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Summary Chronic lymphocytic leukaemia (CLL) is associated with alterations in T cell number, subset distribution and function. Among these changes, an increase in CD4+ T cells was reported. CD4+ T cells are a heterogeneous population and distinct subsets have been described to exert pro‐ and anti‐tumour functions. In CLL, controversial reports describing the dominance of IFNγ‐expressing Th1 T cells or of IL‐4‐producing Th2 T cells exist. Our study shows that blood of CLL patients is enriched in Th1 T cells producing high amounts of IFNγ. Moreover, we observed that their frequency remains relatively stable in CLL patients over a time course of five years. Furthermore, we provide evidence for an accumulation of Th1 T cells in the Eµ‐TCL1 mouse model of CLL. As TBET (encoded by Tbx21) is a crucial transcription factor for Th1 polarization, we generated Tbx21−/− bone marrow chimaeric mice which showed a lower number of IFNγ‐producing Th1 T cells, and used them for adoptive transfer of Eµ‐TCL1 leukaemia. Disease development in these mice was, however, comparable to that in wild‐type controls, excluding a major role for TBET‐expressing Th1 cells in Eµ‐TCL1 leukaemia. Collectively, our data highlight that Th1 T cells accumulate in CLL but reducing their number has no impact on disease development., publishedVersion

Chronic lymphocytic leukemia (CLL) is associated with an accumulation of oligoclonal CD8

Chronic lymphocytic leukaemia (CLL) is associated with alterations in T cell number, subset distribution and function. Among these changes, an increase in CD4

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID-19), a lung disease that may progress to systemic organ involvement and in some cases, death. The identification of the earliest predictors of progressive lung disease would allow for therapeutic intervention in those cases. In an earlier clinical study, individuals with moderate COVID-19 were treated with either arbidol (ARB) or inhaled interferon (IFN)-&alpha, 2b +/&minus, ARB. IFN treatment resulted in accelerated viral clearance from the upper airways and in a reduction in the circulating levels of the inflammatory biomarkers IL-6 and C-reactive protein (CRP). We have extended the analysis of this study cohort to determine whether IFN treatment had a direct effect on virus-induced lung abnormalities and also to ascertain whether any clinical or immune parameters are associated with worsening of lung abnormalities. Evidence is provided that IFN-&alpha, 2b treatment limits the development of lung abnormalities associated with COVID-19, as assessed by CT images. Clinical predictors associated with worsening of lung abnormalities include low CD8+ T cell numbers, low levels of circulating albumin, high numbers of platelets, and higher levels of circulating interleukin (IL)-10, IL-6, and C-reactive protein (CRP). Notably, in this study cohort, IFN treatment resulted in a higher percentage of CD8+ T cells, lower tumor necrosis factor (TNF)-&alpha, levels and, as reported earlier, lower IL-6 levels. Independent of treatment, age and circulating levels of albumin and CRP emerged as the strongest predictors of the severity of lung abnormalities.

Targeting B-cell receptor signaling using the PI3Kδ inhibitor idelalisib is a highly effective treatment option for relapsed/refractory chronic lymphocytic leukemia (CLL) patients. In addition to its direct impact on tumor cells, PI3Kδ inhibition can modulate the activity of regulatory T-cells (Tregs) resulting in enhanced anti-tumoral immune functions which may contribute to the success of PI3Kδ inhibitors in cancer therapy. The role of Tregs in CLL and their modulation by PI3Kδ inhibitors was so far poorly understood. Using the Eµ-TCL1 adoptive transfer model of CLL, we show that disease development induces the accumulation of activated and highly immunosuppressive Tregs. Depletion of CD25+ Tregs using anti-CD25 antibodies resulted in enhanced CD8+ T-cell activation, effector differentiation, and functional capacity. We further show that pharmacological inhibition of PI3Kδ effectively controlled disease and significantly decreased both CD25+ and CD25- Treg numbers, proliferation and activation status in CLL-bearing mice. Nonetheless, this PI3Kδ-mediated decrease in Tregs did not translate into better CD8+ T-cell function, as PI3Kδ inhibition concomitantly abrogated T-cell receptor signaling in CD8+ T-cells leading to decreased activation, effector cell differentiation and proliferation. Collectively, these data highlight the strong immunomodulatory effects of PI3Kδ inhibitors in CLL and are of relevance for a rational design of idelalisib-based combination therapies in CLL.

The focus of the current study was to interrogate the predictive potential of laser-induced autofluorescence (LIAF) by objectively assessing collagen synthesis in burn wound granulation tissues ex vivo. Prior grafting, granulation tissues (20 samples) following burn injury were collected from 17 subjects of age range 18 to 60 years with patient/donor consent and the corresponding autofluorescence spectra were recorded at 325 nm He-Cd laser (≈2 mW) excitations. The resulting endogenous collagen intensity from the above tissue samples was computed by normalizing the nicotinamide adenine dinucleotide levels. In addition, the hydroxyproline content was also estimated biochemically from the same granulation tissues. A comparative assessment of both LIAF and biochemical estimations for endogenous collagen by hydroxyproline resulted in strong positive correlation among them. The above relevant observations suggest that LIAF is equally informative as that of biochemical estimations, in evaluating endogenous collagen content in wound granulation tissues. Thus, it can be concluded that LIAF has the predictive potential, as a noninvasive objective tool to measure the endogenous collagen levels in wound biopsy tissues and provide complementary data conducive for making clinical decisions.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eμ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6C(low) patrolling or nonclassical phenotype. In addition, the percentage of MHC-II(hi) dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

Feng, Weijun et al., Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation., This work was supported by the Helmholtz Association (VH-NG-702), the Deutsche Forschungsgemeinschaft (KA 4472/1-1 for D.K., LI 2140/1-1 for H.-K.L.), the Deutsche Krebshilfe (110226 to H.-K.L.), the ERC (European Research Council) consolidator grant (647055) (to H.-K.L.), the Helmholtz Alliance ‘Preclinical Comprehensive Cancer Center' Grant HA-305 (to J.G., P.L. S.M.P. and H.-K.L.), the DKFZ Intramural Grant (to W.F. and D.K.), the Spanish Government (SAF2010-21017, SAF2013-47343-P, SAF2014-55532-R and FEDER funds, to F.C.-L.), the Andalusian Regional Government (P11-CVI-7948 and FEDER funds, to F.C.-L.), the European Research Council (ERC-CoG-2014-647359, to F.C.-L.) and Predoctoral Studentships from the University of Seville to J.A.L. (PIF-2011). CABIMER is supported by the Andalusian Regional Government (Junta de Andalucía).

Introduction: Genome-wide association studies showed that a single-nucleotide polymorphism (SNP) affecting the transcription factor Eomesodermin (Eomes) is associated with a significantly increased risk to develop chronic lymphocytic leukemia (CLL). Eomes and its paralogue T-bet are known master regulators of CD8+ effector T cells and CD4+ T helper cells and critical for T cell-mediated immune responses against pathogens and cancer. While Eomes has been shown to be essential for effector function of CD8 T cells, its role in CD4 T cells is less well understood. Recent work suggested that Eomes drives the development of IL-10 and IFNγ co-producing, FoxP3-negative, regulatory T cells, named Tr1 cells, a population that was found enriched in inflamed tissues in patients with chronic inflammatory disorders. In CLL, the T cell compartment is altered with an enrichment of effector and memory T cells that were shown to control leukemia development in a mouse model, but also to acquire a dysfunctional or exhausted state. Methods: We investigated Eomes-expressing CD4 and CD8 T cells in blood and lymph node (LN) samples of patients with CLL, as well as in the Eµ-TCL1 mouse model of CLL by flow cytometry, CyTOF, mRNA sequencing, and ex vivo functional assays. The role and function of these cells was explored in bone marrow-chimeric mice harbouring an Eomes-deficient hematopoietic microenvironment that were used for adoptive transfer of TCL1 leukemia, as well as by co-transfer experiments of Eomes- or IL-10R-deficent CD4 or CD8 T cells with TCL1 leukemia cells in Rag2-/- mice lacking B and T cells. Results: We detected an accumulation of Eomes-expressing CD4 and CD8 T cells in CLL patients, which was more severe in LN compared to blood samples, and significantly stronger in CLL LN compared to reactive LN samples as non-cancer control (Fig. 1A). This was in line with an observed expansion of Eomes-positive T cells in the spleen of leukemic Eµ-TCL1 mice and upon adoptive transfer of TCL1 leukemia. Eomes expression in CD8 T cells correlated with the expression of CD69, IFNγ and PD-1, suggesting a link between Eomes and CD8 T cell activation and function in CLL. The importance of Eomes in CD8 T cell-mediated control of CLL was demonstrated in mice that were transplanted with TCL1 leukemia, where Eomes-deficient CD8 T cells failed to control leukemia development. As we detected significantly less Eomes-deficient CD8 T cells in these mice compared to respective wildtype controls, and a lower percentage of them was positive for the proliferation marker Ki-67, we conclude that Eomes drives the differentiation and expansion of CD8 T cells in mice with CLL-like disease. We further explored Eomes-expressing CD4 T cells in CLL by transcriptome analysis, flow cytometry and ex vivo functional assays, and observed increased expression of IFNγ and IL-10, as well as inhibitory receptors, like PD-1, BLIMP-1 and LAG3, features that are described for Tr1 cells. Transfer of Eomes-deficient or wildtype CD4 T cells in Rag2-/- mice that were injected with TCL1 leukemia cells, lead to a comparable expansion of CD4 T cells independent of Eomes. But even though wildtype CD4 T cells were able to control leukemia development in this setting, Eomes-deficient CD4 T cells failed to do so (Fig. 1B). As Eomes is a known driver of IL-10 expression, we tested whether IL-10R signalling in CD4 T cells is involved in the anti-tumor activity by performing respective CD4 T cell transfer experiments comparing this time wildtype with IL-10R-deficent CD4 T cells. Interestingly, lack of IL-10R in CD4 T cells lead to a reduction in anti-tumor control (Fig. 1C) and therefore suggests that IL-10 is involved in Eomes-driven regulation of CD4 T cell-mediated immune control. Conclusions: In summary, we conclude that Eomes is required for CD8 T cell-mediated control of CLL, and Eomes+ PD-1+ IL-10-producing CD4 T cells contribute to adaptive immunity in CLL. The increased risk of developing CLL in individuals harbouring a SNP in the Eomes gene might be therefore explained by a negative impact of this alteration on CD4 and/or CD8 T cell-mediated immune control of CLL. Disclosures Stilgenbauer: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A secondary metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A (ChA), a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by ChA, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell-cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that ChA prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles 30 to 1000 nm in size and represent a novel mechanism of cell communication. By transferring RNA and protein from their cell of origin, they can reprogram target cells and thus are involved in changes within the cellular microenvironment - a key player in CLL pathogenesis. In the current study, we were able to isolate EVs of 20 to 300 nm from blood plasma of CLL patients as well as from supernatant of primary CLL cells in culture. Further, proteomic profiling by Coomassie staining of SDS-PAGE gels and by mass spectrometry revealed an EV-specific protein profile. These findings suggest that EVs represent an important mean of CLL cells to interact with other cells, which might contribute to the establishment of a pro-survival microenvironment for CLL cells.

This paper models the long-run relationship between the Bureau De Change exchange rate and external reserves in Nigeria in a Threshold Vector Error Correction Model (TVECM) framework using daily data that spans from Jan 1, 2014 to Jul 31, 2015. Modeling BDC exchange rate and external reserves within this context can be motivated by the fact that the transition mechanism between the variables is controlled by the degree of BDC exchange rates premium which is within central bank of Nigeria's policy oversight. The supLM test result indicates that there is a non-linear long-run relationship between the series, providing empirical support in favor of a TVECM specification. Thus, Cointegration occurs when the divergence between the two variables is above the threshold point estimate. Two regimes are implied by the model: the 'usual' regime, which accounts for 93.1per cent of the observations and the 'unusual' one, representing about 6.9 per cent of the observations of the sample. We also find that the error correction coefficients for both the bureau de change exchange rate and external reserves equations were not statistically significant at the 5 per cent significance level. While in the second regime, error correction coefficient for the external reserves equation was found to be statistically significant at 10 per cent. This implies that the adjustment mechanism between the two variables flow from external reserves to BDC exchange rate.

To examine the fertilizing ability and DNA damage response of preimplantation stage embryos derived from the γ-irradiated mouse sperm carrying varying amounts of DNA strand-breaks.The DNA damage in the sperm was induced by exposing the testicular area to different doses of γ-radiation. After mating with healthy female mice, sperm zona binding, fertilizing ability of DNA damaged sperm and developmental competence of embryos derived from the DNA damaged sperm were assessed.The in vivo zona binding ability and fertilizing ability of DNA damaged sperm was significantly affected in the 5.0 and 10.0 Gy sperm irradiation groups. Although the development of the embryos derived from the DNA damaged sperm was not significantly affected until day 2.5 post-coitus, further development was significantly altered, as evidenced by the total cell number in the embryos.The sperm carrying DNA strand breaks still has the ability to fertilize the oocyte normally. However, the events like zona-binding and successful fertilization depend on the extent of sperm DNA fragmentation. The study has also showed a great heterogeneity in embryonic development at peri-implantation period with respect to the degree of sperm DNA damage.Gama ışınları ile ışınlanmış ve değişen miktarda DNA zincir kırığı taşıyan fare sperminin dölleme yeteneğini ve bu spermlerden oluşan implantasyon öncesi embriyoların DNA hasarı yanıtını incelemek.Spermde DNA hasarı, testiküler bölgenin farklı dozlarda gama ışınlarına maruz bırakılmasıyla indüklendi. Sağlıklı dişi fareler ile çiftleşmeden sonra, sperm-zona bağlanması, DNA hasarlı spermin dölleme yeteneği ve DNA hasarlı spermden oluşan embriyoların gelişimsel kompetansı değerlendirildi.5.0 ve 10.0 Gy ışın uygulanan gruplarda, DNA hasarlı sperminDNA zincir kırıkları taşıyan sperm hala oositi normal bir şekilde dölleme yeteneğine sahiptir, bununla beraber, zona-bağlanması ve başarılı fertilizasyon gibi olaylar sperm DNA parçalanmasının boyutuna bağlıdır. Çalışma ayrıca peri-implantasyon döneminde embriyonik gelişimde sperm DNA hasarının derecesine ilişkin olarak büyük bir heterojenlik göstermiştir.

Blockade of the programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint augments antitumor immunity and induces durable responses in patients with solid cancers, but data on clinical efficacy in leukemias are sparse. Chronic lymphocytic leukemia (CLL) is associated with a tumor-supportive microenvironment and a dysfunctional immune system, as shown by “exhausted” T cells, defective immunologic synapse formation, and immunosuppressive myeloid cells. These defects involve aberrant expression of PD-L1 and are closely mirrored in the Eµ-TCL1 mouse model for CLL. In this study, we treated mice after adoptive transfer of Eµ-TCL1 CLL with PD-L1–blocking antibodies, which prevented CLL development and was accompanied by a reactivation of immune effector functions. This included restoration of mature macrophages and major histocompatibility complex class II–expressing dendritic cells and prevention of aberrant and exhaustion-like T-cell phenotypes. In addition, PD-L1 blockade restored CD8 T-cell cytotoxicity and immune synapse formation and normalized T-cell cytokines and proliferation ex vivo and in vivo. Our data demonstrate that early PD-L1 blockade effectively corrects leukemia-induced immune dysfunction and thus prevents CLL development in mice. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies with CLL patients, ideally in combination with novel compounds to help eliminate CLL.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy that is stringently associated with a tumor-supportive microenvironment and defective anti-tumor immunity. T cells from CLL patients show features of exhaustion, including expression of PD-1, and are highly impaired in their activity. Our previous work showed that immune checkpoint blockade using anti-PD-L1 effectively prevents disease and restores T-cell activity in the Eμ-TCL1 mouse model of CLL. Development of disease in these mice was shown to be associated with a relative loss of naïve T cells and an enrichment of effector T cells displaying an exhausted phenotype. Our work showed that T cells in the Eμ-TCL1 mice express high levels of the chemokine receptor CXCR3 accompanied with elevated serum levels of the corresponding chemokines CXCL9 and CXCL10, suggesting a role for CXCR3 in T-cell skewing in CLL. Intracellular flow cytometric analysis showed an enrichment of CXCL9+ CD45+ hematopoietic cells in leukemic spleens which were identified as Ly6C+ monocytes. Along this line, depletion of phagocytic cells by Clodronate Liposomes resulted in significantly decreased serum levels of CXCL9, a reversal of T-cell skewing and slowed down development of CLL in mice. To evaluate the role of this receptor in CLL in vivo, adoptive transfer of murine CLL cells in CXCR3 knockout mice is currently ongoing. We further perform an in-depth phenotypical analysis of T-cell subsets in blood samples of CLL patients from different prognostic subgroups to characterize T-cell aberrations along with disease progression. Taken together, our data suggest a pathomechanistic role for CXCL9, CXCL10 and their receptor CXCR3 in CLL. Their potential as novel drug targets needs to be explored in the future.

Background: Clinical studies have demonstrated that targeted immunotherapy using PD-1/PD-L1 antibodies induces tumor regression and prolongs disease stabilization in advanced solid cancers. Data on the clinical efficacy in hematological malignancies is largely missing, even though PD-L1/PD-1 interactions have been described as major mediators of immune dysfunction in several leukemias and lymphomas. They are therefore ideal to study if PD-L1/PD-1 blockade has the potential to control disease by restoring anti-tumor immune responses. Several groups showed that chronic lymphocytic leukemia (CLL) provokes immune evasion via PD-L1/PD-1 inhibitory signaling, and that this is very closely mirrored in the Eµ-TCL1 (TCL1) murine model for CLL. Our recent data suggest that in this model, aberrant PD-L1 expression in myeloid cells contributes to the immune defect in CLL. We further demonstrated that the T cell and myeloid cell immune defects in ageing leukemic mice can be induced in young wild-type (WT) mice by adoptive transfer (AT) of murine CLL. In the current study, we used the AT model to test if in vivoPD-L1 blockade corrects leukemia-induced cellular immune dysfunction in myeloid and T cells and enhances anti-tumor immunity. Methods: WT mice transplanted with 4x107 TCL1 splenocytes were randomized to treatment with 10 mg/kg α-murine-PD-L1 (n=15) or isotype antibody (n=10), which was administered i.p. every 3 days starting 1 day after AT, and sacrificed 31 days later. Matched non-transplanted WT mice (n=6) served as additional controls. Immune cell subsets, expression of immune checkpoint markers and T cell effector functions were analyzed by multicolor flow cytometry using cells isolated form spleen, peripheral blood (PB), bone marrow (BM) and peritoneal cavity (PC). Cell proliferation was measured by EdU incorporation in vivo. Immune synapse (IS) formation was assessed by confocal microscopy. Serum cytokines were quantified by multiplex bead arrays. Results: We first confirmed successful engraftment and presence of disease by immunohistochemistry. Compared to isotype controls, α-PD-L1 treated mice had significantly lower spleen weights (median 0.2 g vs 0.9 g, p Conclusion: Our in vivodata demonstrate that early PD-L1 blockade very effectively controls CLL development and enables complex effector function of myeloid and T cells, thus restoring anti-tumor immune responses. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies, potentially in combination with novel substances. BH/FM and MS/JGG contributed equally to first and last authorship. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that is characterized by apoptosis resistance and dysfunctional immune system. The chronic nature and slow development of the disease indicates a contribution of CLL-induced inflammation in the disease course. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in an in vivo system. Here, we used the well-established CLL mouse model, Eµ-TCL1 mice (TCL1), to characterize changes within myeloid cell populations along with CLL development and the influence of their depletion on disease progression and immune dysfunction. We have recently shown that CLL development in TCL1 mice is associated with massive changes within myeloid cell populations. In the peritoneal cavity (PC) of leukemic mice we observed an infiltration of monocytes and an M2-like skewing of macrophages according to phenotypical and signaling signatures. Along this line, monocytes infiltrated the spleens of leukemic animals, both in primary CLL and adoptive transfer models, which is most likely due to high CCL2 serum levels. These monocytes lost the inflammatory Ly6Chi subset and were severely skewed towards Ly6Clow patrolling monocytes, accompanied by high expression of adhesion and angiogenic molecules like ICAM1, PECAM1 and MMP14. Gene expression profiling of splenic myeloid cells from TCL1 mice revealed an enrichment of various genes involved in dendritic cell (DC) maturation and MHC-II-mediated antigen presentation. However, the numbers of MHC-IIhi mature DCs and macrophages were significantly decreased, suggesting a monocyte differentiation arrest leading to impaired anti-tumor immune response. The observed transcriptional upregulation of multiple inflammatory cytokines like TNF-α, CXCL9, CXCL10 and CXCL16 in monocytes was confirmed by serum cytokine arrays, and is likely due to the overexpression of the pro-inflammatory regulator TREM-1. In addition, TCL1 monocytes upregulated the expression of several inhibitory molecules like PD-L1, IL-10, IL1ra and IL4i1 suggesting an impaired immune function. While CLL-induced immune dysfunction is a well-established phenomenon, the contribution of myeloid cells in this context was not clear. We therefore sought to determine the in vivo effects of myeloid cell depletion on CLL development and its associated immune defects. For that purpose we used liposomal clodronate to selectively ablate macrophages and monocytes from young wild-type mice adoptively transferred with murine CLL. Our data clearly show control of CLL development in clodronate-treated mice relative to control liposomes as demonstrated by decreased spleen weight (1.09 vs. 0.54 g, p < 0.0001) and a significant drop in tumor load, defined as CD5+CD19+ cells, in spleen (60.58% vs. 42.25%), peripheral blood (43% vs 11.8%), PC (66.2% vs 3.1%), lymph nodes (4.9% vs 1.2%) and bone marrow (1.9% vs 0.8%). In addition, we observed changes in immune effector cells in response to myeloid cell depletion suggesting better immune status in treated mice. Interestingly, the loss of macrophages/monocytes was compensated by increased splenic monocyte proliferation as shown by EdU incorporation in vivo. In contrast to control mice, the repopulating monocytes upon clodronate treatment were largely inflammatory Ly6Chi monocytes. In summary, our data show that skewing of myeloid cells actively contributes to CLL development via; 1) enhancing the survival of leukemic cells, and 2) suppressing anti-tumor immune functions. In the absence of monocytes and macrophages, disease development is delayed in mice adoptively transferred with murine CLL. Therefore, we suggest that targeting non-malignant myeloid cells in CLL might serve as a novel strategy for CLL immunotherapy. Disclosures No relevant conflicts of interest to declare.

The importance of the tumor microenvironment in the development of B-cell chronic lymphocytic lekuemia (CLL) is now widely accepted. Previous studies within our and other groups revealed the establishment of an inflammatory milieu in CLL characterized by enhanced expression and secretion of several cytokines and their receptors. Using 250 CLL serum samples and 50 age-matched controls, we found significantly increased levels of the TNFα receptors TNFR1 and TNFR2 in the patients that positively correlated with an adverse clinical outcome. Based on these findings we aimed to investigate the functional role of TNFR signaling in CLL development. In contrast to healthy B cells that do not express TNFα receptors, we detected TNFR1 expression in CLL cells upon survival-inducing culture conditions and in lymph node sections of 80 CLL patients, where TNFR1 signals co-localized to the B cell marker CD20 and were mainly present within proliferation centers. These findings were confirmed in Eµ-TCL1 mice, a well-established mouse model for CLL. Here, CLL cells in the peripheral blood were negative for TNFR1. However, the cells upregulated the receptor upon entering the spleen where they get into contact with accessory cells, receive survival stimuli and undergo proliferation. In addition, increased levels of soluble TNFR1 in the serum were confirmed in the mice. After ligand binding, TNFR1 can activate two different signaling pathways: (1) the extrinsic apoptosis cascade through its death receptor domain, or (2) survival-inducing NFκB signaling. The latter pathway can be blocked by wogonin, a naturally occurring monoflavonoid. In a multitude of in vitro and in vivo studies, wogonin has been shown to exert anti-oxidant, anti-inflammatory and anti-tumor activities. By ex vivo treatment of CLL cells with TNFα we observed NFκB activation which was reversed by TNFα-blocking antibody, suggesting TNFR1-mediated survival signaling in CLL. Therefore, we aimed to test whether wogonin can prevent this signaling in CLL cells and might therefore represent a novel potential drug for CLL. In CLL cocultures, wogonin treatment resulted in a concentration-dependent apoptosis induction, which was significantly increased by the addition of TNFα. To test the effects of wogonin in vivo, we transplanted isogenic, immunocompetent wild-type mice with CLL cells from leukemic TCL1 animals and treated them daily with 40 mg/kg wogonin. When treatment was started 2 days after transplantation, wogonin significantly reduced spleen weights and lead to a reduced CLL content in the spleen, the bone marrow and the peritoneal cavity. If treatment was started in advanced disease stage, wogonin slightly lowered spleen weight and the CLL content in the spleen, whereas the percentage of CLL cells in the peripheral blood was increased. Interestingly, here wogonin treatment resulted in a loss of cell surface TNFR1 expression in splenic CLL cells and increased TNFR1 levels in the serum. These data suggest that wogonin induces a redistribution of CLL cells in vivo, preventing their homing to lymphoid organs and that loss of TNFR1 expression might be involved in this process. In summary, our results show that TNFR1 signaling is involved in CLL cell activation and survival. Targeting this pathway with wogonin reduces CLL cell viability in vitro and leukemia development in TCL1 mice. Disclosures No relevant conflicts of interest to declare.