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Hematopoiesis in patients with cancer is characterized by reduced production of red blood cells and an increase in myelopoiesis, which contributes to the immunosuppressive environment in cancer. Some tumors produce growth factors that directly stimulate myelopoiesis such as G-CSF or GM-CSF. However, for a majority of tumors that do not directly secrete hematopoietic growth factors, the mechanisms involved in the activation of myelopoiesis are poorly characterized. In this study, we document in different murine tumor models activated hematopoiesis with increased proliferation of long-term and short-term hematopoietic stem cells and myeloid progenitor cells. As a consequence, the frequency of myeloid-derived suppressor cells and its ratio to CD8+ T cells increased in tumor-bearing mice. Activation of hematopoiesis and myeloid differentiation in tumor-bearing mice was induced by TNFα, which was mainly secreted by activated CD4+ T cells. Therefore, the activated adaptive immune system in cancer induces emergency myelopoiesis and immunosuppression.Significance:These findings characterize a regulatory circuit linking activated T cells to suppression of tumor-specific immune responses, providing a conceptual advance in the understanding of emergency-hematopoiesis in cancer and opening new targets for therapeutic approaches.

Table S1: suppl. cell cycle analysis to Fig 3; Table S2: suppl. BrDU and AnnexinV analysis to Fig 3; Table S3: suppl. analysis of the compositions of HSPC compartments to Fig 4; Figure S1: suppl. to Fig 1; Figure S2: suppl. to Fig 4; Figure S3: suppl. to Fig 6; Figure S4: suppl. to Fig 7

The production of blood cells during steady-state and increased demand depends on the regulation of hematopoietic stem cell (HSC) self-renewal and differentiation. Similarly, the balance between self-renewal and differentiation of leukemia stem cells (LSCs) is crucial in the pathogenesis of leukemia. Here, we document that the TNF receptor superfamily member lymphotoxin-β receptor (LTβR) and its ligand LIGHT regulate quiescence and self-renewal of murine and human HSCs and LSCs. Cell-autonomous LIGHT/LTβR signaling on HSCs reduces cell cycling, promotes symmetric cell division and prevents primitive HSCs from exhaustion in serial re-transplantation experiments and genotoxic stress. LTβR deficiency reduces the numbers of LSCs and prolongs survival in a murine chronic myeloid leukemia (CML) model. Similarly, LIGHT/LTβR signaling in human G-CSF mobilized HSCs and human LSCs results in increased colony forming capacity in vitro. Thus, our results define LIGHT/LTβR signaling as an important pathway in the regulation of the self-renewal of HSCs and LSCs., Regulation of self-renewal is critical during steady state and stress in haematpoietic stem cells (HSCs), and underlies leukaemia pathology. Here, the authors show that LIGHT and its receptor LTβR promote quiescence and self-renewal of HSCs and that LTβR deficiency promotes survival in a mouse leukaemia model.

Background Over the past few years, immune checkpoint inhibitors have changed the therapeutic landscape of non-small-cell lung cancer (NSCLC). Response to immune checkpoint inhibitors correlates with a pre-existing anti-tumoral immune response. Checkpoint inhibitors have been introduced as second-line therapy and are only very recently used as monotherapy or in combination with chemotherapy as first-line treatment of NSCLC. However, the effect of conventional first-line platinum-based chemotherapy on the immune infiltrate in the tumor is largely unknown. Methods We measured the gene expression of a custom set of 201 cancer- and immune-related genes in 100 NSCLC tumor biopsies collected before chemotherapy and 33 re-biopsies after platinum-based chemotherapy at the time point of progression. For 29 patients matched pre- and post-chemotherapy samples could be evaluated. Results We identified a cluster of 47 co-expressed immune genes, including PDCD1 (PD1) and CD274 (PD-L1), along with three other co-expression clusters. Chemotherapy decreased the average gene expression of the immune cluster while no effect was observed on the other three cluster. Within this immune cluster, CTLA4, LAG3, TNFRSF18, CD80 and FOXP3 were found to be significantly decreased in patient-matched samples after chemotherapy. Conclusion Our results suggest that conventional platinum-based chemotherapy negatively impacts the immune microenvironment at the time point of secondary progression.

Cell lines are essential tools to standardize and compare experimental findings in basic and translational cancer research. The current dogma states that cancer stem cells feature an increased tumor initiation capacity and are also chemoresistant. Here, we identified and comprehensively characterized three morphologically distinct cellular subtypes in the non–small cell lung cancer cell line A549 and challenge the current cancer stem cell dogma. Subtype-specific cellular morphology is maintained during short-term culturing, resulting in the formation of holoclonal, meroclonal, and paraclonal colonies. A549 holoclone cells were characterized by an epithelial and stem-like phenotype, paraclone cells featured a mesenchymal phenotype, whereas meroclone cells were phenotypically intermediate. Cell-surface marker expression of subpopulations changed over time, indicating an active epithelial-to-mesenchymal transition (EMT), in vitro and in vivo. EMT has been associated with the overexpression of the immunomodulators PD-L1 and PD-L2, which were 37- and 235-fold overexpressed in para- versus holoclone cells, respectively. We found that DNA methylation is involved in epigenetic regulation of marker expression. Holoclone cells were extremely sensitive to cisplatin and radiotherapy in vitro, whereas paraclone cells were highly resistant. However, inhibition of the receptor tyrosine kinase AXL, whose expression is associated with an EMT, specifically targeted the otherwise highly resistant paraclone cells. Xenograft tumor formation capacity was 24- and 269-fold higher in holo- than mero- and paraclone cells, respectively. Our results show that A549 subpopulations might serve as a unique system to explore the network of stemness, cellular plasticity, tumor initiation capacity, invasive and metastatic potential, and chemo/radiotherapy resistance.

Co-stimulatory signals, cytokines and transcription factors regulate the balance between effector and memory cell differentiation during T cell activation. Here, we analyse the role of the TRAF2-/NCK-interacting kinase (TNIK), a signaling molecule downstream of the tumor necrosis factor superfamily receptors such as CD27, in the regulation of CD8+ T cell fate during acute infection with lymphocytic choriomeningitis virus. Priming of CD8+ T cells induces a TNIK-dependent nuclear translocation of β-catenin with consecutive Wnt pathway activation. TNIK-deficiency during T cell activation results in enhanced differentiation towards effector cells, glycolysis and apoptosis. TNIK signaling enriches for memory precursors by favouring symmetric over asymmetric cell division. This enlarges the pool of memory CD8+ T cells and increases their capacity to expand after re-infection in serial re-transplantation experiments. These findings reveal that TNIK is an important regulator of effector and memory T cell differentiation and induces a population of stem cell-like memory T cells., Coordinate expression of multiple factors play critical roles in the regulation between effector and memory CD8+ T cell differentiation. Here the authors show upon acute viral infection TNIK is critically required as a regulator of effector and memory T cell differentiation.

Hematopoiesis in patients with cancer is characterized by reduced production of red blood cells and an increase in myelopoiesis, which contributes to the immunosuppressive environment in cancer. Some tumors produce growth factors that directly stimulate myelopoiesis such as G-CSF or GM-CSF. However, for a majority of tumors that do not directly secrete hematopoietic growth factors, the mechanisms involved in the activation of myelopoiesis are poorly characterized. In this study, we document in different murine tumor models activated hematopoiesis with increased proliferation of long-term and short-term hematopoietic stem cells and myeloid progenitor cells. As a consequence, the frequency of myeloid-derived suppressor cells and its ratio to CD8+ T cells increased in tumor-bearing mice. Activation of hematopoiesis and myeloid differentiation in tumor-bearing mice was induced by TNFα, which was mainly secreted by activated CD4+ T cells. Therefore, the activated adaptive immune system in cancer induces emergency myelopoiesis and immunosuppression. Significance: These findings characterize a regulatory circuit linking activated T cells to suppression of tumor-specific immune responses, providing a conceptual advance in the understanding of emergency-hematopoiesis in cancer and opening new targets for therapeutic approaches.

Oncogenic KRAS mutations comprise the largest subset of lung cancer defined by genetic alterations, but in the clinic no targeted therapies are available that effectively control mutational KRAS activation. Consequently, patients with KRAS-driven tumors are routinely treated with cytotoxic chemotherapy, which is often transiently effective owing to development of drug resistance. In this study, we show that hyperactivated mammalian target of rapamycin (mTOR) pathway is a characteristic hallmark of KRAS-mutant lung adenocarcinoma after chemotherapy treatment, and that KRAS-mutant lung cancer cells rely on persistent mTOR signaling to resist chemotherapeutic drugs. Coherently, mTOR inhibition circumvents the refractory phenotype and restores sensitivity of resistant KRAS-mutant lung cancer cells to chemotherapy. Importantly, drug combinations of clinically approved mTOR inhibitors and chemotherapy drugs synergize in inhibiting cell proliferation of KRAS-mutant cancer cells in vitro and in vivo, and the efficacy of this combination treatment correlates with the magnitude of mTOR activity induced by chemotherapy alone. These results pinpoint mTOR as a mechanism of resistance to chemotherapy in KRAS-mutant lung cancer and validate a rational and readily translatable strategy that combines mTOR inhibitors with standard chemotherapy to treat KRAS-mutant adenocarcinoma, the most common and deadliest lung cancer subset.

Chronic myeloid leukemia (CML) is a typical stem-cell driven malignancy, driven by leukemia stem cells (LSCs). LSCs are resistant to conventional therapies. This resistance is mediated by cell-intrinsic mechanisms and interactions with the microenvironment. LSCs depend on signals from a specialized microenvironment, a so called niche, to maintain their stem cell characteristics. In CML the bone marrow (BM) as a niche is well-investigated and several therapeutic targets, which aim at LSCs by interrupting their interaction with the BM-niche are under investigation. However, even though splenomegaly is a hallmark of CML the contribution of the splenic microenvironment to CML development has not been studied so far. This project aims to investigate the role of the splenic microenvironment as an independent secondary LSC niche and its contribution to disease development. To induce a CML-like disease in mice we retrovirally transduced FACS-sorted Lineage- Sca-1+ cKit+ BM cells with pMSCV-p210BCR/ABL-IRES-GFP and injected the transduced cells into non-irradiated mice. To find out if the spleen contributes to disease development we induced CML in splenectomized and sham operated mice. Splenectomized mice survived significantly longer compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20% of the splenectomized mice surviving longer than 90 days. Moreover, the number of LSCs in the BM of splenectomized mice was reduced 3.7-fold (p=0.002). Flowcytometric analysis of the spleen and BM compartments of CML bearing mice revealed that the majority of the leukemic stem and progenitor cells (LSPCs) were located in the spleen (19-fold more LSCs in the spleen; p =0.007). Moreover we found the leukemic compartment in the spleen to be enriched for LSPCs compared to the BM (20 % spleen vs. 10 % BM; % LSPCs of total leukemic cells; p=0.01). To confirm this phenotypic observation functionally we performed a limiting dilution transplantation of leukemic cells from spleen and BM. In line with the phenotypic observation we found a higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02). We next analyzed the gene expression of LSPCs from spleen and BM. We found that the gene expression profile of splenic LSPCs showed higher expression of stemness-related genes and reduced expression of myeloid differentiation genes compared to BM LSPCs, indicating that the spleen is more supportive of primitive LSPCs. Knowing that the spleen contributes to disease development by providing an alternate niche for LSCs we next analyzed the spleens using confocal microscopy. We found that the LSCs resided exclusively in the red pulp. Previous studies have shown that HSCs reside in direct contact with red pulp macrophages (RPMs) during extramedullary hematopoiesis (Dutta et al., JEM, 2015). In addition we found that in spleens from human CML patients CD34+ leukemia cells localized together with macrophages (p=0.001). Furthermore we could show that RPMs are capable of producing both SCF and G-CSF. To test the role of RPMs as a potential niche component in vitro we co-incubated LSCs and RPMs overnight before plating the LSCs in a colony formation assay. We found that the co-incubation with RPMs improved the colony formation capacity of LSCs (CFUs 166 vs. 138; p=0.0356). To test the role of RPMs in vivo we depleted macrophages in CML mice using clodronate liposomes. This resulted in significantly reduced splenomegaly (867mg vs. 249mg; p In summary, we found that the spleen provides an alternate niche for LSCs, thereby contributing to CML development. Compared to the BM niche the splenic niche is more supportive of primitive LSPCs, as shown by the higher frequency of LSCSs found in the spleen and the higher expression of stemness related genes in splenic LSCs compared to BM LSCs. Moreover we identified CD24low RPMs as a unique and central component of the splenic LSC niche. Even though we could show that RPMs are capable of producing SCF and G-CSF the exact mechanisms by which RPMs support LSCs remains to be investigated. Disclosures No relevant conflicts of interest to declare.

Cytotoxic T cells increase leukemia stem cell numbers in a murine model of chronic myeloid leukemia., Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8+ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34+ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.