Lack of Functional P110δ Affects Expression of Activation Marker CD80 but Does Not Influence Functions of Neutrophils.

Neutrophils are specialized immune cells that are essential constituents of the innate immune response. They defend the organism against pathogens through various mechanisms. It was reported that phosphatidylinositols are key players in neutrophil functions, especially in the activity of class-I phosphoinositide 3-kinases (PI3Ks). P110δ, one of the PI3K subunits, is mostly expressed in immune cells, and its activity plays an important role in inflammatory responses. The aim of this study was to investigate the role of p110δ in neutrophil antimicrobial functions, activation status and cytokine production. To this end, we used bone marrow and splenic neutrophils isolated from a murine model expressing catalytically inactive p110δD910A/D910A. The level of phagocytosis and degranulation, the expressions of activation markers and cytokine production were determined by flow cytometry. ROS generation and NET release were assessed by fluorometry and fluorescent microscopy. We observed a significantly higher percentage of CD80-positive cells among the splenic granulocytes and found granulocytes subpopulations of differing phenotypes between WT and p110δD910A/D910A mice by multiparametric tSNE analysis. Moreover, we detected some differences in the expressions of activation markers, intracellular production of cytokines and bacterial killing. However, we did not observe any alterations in the selected neutrophil functions in p110δ mutant mice. Altogether, our data suggest that the catalytic p110 subunit(s), other than p110δ, is a key player in most neutrophil functions in mice. A follow-up study to correlate these in vitro results with in vivo observations is highly recommended.

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps.

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.

The Role of Neutrophils in the Pathogenesis of Chronic Lymphocytic Leukemia.

Tumor-associated neutrophils appear to be a crucial element of the tumor microenvironment that actively participates in the development and progression of cancerous diseases. The increased lifespan, plasticity in changing of phenotype, and functions of neutrophils influence the course of the disease and may significantly affect survival. In patients with chronic lymphocytic leukemia (CLL), disturbances in neutrophils functions impede the effective immune defense against pathogens. Therefore, understanding the mechanism underlying such a phenomenon in CLL seems to be of great importance. Here we discuss the recent reports analyzing the phenotype and functions of neutrophils in CLL, the most common leukemia in adults. We summarize the data concerning both the phenotype and the mechanisms by which neutrophils directly support the proliferation and survival of malignant B cells.

Inhibition of IDO leads to IL-6-dependent systemic inflammation in mice when combined with photodynamic therapy.

It was previously reported that the activation of antitumor immune response by photodynamic therapy (PDT) is crucial for its therapeutic outcome. Excessive PDT-mediated inflammation is accompanied by immunosuppressive mechanisms that protect tissues from destruction. Thus, the final effect of PDT strongly depends on the balance between the activation of an adoptive arm of immune response and a range of activated immunosuppressive mechanisms. Here, with flow cytometry and functional tests, we evaluate the immunosuppressive activity of tumor-associated myeloid cells after PDT. We investigate the antitumor potential of PDT combined with indoleamine 2,3-dioxygenase 1 (IDO) inhibitor in the murine 4T1 and E0771 orthotopic breast cancer models. We found that the expression of IDO, elevated after PDT, affects the polarization of T regulatory cells and influences the innate immune response. Our results indicate that, depending on a therapeutic scheme, overcoming IDO-induced immunosuppressive mechanisms after PDT can be beneficial or can lead to a systemic toxic reaction. The inhibition of IDO, shortly after PDT, activates IL-6-dependent toxic reactions that can be diminished by the use of anti-IL-6 antibodies. Our results emphasize that deeper investigation of the physiological role of IDO, an attractive target for immunotherapies of cancer, is of great importance.

Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species.

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils' antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents.

BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells. METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples. RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate. CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells.

A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy.

BACKGROUND: Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. METHODS: In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. RESULTS: Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. CONCLUSIONS: Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.

New insights into redox homeostasis as a therapeutic target in B-cell malignancies.

PURPOSE OF REVIEW: The goal of this review is to summarize recent advances in our understanding of the regulation of redox homeostasis and the subtype-specific role of antioxidant enzymes in B-cell-derived malignancies. Furthermore, it presents selected prooxidative therapeutic strategies against B-cell neoplasms. RECENT FINDINGS: Recent reports have shown that the disturbed redox homeostasis in B-cell malignancies is regulated by cancer-specific signaling pathways and therefore varies between the individual subtypes. For instance, in a subtype of diffuse large B-cell lymphoma with increased oxidative phosphorylation, elevated reactive oxygen species are accompanied by higher levels of thioredoxin and glutathione and inhibition of either of these systems is selectively toxic to this subtype. In addition, growing number of small molecule inhibitors targeting antioxidant enzymes, such as auranofin, SK053, adenanthin, or decreasing glutathione level, such as imexon, buthionine sulfoximine, and L-cysteinase, trigger specific cytotoxic effects against B-cell malignancies. Lastly, attention is drawn to recent reports of effective treatment modalities involving prooxidative agents and interfering with redox homeostasis provided by stromal cells. SUMMARY: Recent findings reveal important differences in redox homeostasis within the distinct subsets of B-cell-derived malignancies that can be therapeutically exploited to improve existing treatment and to overcome drug resistance.

Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells.

Natural killer (NK) cells are considered critical components of the innate and adaptive immune responses. Deficiencies in NK cell activity are common, such as those that occur in cancer patients, and they can be responsible for dysfunctional immune surveillance. Persistent oxidative stress is intrinsic to many malignant tumours, and numerous studies have focused on the effects of reactive oxygen species on the anti-tumour activity of NK cells. Indeed, investigations in animal models have suggested that one of the most important thiol-dependent antioxidant enzymes, peroxiredoxin 1 (PRDX1), is essential for NK cell function. In this work, our analysis of the transcriptomic expression pattern of antioxidant enzymes in human NK cells has identified PRDX1 as the most prominently induced transcript out of the 18 transcripts evaluated in activated NK cells. The change in PRDX1 expression was followed by increased expression of two other enzymes from the PRDX-related antioxidant chain: thioredoxin and thioredoxin reductase. To study the role of thiol-dependent antioxidants in more detail, we applied a novel compound, adenanthin, to induce an abrupt dysfunction of the PRDX-related antioxidant chain in NK cells. In human primary NK cells, we observed profound alterations in spontaneous and antibody-dependent NK cell cytotoxicity against cancer cells, impaired degranulation, and a decreased expression of activation markers under these conditions. Collectively, our study pinpoints the unique role for the antioxidant activity of the PRDX-related enzymatic chain in human NK cell functions. Further understanding this phenomenon will prospectively lead to fine-tuning of the novel NK-targeted therapeutic approaches to human disease.

Immunological aspects of antitumor photodynamic therapy outcome.

Photodynamic therapy (PDT) of cancer is an efficient and promising therapeutic modality approved for the treatment of several types of tumors and non-malignant diseases. It involves administration of a non-toxic photosensitizer followed by illumination of the tumor site with a harmless visible light. A light activated photosensitizer can transfer its energy directly to molecular oxygen, leading to production of highly toxic reactive oxygen species (ROS). Antitumor effects of PDT result from the combination of three independent mechanisms involving direct cytotoxicity to tumor cells, destruction of tumor vasculature and induction of the acute local inflammatory response. PDT-mediated inflammatory reaction is accompanied by tumor infiltration of the leukocytes, enhanced production of pro-inflammatory factors and cytokines. Photodynamic therapy is able to effectively stimulate both the innate and the adaptive arm of the immune system. In consequence, this regimen can lead to development of systemic and specific antitumor immune response. However, there are limited studies suggesting that under some specific circumstances, PDT on its own may exert some immunosuppressive effects leading to activation of immunosuppressive cells or cytokines production. In this report we briefly review all immunological aspects of PDT treatment.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

The Exploitation of the Glycosylation Pattern in Asthma: How We Alter Ancestral Pathways to Develop New Treatments.

Asthma has reached epidemic levels, yet progress in developing specific therapies is slow. One of the main reasons for this is the fact that asthma is an umbrella term for various distinct subsets. Due to its high heterogeneity, it is difficult to establish biomarkers for each subset of asthma and to propose endotype-specific treatments. This review focuses on protein glycosylation as a process activated in asthma and ways to utilize it to develop novel biomarkers and treatments. We discuss known and relevant glycoproteins whose functions control disease development. The key role of glycoproteins in processes integral to asthma, such as inflammation, tissue remodeling, and repair, justifies our interest and research in the field of glycobiology. Altering the glycosylation states of proteins contributing to asthma can change the pathological processes that we previously failed to inhibit. Special emphasis is placed on chitotriosidase 1 (CHIT1), an enzyme capable of modifying LacNAc- and LacdiNAc-containing glycans. The expression and activity of CHIT1 are induced in human diseased lungs, and its pathological role has been demonstrated by both genetic and pharmacological approaches. We propose that studying the glycosylation pattern and enzymes involved in glycosylation in asthma can help in patient stratification and in developing personalized treatment.

Prenyltransferases regulate CD20 protein levels and influence anti-CD20 monoclonal antibody-mediated activation of complement-dependent cytotoxicity.

Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

Regulatory T cells contribute to the immunosuppressive phenotype of neutrophils in a mouse model of chronic lymphocytic leukemia.

BACKGROUND: Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. METHODS: In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. RESULTS: The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the "healthy" phenotype of neutrophils. CONCLUSIONS: Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Evaluation of the Antitumor Immune Response Following Photofrin-Based PDT in Combination with the Epigenetic Agent 5-Aza-2'-Deoxycytidine.

Photofrin-based photodynamic therapy (PDT) is approved for clinical use by the US Food and Drug Administration and the European Medicines Agency and is among the most widely used photosensitizer for the treatment of cancer. It was broadly reported that both the innate and the adaptive arms of immune response can be activated by PDT and play a critical role in the anticancer outcome of this treatment. PDT leads to the induction of acute local inflammation that includes leukocyte infiltration as well as increased activation and production of pro-inflammatory factors and cytokines. These events can lead to the development of systemic and specific antitumor immune response. Combining Photofrin-PDT with the epigenetic agent 5-aza-2'-deoxycytidine results in potentiated antitumor effects in vivo. Understanding the molecular mechanisms underlying this phenomenon would be invaluable for clinical development of this therapeutic approach. This chapter describes a detailed protocol allowing evaluation of specific antitumor immune response induced by PDT.

PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells.

BACKGROUND: Immune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets. METHODS: New atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1-CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2-CAR T cells were used for combination with PD-L1-CAR T cells against MCF-7 cells. RESULTS: PD-L1-CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1-CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1-CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1-CAR cells. Accordingly, PD-L1-CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1-CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2-CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1-CAR T cells when used in combination. CONCLUSIONS: In summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1-CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1-CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1-CAR strategy into clinical studies.

A Specific CD44lo CD25lo Subpopulation of Regulatory T Cells Inhibits Anti-Leukemic Immune Response and Promotes the Progression in a Mouse Model of Chronic Lymphocytic Leukemia.

Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.

Potent, p53-independent induction of NOXA sensitizes MLL-rearranged B-cell acute lymphoblastic leukemia cells to venetoclax.

The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.