Der Einfluss von Antithymozytenglobulin auf humane Monozyten und dendritische Zellen

Die allogene Transplantation hämatopoetischer Stammzellen hat eine große Bedeutung in der Therapie maligner hämatologischer Erkrankungen. Allerdings wird der Erfolg dieser Therapieoption durch die Entwicklung einer Graft-versus-Host-Disease (GvHD) beschränkt. Prophylaktisch werden daher immunsuppressive Medikamente wie Antithymozytenglobulin (ATG) eingesetzt. Der am besten dokumentierte Effekt von ATG ist die Depletion von T-Zellen via Komplement-abhängiger Lyse oder alternativ via Apoptose und Phagozytose. In der vorliegenden Arbeit wurde untersucht, welche Effekte ATG auf humane Monozyten und dendritische Zellen (DCs) hat. Zunächst konnte nachgewiesen werden, dass ATG im Gegensatz zu einer polyklonalen IgG-Kontrolle zu einer signifikant gesteigerten Expression des Enzyms Indolamin-2,3-Dioxygenase (IDO) in DCs führt. Die Expression von IDO in DCs hat immunregulatorische Effekte, wie z.B. die Induktion regulatorischer T-Zellen. Weiterhin konnte auf mRNA-Ebene eine verstärkte Expression der Kynureninase, einem weiteren Enzym des Tryptophan-Stoffwechsels, beobachtet werden. In Monozyten hatte ATG dagegen keine signifikanten Effekte auf die untersuchten Enzyme. Weiterhin konnte dargelegt werden, dass ATG die mRNA-Expression des Enzyms 25-Hydroxyvitamin D3 1α-Hydroxylase induziert. Dieses Enzym katalysiert die Umwandlung von 25-Hydroxy-Vitamin D3 zu physiologisch aktivem 1,25-Dihydroxy-Vitamin D3 [1,25(OH)2D3]. In einem nächsten Schritt konnte nachgewiesen werden, dass ATG zu einer signifikanten Zunahme von 1,25(OH)2D3 im Überstand der DCs führt. Von 1,25(OH)2D3 ist bekannt, dass es die Reifung und Differenzierung von DCs und somit ihre Fähigkeit T-Zellen zu stimulieren, inhibiert. Zudem konnte gezeigt werden, dass ATG in humanen DCs zu einer signifikant gesteigerten Sekretion von IL-10, ohne gleichzeitige Produktion von IL-12 führt. Diese Kombination ist ein typisches Charakteristikum für tolerogene DCs. In Monozyten hatte ATG keinen Effekt auf die Zytokin-Sekretion. Eine Inkubation der DCs mit allogenen T-Zellen zeigte, dass mit ATG stimulierte DCs nicht in der Lage sind allogene T-Zellen zur Proliferation anzuregen. Weiterhin lieferte diese Methode deutliche Hinweise darauf, dass die mit ATG stimulierten DCs in der Lage sind, die Proliferation von T-Zellen, welche durch reife DCs induziert wurde, zu inhibieren. In summa legen die Daten der vorliegenden Arbeit nahe, dass ATG im Gegensatz zu einer polyklonalen IgG-Kontrolle dazu in der Lage ist, einen tolerogenen DC-Phänotyp zu induzieren. Dieser Mechanismus könnte für die unter ATG beschriebene Induktion regulatorischer T-Zellen verantwortlich sein. Neben dem bekannten Effekt der T-Zell-Depletion könnte ATG seine immunsuppressive Wirkung auch durch die oben beschriebenen Mechanismen vermitteln. Ob diese Effekte in vivo allerdings tatsächlich von Bedeutung sind, muss erst durch weitere Untersuchungen bestätigt werde

Functional analysis of structural variants in single cells using Strand-seq

Somatic structural variants (SVs) are widespread in cancer, but their impact on disease evolution is understudied due to a lack of methods to directly characterize their functional consequences. We present a computational method, scNOVA, which uses Strand-seq to perform haplotype-aware integration of SV discovery and molecular phenotyping in single cells by using nucleosome occupancy to infer gene expression as a readout. Application to leukemias and cell lines identifies local effects of copy-balanced rearrangements on gene deregulation, and consequences of SVs on aberrant signaling pathways in subclones. We discovered distinct SV subclones with dysregulated Wnt signaling in a chronic lymphocytic leukemia patient. We further uncovered the consequences of subclonal chromothripsis in T cell acute lymphoblastic leukemia, which revealed c-Myb activation, enrichment of a primitive cell state and informed successful targeting of the subclone in cell culture, using a Notch inhibitor. By directly linking SVs to their functional effects, scNOVA enables systematic single-cell multiomic studies of structural variation in heterogeneous cell populations

Comparing the value of mono- vs coculture for high-throughput compound screening in hematological malignancies

Large-scale compound screens are a powerful model system for understanding variability of treatment response and discovering druggable tumor vulnerabilities of hematological malignancies. However, as mostly performed in a monoculture of tumor cells, these assays disregard modulatory effects of the in vivo microenvironment. It is an open question whether and to what extent coculture with bone marrow stromal cells could improve the biological relevance of drug testing assays over monoculture. Here, we established a high-throughput platform to measure ex vivo sensitivity of 108 primary blood cancer samples to 50 drugs in monoculture and coculture with bone marrow stromal cells. Stromal coculture conferred resistance to 52% of compounds in chronic lymphocytic leukemia (CLL) and 36% of compounds in acute myeloid leukemia (AML), including chemotherapeutics, B-cell receptor inhibitors, proteasome inhibitors, and Bromodomain and extraterminal domain inhibitors. Only the JAK inhibitors ruxolitinib and tofacitinib exhibited increased efficacy in AML and CLL stromal coculture. We further confirmed the importance of JAK-STAT signaling for stroma-mediated resistance by showing that stromal cells induce phosphorylation of STAT3 in CLL cells. We genetically characterized the 108 cancer samples and found that drug-gene associations strongly correlated between monoculture and coculture. However, effect sizes were lower in coculture, with more drug-gene associations detected in monoculture than in coculture. Our results justify a 2-step strategy for drug perturbation testing, with large-scale screening performed in monoculture, followed by focused evaluation of potential stroma-mediated resistances in coculture

Ex vivo drug response profiling for response and outcome prediction in hematologic malignancies: the prospective non-interventional SMARTrial

Ex vivo drug response profiling is a powerful tool to study genotype-drug response associations and is being explored as a tool set for precision medicine in cancer. Here we conducted a prospective non-interventional trial to investigate feasibility of ex vivo drug response profiling for treatment guidance in hematologic malignancies (SMARTrial, NCT03488641 ). The primary endpoint to provide drug response profiling reports within 7 d was met in 91% of all study participants (N = 80). Secondary endpoint analysis revealed that ex vivo resistance to chemotherapeutic drugs predicted chemotherapy treatment failure in vivo. We confirmed the predictive value of ex vivo response to chemotherapy in a validation cohort of 95 individuals with acute myeloid leukemia treated with daunorubicin and cytarabine. Ex vivo drug response profiles improved ELN-22 risk stratification in individuals with adverse risk. We conclude that ex vivo drug response profiling is clinically feasible and has the potential to predict chemotherapy response in individuals with hematologic malignancies beyond clinically established genetic markers

EOMES and IL-10 regulate antitumor activity of T regulatory type 1 CD4 + T cells in chronic lymphocytic leukemia

The transcription factor eomesodermin (EOMES) promotes interleukin (IL)-10 expression in CD4(+) T cells, which has been linked to immunosuppressive and cytotoxic activities. We detected cytotoxic, programmed cell death protein-1 (PD-1) and EOMES co-expressing CD4(+) T cells in lymph nodes (LNs) of patients with chronic lymphocytic leukemia (CLL) or diffuse large B-cell lymphoma. Transcriptome and flow cytometry analyses revealed that EOMES does not only drive IL-10 expression, but rather controls a unique transcriptional signature in CD4(+) T cells, that is enriched in genes typical for T regulatory type 1 (T(R)1) cells. The T(R)1 cell identity of these CD4(+) T cells was supported by their expression of interferon gamma and IL-10, as well as inhibitory receptors including PD-1. T(R)1 cells with cytotoxic capacity accumulate also in Eµ-TCL1 mice that develop CLL-like disease. Whereas wild-type CD4(+) T cells control TCL1 leukemia development after adoptive transfer in leukopenic Rag2(−/)(−) mice, EOMES-deficient CD4(+) T cells failed to do so. We further show that T(R)1 cell-mediated control of TCL1 leukemia requires IL-10 receptor (IL-10R) signaling, as Il10rb-deficient CD4(+) T cells showed impaired antileukemia activity. Altogether, our data demonstrate that EOMES is indispensable for the development of IL-10-expressing, cytotoxic T(R)1 cells, which accumulate in LNs of CLL patients and control TCL1 leukemia in mice in an IL-10R-dependent manner

Antithymocyte Globulin Induces a Tolerogenic Phenotype in Human Dendritic Cells

Antithymocyte globulin (ATG) is used in the prevention of graft-versus-host disease during allogeneic hematopoietic stem cell transplantation. It is generally accepted that ATG mediates its immunosuppressive effect primarily via depletion of T cells. Here, we analyzed the impact of ATG-Fresenius (now Grafalon (R)) on human monocyte-derived dendritic cells (DC). ATG induced a semi-mature phenotype in DC with significantly reduced expression of CD14, increased expression of HLA-DR, and intermediate expression of CD54, CD80, CD83, and CD86. ATG-DC showed an increase in IL-10 secretion but no IL-12 production. In line with this tolerogenic phenotype, ATG caused a significant induction of indoleamine 2,3-dioxygenase expression and a concomitant increase in levels of tryptophan metabolites in the supernatants of DC. Further, ATG-DC did not induce the proliferation of allogeneic T cells in a mixed lymphocyte reaction but actively suppressed the T cell proliferation induced by mature DC. These data suggest that besides its well-known effect on T cells, ATG modulates the phenotype of DC in a tolerogenic way, which might constitute an essential part of its immunosuppressive action in vivo

Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll-like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL-infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell-extrinsic mechanisms of drug resistance and disease progression

Anti-Thymocyte Globulin Treatment Augments 1,25-Dihydroxyvitamin D3 Serum Levels in Patients Undergoing Hematopoietic Stem Cell Transplantation

Application of anti-thymocyte globulin (ATG) is a widely used strategy for the prevention of graftversus-host disease (GvHD). As vitamin D3 serum levels are also discussed to affect hematopoietic stem cell transplantation (HSCT) outcome and GvHD development, we analysed a possible interplay between ATG treatment and serum levels of 25-hydroxyvitamin D3and 1,25-dihydroxyvitaminD3in 4HSCT cohorts withdifferent vitaminD3supplementation. ATG is significantly associated with higher serum level of 1,25 dihydroxyvitamin D3 around HSCT (day -2 to 7, peri-transplant), however only in patients with adequate levels of its precursor 25-hydroxyvitamin D3. ATG exposure had no impact on overall survival in patients supplemented with high dose vitamin D3, but was associated with higher risk of one-year treatment-related mortality (log rank test p=0.041) in patients with no/low vitamin D3 supplementation. However, the difference failed to reach significance applying a Cox-model regression without and with adjustment for baseline risk factors (unadjusted P=0,058, adjusted p=0,139). To shed some light on underlying mechanisms, we investigated the impact of ATG on 1,25-DihydroxyvitaminD3 production by human dendritic cells (DCs) in vitro.ATGincreased gene expression ofCYP27B1, the enzyme responsible for the conversion of 25-hydroxyvitamin D3 into 1,25-dihydroxyvitamin D3, which was accompanied by higher 1,25-dihydroxyvitamin D3levels in ATG-treatedDCculture supernatants.Our data demonstrate a cooperative effect of 25-hydroxyvitamin D3 and ATG in the regulation of 1,25-dihydroxyvitamin D3 production. This finding may be of importance in the context of HSCT, where early high levels of 1,25- dihydroxyvitamin D3 levels have been shown to be predictive for lower transplant related mortality and suggest that vitamin D3 supplementation may especially be important in patients receiving ATG for GvHD prophylaxis

FlowCytometryData_LN_B-NHL

LN-derived cells were thawed and stained for viability using a fixable viability dye e506 (Thermo Fisher Scientific, #65-0866-14) and for different surface markers depending on the experimental set-up. The following surface antibodies were used: anti-CD3-PerCP-Cy5.5, anti-CD4-PE-Dazzle, anti-CD8-APC-Cy7, anti-CD45RA-FITC, anti-CD25-BV421, anti-CD31-BV605, anti-CXCR5-BV711, anti-TIM3-BV711, anti-CD278-BV605, anti-PD1-PE-Cy7, anti-CD69-AF700, anti-CD244-BV421 (all BioLegend). For subsequent intracellular staining, cells were fixed and permeabilized with the intracellular fixation/permeabilization buffer set (Thermo Fisher Scientific, #88-8824-00) and stained with anti-Ki67-BV785, anti-FoxP3-AF647, anti-IKZF3-PE or adequate isotype controls (Thermo Fisher Scientific, BD Biosciences). Then, cells were analyzed using an LSR Fortessa (BD Biosciences) and FACSDiva (BD Biosciences, version 8). For analysis and gating of flow cytometry data FlowJo (v10.8.0) was used.Compensation was done prior measurement in FACSDiva. Corresponding compensation controls are provided for each measurement. </p

Recent advances in understanding and managing hairy cell leukemia [version 1; referees: 4 approved]

Hairy cell leukemia is a rare B-cell malignancy that is characterized by an indolent course. It was initially described as a distinct entity in 1958. Before the establishment of modern treatment, median survival was only 4 years. Since then, major advances in the treatment and understanding of the biology and genomic landscape of hairy cell leukemia have been made. This review summarizes the present understanding of hairy cell leukemia with particular focus on the development of novel and targeted approaches to treatment