLA-class II mismatches combined with HLA-class I matches resulted in a superior OS of 92%, as did a negative vs positive CTLp test, that is, 65 vs 33%. Analysis of other variables showed a poorer OS in patients > or =10 yrs vs <10 yrs, that is, 54 vs 73%, and in male recipients of a female donor graft, that is, 53 vs 69% for other combinations. UD-BMT can be optimized by permitting HLA-class I-matched and/or CTLp-negative donors, and probably by choosing male donors for male recipients.

New tools to monitor the impact of viral infection on the alloreactive T-cell repertoire.

Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.

HLA-DP as specific target for cellular immunotherapy in HLA class II-expressing B-cell leukemia.

Mismatching for human leukocyte antigen (HLA)-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a decreased risk of disease relapse, indicating that HLA-DP may represent a target for graft-versus-leukemia (GVL) reactivity in HLA class II-expressing hematological malignancies. To investigate whether HLA-DP-specific T cells could mediate GVL reactivity following HLA-DPB1-mismatched URD-SCT and donor lymphocyte infusion (DLI), we analyzed the immune response in a patient with leukemic lymphoplasmacytic lymphoma responding to DLI without graft-versus-host disease. The emergence of leukemia-reactive CD4+ T cells during the clinical immune response was demonstrated by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot(ELISPOT)analysis. Following clonal isolation of these leukemia-reactive CD4+ T cells, blocking studies, panel studies and retroviral transduction experiments of both mismatched HLA-DPB1 alleles identified HLA-DPB1(*)0201 and HLA-DPB1(*)0301 as the targets of this immune response. The HLA-DPB1-specific CD4+ T-cell clones were capable of recognizing and lysing several HLA-DP-expressing myeloid and lymphoid hematological malignant cells. Since HLA-DP expression is mainly restricted to hematopoietic cells, HLA-DP may be used as a specific target for immunotherapy following T-cell-depleted URD-SCT. Therefore, in patients with HLA class II-expressing hematological malignancies HLA-DP-mismatched SCT may be preferable over fully matched SCT allowing DLI to induce a GVL effect.

T-cell receptor gene transfer for treatment of leukemia.

The therapeutic efficacy of donor lymphocyte infusions has been proven for patients with relapsed hematologic malignancies after allogeneic stem cell transplantation. The beneficial effect of donor lymphocytes, however, is often accompanied by graft-versus-host-disease (GvHD). Adoptive transfer of antigen (Ag)-specific T-cell lines may eradicate the relapsed hematological malignancy, and may separate the anti-leukemic effect from GvHD. The main drawback of adoptive therapy of defined T-cell populations is the difficulty in producing sufficient quantities of these Ag-specific T cells. In addition, the specificity of the infused T cells is difficult to control. As the T-cell receptor (TCR) solely determines the specificity of T cells, transfer of relevant TCR genes into appropriate T-cell populations may provide a potent therapeutic reagent. With this strategy, donor-derived T-cell populations would be equipped with a TCR of defined specificity in short-term in vitro procedures, and infusion of the redirected cells would result in T-cell reactivity against the defined Ag. In this review we discuss the current status of TCR gene transfer for the treatment of hematological malignancies.

Monitoring of indirect allorecognition: wishful thinking or solid data?

Monitoring of T cells involved in the alloimmune response after transplantation requires the availability of reliable in vitro assays for the detection of T cells with both direct and indirect allospecificity. While generally accepted assays exist to measure helper and cytotoxic T cells involved in direct allorecognition, consensus about an assay for monitoring indirect T-cell allorecognition in clinical transplantation is lacking. Many studies claim a relationship between the reactivity of T cells with indirect allospecificity and graft rejection, but different protocols are used and essential controls are often lacking. In this review, the disadvantages and pitfalls of the current approaches are discussed, in some cases supported by the results of our own in vitro experiments. We conclude that an international workshop is necessary to establish and validate a uniform, robust and reliable assay for the monitoring of transplant recipients and to study the actual role of indirect allorecognition in acute and chronic rejection.

Highly diverged MHC class I mismatches are acceptable for haematopoietic stem cell transplantation.

A fully major histocompatilbility complex (MHC) matched donor is not available for the majority of patients in need of a haematopoietic stem cell transplantation (SCT), which illustrates the need for a tool to define acceptable MHC disparities. Previously, we noticed that a variety of single MHC class I mismatched allogeneic donor-recipient pairs did not elicit an allogeneic cytotoxic-lymphocyte (CTL) response in vitro if the MHC amino-acid sequences had five or more differences in the alpha-helices plus five or more differences in the beta-sheet (> or =5alpha5beta) (7). To address the clinical relevance of this observation, we analysed CTL precursor (CTLp) assay outcome and SCT outcome in 53 Dutch recipients of a single MHC class I mismatched graft from an unrelated donor. Overall patient survival was 44% after 4 years. In multivariate analysis, recipients of a > or =5alpha5beta mismatched graft with negative CTLp frequencies in vitro before transplantation demonstrated superior survival: survival at 4 years was 80% as compared to 47% in recipients of other mismatched grafts with negative CTLp frequencies (hazard ratio=0.131; 95% CI=(0.03-0.61); P=0.009). This option of acceptable mismatches may enlarge the pool of potentially acceptable stem cell donors.

Involvement of caspase-8 in chemotherapy-induced apoptosis of patient derived leukemia cell lines independent of the death receptor pathway and downstream from mitochondria.

Resistance of leukemic cells to chemotherapy frequently occurs in patients with acute leukemia, which may be caused by alterations in common apoptotic pathways. Controversy exists whether cytostatic agents induce the mitochondrial or death receptor pathway of apoptosis. In the mitochondrial pathway cytochrome C release and caspase-9 activation play a central role in the induction of apoptosis, while formation of a Death Inducing Signaling Complex (DISC) and caspase-8 activation have been reported to be essential in death receptor-induced apoptosis. Here, we show in human derived myeloid and lymphoblastic leukemia cell lines that caspase-8 plays a more important role than previously expected in apoptosis mediated via the mitochondrial pathway. We demonstrated in these malignant cells chemotherapy-induced apoptosis independent of the death receptor pathway, since blocking this pathway using a retroviral construct encoding Flice inhibitory protein (FLIP) did not inhibit drug-induced apoptosis or caspase-8 activation, while overexpression of Bcl-2 completely inhibited both events. Furthermore, we showed that activation of caspase-8 by cytostatic agents occurred downstream from mitochondria. Since caspase-8 plays a central role in both death receptor- and chemotherapy-induced apoptosis of malignant cells from patients with acute leukemia, therapeutic strategies focusing at modulation and activation of caspase-8 may be successful in the treatment of drug-resistant malignancies.

Hematopoietic stem cell donor selection: the Europdonor experience.

International collaboration is essential for the optimal selection of unrelated hematopoietic stem cell donors. This review focuses on the benefit of a joint worldwide donor file called Bone Marrow Donors Worldwide and the experience of the Europdonor Foundation in selecting strategies to identify the best human leukocyte antigen-matched donor in the shortest time.