The Alcatraz-Strategy: a roadmap to break the connectivity barrier in malignant brain tumours.

In recent years, the discovery of functional and communicative cellular tumour networks has led to a new understanding of malignant primary brain tumours. In this review, the authors shed light on the diverse nature of cell-to-cell connections in brain tumours and propose an innovative treatment approach to address the detrimental connectivity of these networks. The proposed therapeutic outlook revolves around three main strategies: (a) supramarginal resection removing a substantial portion of the communicating tumour cell front far beyond the gadolinium-enhancing tumour mass, (b) morphological isolation at the single cell level disrupting structural cell-to-cell contacts facilitated by elongated cellular membrane protrusions known as tumour microtubes (TMs), and (c) functional isolation at the single cell level blocking TM-mediated intercellular cytosolic exchange and inhibiting neuronal excitatory input into the malignant network. We draw an analogy between the proposed therapeutic outlook and the Alcatraz Federal Penitentiary, where inmates faced an impassable sea barrier and experienced both spatial and functional isolation within individual cells. Based on current translational efforts and ongoing clinical trials, we propose the Alcatraz-Strategy as a promising framework to tackle the harmful effects of cellular brain tumour networks.

The Potential Role of the Extracellular Matrix Glycoprotein Reelin in Glioblastoma Biology.

Glioblastoma, the most common and lethal primary adult brain tumor, cannot be successfully removed surgically due to its highly invasive nature. Therapeutically, approaches must be aimed at a systemic brain disease and not merely at a tumor located within the brain, unless a successful containment strategy can be found. Reelin, an extracellular matrix glycoprotein, plays an important role in neuronal migration and serves here as a natural stop signal. Interestingly, the expression of reelin is negatively associated with tumor grade and, within glioblastoma, correlates with increased overall survival. To further elucidate a potential biological reason for these findings, we looked at the cellular behavior of glioblastoma cell lines grown on a pure fibronectin matrix or a matrix with reelin inserts. While reelin had no significant effects on cellular metabolism, proliferation, or resistance to chemotherapeutic agents, it did significantly affect the cells' interaction with fibronectin. Both matrix attachment and detachment were modulated by reelin, and thus, the invasion and motility of cells interacting with a reelin-containing matrix were altered. The data presented in this work strongly suggest that reelin might be a potential modulator of underlying molecular mechanisms that contribute to glioblastoma invasion.

Chemotherapy for a secondary malignancy nearly restores complete chimerism in an SCID-patient after HSCT.

For patients with inborn errors of immunity (IEI) and other inborn diseases, mixed donor chimerism is a well-accepted outcome of hematopoietic stem cell transplantation (HSCT). Cytoreductive chemotherapy for a secondary malignancy is a potential challenge for the stability of the graft function after HSCT. We report on a boy with X-SCID who developed Ewing sarcoma ten years after HSCT which was successfully treated with cytoreductive chemotherapy, surgery and local radiation. Surprisingly, this treatment had a positive impact on mixed chimerism with an increase of donor-cell proportions from 40% for neutrophils and 75% for non-T-mononuclear cells (MNCs) to >90% for both. T-cell counts remained stable with 100% of donor origin. This is -to our knowledge- the first report on the impact of cytoreductive chemotherapy on post-HSCT mixed chimerism and provides an important first impression for future patients.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Inhibition of Survivin Homodimerization Decreases Neuroblastoma Cell Growth.

Increased expression of BIRC5/survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB), the most common extracranial tumor of childhood. Transcriptional inhibitors of survivin have been tested in adult cancers and inhibitors of survivin homodimerization are emerging. We compared genetic inhibition of survivin transcription with the inhibition of survivin homodimerization by S12 and LQZ-7I, chosen from a larger panel of survivin dimerization inhibitors with activity against NB cells. Mice hemizygous for Birc5 were crossed with NB-prone TH-MYCN mice to generate Birc5+/-/MYCNtg/+ mice. The marked decrease of survivin transcription in these mice did not suffice to attenuate the aggressiveness of NB, even when tumors were transplanted into wild-type mice to assure that immune cell function was not compromised by the lack of survivin. In contrast, viability, clonogenicity and anchorage-independent growth of NB cells were markedly decreased by S12. S12 administered systemically to mice with subcutaneous NB xenotransplants decreased intratumoral hemorrhage, albeit not tumor growth. LQZ-7I, which directly targets the survivin dimerization interface, was efficacious in controlling NB cell growth in vitro at markedly lower concentrations compared to S12. LQZ-7I abrogated viability, clonogenicity and anchorage-independent growth, associated with massively distorted mitotic spindle formation. In vivo, LQZ-7I effectively reduced tumor size and cell proliferation of NB cells in CAM assays without apparent toxicity to the developing chick embryo. Collectively, these findings show that inhibiting survivin homodimerization with LQZ-7I holds promise for the treatment of NB and merits further investigation.

The survivin-ran inhibitor LLP-3 decreases oxidative phosphorylation, glycolysis and growth of neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB), the most common extracranial solid malignancy in children, carries a poor prognosis in high-risk disease, thus requiring novel therapeutic approaches. Survivin is overexpressed in NB, has pro-mitotic and anti-apoptotic functions, and impacts on oxidative phosphorylation (OXPHOS) and aerobic glycolysis. The subcellular localization and hence function of survivin is directed by the GTPase Ran. AIM: To determine efficacy and modes of action of the survivin-Ran inhibitor LLP-3 as a potential novel therapy of NB. METHODS: Survivin and Ran mRNA expression in NB tumors was correlated to patient survival. Response to LLP-3 in NB cell lines was determined by assays for viability, proliferation, apoptosis, clonogenicity and anchorage-independent growth. Interaction of survivin and Ran was assessed by proximity-linked ligation assay and their subcellular distribution by confocal immunofluorescence microscopy. Expression of survivin, Ran and proteins important for OXPHOS and glycolysis was determined by Western blot, hexokinase activity by enzymatic assay, interaction of survivin with HIF-1α by co-IP, and OXPHOS and glycolysis by extracellular flux analyzer. RESULTS: High mRNA expression of survivin and Ran is correlated with poor patient survival. LLP-3 decreases viability, induces apoptosis, and inhibits clonogenic and anchorage-independent growth in NB cell lines, including those with MYCN amplification, and mutations of p53 and ALK. LLP-3 inhibits interaction of survivin with Ran, decreasing their concentration both in the cytoplasm and the nucleus. LLP-3 impairs flexibility of energy metabolism by inhibiting both OXPHOS and glycolysis. Metabolic inhibition is associated with mitochondrial dysfunction and attenuated hexokinase activity but is independent of HIF-1α. CONCLUSION: LLP-3 attenuates interaction and concentration of survivin and Ran in NB cells. It controls NB cells with diverse genetic alterations, associated with inhibition of OXPHOS, aerobic glycolysis, mitochondrial function and HK activity. Thus, LLP-3 warrants further studies as a novel drug against NB.

Immune-epithelial cell cross-talk enhances antiviral responsiveness to SARS-CoV-2 in children.

The risk of developing severe COVID-19 rises dramatically with age. Schoolchildren are significantly less likely than older people to die from SARS-CoV-2 infection, but the molecular mechanisms underlying this age-dependence are unknown. In primary infections, innate immunity is critical due to the lack of immune memory. Children, in particular, have a significantly stronger interferon response due to a primed state of their airway epithelium. In single-cell transcriptomes of nasal turbinates, we find increased frequencies of immune cells and stronger cytokine-mediated interactions with epithelial cells, resulting in increased epithelial expression of viral sensors (RIG-I, MDA5) via IRF1. In vitro, adolescent peripheral blood mononuclear cells produce more cytokines, priming A549 cells for stronger interferon responses to SARS-CoV-2. Taken together, our findings suggest that increased numbers of immune cells in the airways of children and enhanced cytokine-based interactions with epithelial cells tune the setpoint of the epithelial antiviral system. Our findings shed light on the molecular basis of children's remarkable resistance to COVID-19 and may suggest a novel concept for immunoprophylactic treatments.

Automated production of specific T cells for treatment of refractory viral infections after allogeneic stem cell transplantation.

Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.

Targeted parallel DNA sequencing detects circulating tumor-associated variants of the mitochondrial and nuclear genomes in patients with neuroblastoma.

BACKGROUND: The utility for liquid biopsy of tumor-associated circulating single-nucleotide variants, as opposed to mutations, of the mitochondrial (mt) and nuclear genomes in neuroblastoma (NB) is unknown. PROCEDURE: Variants of the mt and nuclear genomes from tumor, blood cells, and consecutive plasma samples of five patients with metastatic NB that relapsed or progressed were analyzed. Targeted parallel sequencing results of the mt genome, and of the coding region of 139 nuclear genes and 22 miRNAs implicated in NB, were correlated with clinical imaging and laboratory data. RESULTS: All tumors harbored multiple somatic mt and nuclear single nucleotide variants with low allelic frequency, most of them not detected in the circulation. In one patient a tumor-associated mt somatic variant was detected in the plasma before and during progressive disease. In a second patient a circulating nuclear tumor-associated DNA variant heralded clinical relapse. In all patients somatic mt and nuclear variants not evident in the tumor biopsy at time of diagnosis were found circulating at varying timepoints. This suggests either tumor heterogeneity, evolution of tumor variants or a confounding contribution of normal tissues to somatic variants in patient plasma. The number and allelic frequency of the circulating variants did not reflect the clinical course of the tumors. Mutational signatures of mt and nuclear somatic variants differed. They varied between patients and were detected in the circulation without mirroring the patients' course. CONCLUSIONS: In this limited cohort of NB patients clinically informative tumor-associated mt and nuclear circulating variants were detected by targeted parallel sequencing in a minority of patients.

Unblinding the watchmaker: cancer treatment and drug design in the face of evolutionary pressure.

INTRODUCTION: Death due to cancer is mostly associated with therapy ineffectiveness, i.e. tumor cells no longer responding to treatment. The underlying dynamics that facilitate this mutational escape from selective pressure are well studied in several other fields and several interesting approaches exist to combat this phenomenon, for example in the context of antibiotic-resistance in bacteria. AREAS COVERED: Ninety percent of all cancer-related deaths are associated with treatment failure. Here, we discuss the common treatment modalities and prior attempts to overcome acquired resistance to therapy. The underlying molecular mechanisms are discussed and the implications of emerging resistance in other systems, such as bacteria, are discussed in the context of cancer. EXPERT OPINION: Reevaluating emerging therapy resistance in tumors as an evolutionary mechanism to survive in a rapidly and drastically altering fitness landscape leads to novel treatment strategies and distinct requirements for new drugs. Here, we propose a scheme of considerations that need to be applied prior to the discovery of novel therapeutic drugs.

PPP2CA Is a Novel Therapeutic Target in Neuroblastoma Cells That Can Be Activated by the SET Inhibitor OP449.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and has a poor prognosis in high-risk cases, requiring novel therapies. Pathways that depend on phospho-signaling maintain the aggressiveness of NB. Protein phosphatase 2 (PP2A) with its catalytic subunit PPP2CA is a major phosphatase in cancer cells, including NB. We show that reduction of PPP2CA by knock-down decreased growth of NB cells and that complete ablation of PPP2CA by knock-out was not tolerated. Thus, NB cells are addicted to PPP2CA, an addiction augmented by MYCN activation. SET, a crucial endogenous inhibitor of PP2A, was overexpressed in poor-prognosis NB. The SET inhibitor OP449 effectively decreased the viability of NB cells, independent of their molecular alterations and in line with a tumor suppressor function of PPP2CA. The contrasting concentration-dependent functions of PPP2CA as an essential survival gene at low expression levels and a tumor suppressor at high levels are reminiscent of other genes showing this so-called Goldilocks phenomenon. PP2A reactivated by OP449 decreased activating phosphorylation of serine/threonine residues in the AKT pathway. Conversely, induced activation of AKT led to partial rescue of OP449-mediated viability inhibition. Dasatinib, a kinase inhibitor used in relapsed/refractory NB, and OP449 synergized, decreasing activating AKT phosphorylations. In summary, concomitantly reactivating phosphatases and inhibiting kinases with a combination of OP449 and dasatinib are promising novel therapeutic approaches to NB.

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

Diisothiocyanate-Derived Mercapturic Acids Are a Promising Partner for Combination Therapies in Glioblastoma.

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma.

Bcl-XL but Not Bcl-2 Is a Potential Target in Medulloblastoma Therapy.

Medulloblastoma (MB) is the most common solid tumour in children and, despite current treatment with a rather aggressive combination therapy, accounts for 10% of all deaths associated with paediatric cancer. Breaking the tumour cells' intrinsic resistance to therapy-induced cell death should lead to less aggressive and more effective treatment options. In other tumour entities, this has been achieved by modulating the balance between the various pro- and anti-apoptotic members of the Bcl-2 family with small molecule inhibitors. To evaluate the therapeutic benefits of ABT-199 (Venetoclax), a Bcl-2 inhibitor, and ABT-263 (Navitoclax), a dual Bcl-XL/Bcl-2 inhibitor, increasingly more relevant model systems were investigated. Starting from established MB cell lines, progressing to primary patient-derived material and finally an experimental tumour system imbedded in an organic environment were chosen. Assessment of the metabolic activity (a surrogate readout for population viability), the induction of DNA fragmentation (apoptosis) and changes in cell number (the combined effect of alterations in proliferation and cell death induction) revealed that ABT-263, but not ABT-199, is a promising candidate for combination therapy, synergizing with cell death-inducing stimuli. Interestingly, in the experimental tumour setting, the sensitizing effect of ABT-263 seems to be predominantly mediated via an anti-proliferative and not a pro-apoptotic effect, opening a future line of investigation. Our data show that modulation of specific members of the Bcl-2 family might be a promising therapeutic addition for the treatment of MB.

Synergistic activity of combined inhibition of anti-apoptotic molecules in B-cell precursor ALL.

Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.

Biomarkers of DNA Damage Response Enable Flow Cytometry-Based Diagnostic to Identify Inborn DNA Repair Defects in Primary Immunodeficiencies.

DNA damage is a constant event in every cell caused by exogenous factors such as ultraviolet and ionizing radiation (UVR/IR) and intercalating drugs, or endogenous metabolic and replicative stress. Proteins of the DNA damage response (DDR) network sense DNA lesions and induce cell cycle arrest, DNA repair, and apoptosis. Genetic defects of DDR or DNA repair proteins can be associated with immunodeficiency, bone marrow failure syndromes, and cancer susceptibility. Although various diagnostic tools are available to evaluate DNA damage, their quality to identify DNA repair deficiencies differs enormously and depends on affected pathways. In this study, we investigated the DDR biomarkers γH2AX (Ser139), p-ATM (Ser1981), and p-CHK2 (Thr68) using flow cytometry on peripheral blood cells obtained from patients with combined immunodeficiencies due to non-homologous end-joining (NHEJ) defects and ataxia telangiectasia (AT) in response to low-dose IR. Significantly reduced induction of all three markers was observed in AT patients compared to controls. However, delayed downregulation of γH2AX was found in patients with NHEJ defects. In contrast to previous reports of DDR in cellular models, these biomarkers were not sensitive enough to identify ARTEMIS deficiency with sufficient reliability. In summary, DDR biomarkers are suitable for diagnosing NHEJ defects and AT, which can be useful in neonates with abnormal TREC levels (T cell receptor excision circles) identified by newborn screening. We conclude that DDR biomarkers have benefits and some limitations depending on the underlying DNA repair deficiency.

Evaluation of vecabrutinib as a model for noncovalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia.

Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib.

CHD5 inhibits metastasis of neuroblastoma.

CHD5, a tumor suppressor at 1p36, is frequently lost or silenced in poor prognosis neuroblastoma (NB) and many adult cancers. The role of CHD5 in metastasis is unknown. We confirm that low expression of CHD5 is associated with stage 4 NB. Forced expression of CHD5 in NB cell lines with 1p loss inhibited key aspects of the metastatic cascade in vitro: anchorage-independent growth, migration, and invasion. In vivo, formation of bone marrow and liver metastases developing from intravenously injected NB cells was delayed and decreased by forced CHD5 expression. Genome-wide mRNA sequencing revealed reduction of genes and gene sets associated with metastasis when CHD5 was overexpressed. Known metastasis-suppressing genes preferentially upregulated in CHD5-overexpressing NB cells included PLCL1. In patient NB, low expression of PLCL1was associated with metastatic disease and poor survival. Knockdown of PLCL1 and of p53 in IMR5 NB cells overexpressing CHD5 reversed CHD5-induced inhibition of invasion and migration in vitro. In summary, CHD5 is a metastasis suppressor in NB.

Graft-Versus-Host Disease Prevention by In Vitro-Generated Myeloid-Derived Suppressor Cells Is Exclusively Mediated by the CD11b+CD11c+ MDSC Subpopulation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c- MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c- MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c- MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.