Does delayed EBV infection contribute to rising childhood cancers?

Childhood cancer is on the rise in high-income countries. Epidemiological studies suggest that reduced exposure to common infections in early life is to blame. However, no specific infection responsible for protection against cancer has been identified, and the underlying mechanisms remain a matter of speculation. Recent findings that Epstein-Barr virus (EBV) can induce antitumor immunity lead us to hypothesize that the delay in EBV infection in such countries might contribute to the increase in childhood cancers.

M-Sec induced by HTLV-1 mediates an efficient viral transmission.

Human T-cell leukemia virus type 1 (HTLV-1) infects target cells primarily through cell-to-cell routes. Here, we provide evidence that cellular protein M-Sec plays a critical role in this process. When purified and briefly cultured, CD4+ T cells of HTLV-1 carriers, but not of HTLV-1- individuals, expressed M-Sec. The viral protein Tax was revealed to mediate M-Sec induction. Knockdown or pharmacological inhibition of M-Sec reduced viral infection in multiple co-culture conditions. Furthermore, M-Sec knockdown reduced the number of proviral copies in the tissues of a mouse model of HTLV-1 infection. Phenotypically, M-Sec knockdown or inhibition reduced not only plasma membrane protrusions and migratory activity of cells, but also large clusters of Gag, a viral structural protein required for the formation of viral particles. Taken together, these results suggest that M-Sec induced by Tax mediates an efficient cell-to-cell viral infection, which is likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag.

Efficacy and mechanism of the anti-CD38 monoclonal antibody Daratumumab against primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare, aggressive B cell non-Hodgkin's lymphoma of the body cavities with malignant effusions. The prognosis is poor, and no optimal treatment has been established. CD38 is a type II transmembrane glycoprotein known to overexpress in multiple myeloma (MM). Daratumumab (DARA), a human CD38-targeting monoclonal antibody (mAb), is approved for MM treatment. In this study, we found expression of CD38 on PEL cells and assessed the anti-PEL activity of DARA. We found that both KHYG-1 and N6 (CD16-transfected KHYG-1) NK cell lines showed direct killing activity against PEL cells with induction of CD107a, and NK-mediated cytotoxicity by N6NK (CD16+) cells increased with DARA treatment. We confirmed direct NK activity and antibody-dependent cell cytotoxicity (ADCC) by expanded NK cells, indicating that DARA has high ADCC activity. We elucidated the antibody-dependent cell phagocytosis (ADCP) by using human monocyte-derived macrophages (MDMs) and mouse peritoneal macrophages. DARA also showed potent complement-dependent cytolysis (CDC) toward PEL. DARA also induced PEL cell death in the presence of a cross-linking antibody. Moreover, treatment with DARA inhibited tumor growth in a PEL xenograft mouse model. These results provide preclinical evidence that Ab targeting of CD38 could be an effective therapeutic strategy for the treatment of PEL.

Elotuzumab, a potential therapeutic humanized anti-SLAMF7 monoclonal antibody, enhances natural killer cell-mediated killing of primary effusion lymphoma cells.

Primary effusion lymphoma (PEL) is a rare aggressive B-cell non-Hodgkin's lymphoma with no optimal treatment. Signaling lymphocytic activation molecule-F7 (SLAMF7, CD319), a type I transmembrane glycoprotein highly expressed in multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. SLAMF7 also expresses on several hematopoietic lineages including NK cells. Elotuzumab (Elo), a humanized antibody targeting SLAMF7, is approved by FDA for MM treatment. In this study, we analyzed the expression of SLAMF7 on seven PEL cell lines. All PEL cells and NK cells showed high expression of SLAMF7. NK cells were enriched from PBMCs of healthy donors by MACS and expanded by co-culturing with MHC-class I negative K562 cells in the presence of IL-2 and IL-15. Expanded NK cells showed direct killing, and Elo demonstrated potent ADCC against PEL in an Effector:Target (E:T) dependent manner. Surface expression of CD107a on NK cells also increased in the process of ADCC. We also examined SLAMF7 expression of NK subpopulations and found that the CD56+CD16+ NK subpopulation demonstrated the highest SLAMF7 expression. Full-length-Elo but not F(ab')2-Elo exerts direct engagement to the expressing SLAMF7 on NK cells, promotes CD107a expression, and further augments NK cytotoxicity toward PEL. Elo enhanced survival of PEL-bearing immunodeficient mice with adoptive transfer of human NK cells. Taken together, our results show that NK cells play roles in PEL killing, and Elo causes ADCC/SLAMF7 ligation to boost NK cytotoxicity against PEL, offering promising preclinical evidence of Elo as a therapeutic monoclonal antibody treatment for PEL.

Tyrosine kinase/phosphatase inhibitors decrease dengue virus production in HepG2 cells.

Dengue virus is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. High rates of dengue virus replication and virion production are related to disease severity. To identify anti-DENV compounds, we performed cell-based ELISA testing to detect the level of DENV E protein expression. Among a total of 83 inhibitors, eight were identified as inhibitors with antiviral activity. Epidermal growth factor receptor inhibitor II (EGFR/ErbB-2/ErbB-4 inhibitor II) and protein tyrosine phosphatase inhibitor IV (PTP inhibitor IV) significantly inhibited dengue virus production and demonstrated low toxicity in hepatocyte cell lines. Our results suggest the efficacy of tyrosine kinase/phosphatase inhibitors in decreasing dengue virus production in HepG2 cells.

Dengue virus disrupts Daxx and NF-κB interaction to induce CD137-mediated apoptosis.

Dengue virus (DENV) is a positive-strand RNA virus of the Flavivirus family with 4 different serotypes. Clinical manifestations of DENV infection include dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Following DENV infection, apoptosis of hepatic cells is observed both in vitro and in vivo. However, the molecular mechanisms revealing how viral components affect cellular apoptosis remain unclear. In the present study, the role of death domain-associated protein 6 (Daxx) in DENV-mediated apoptosis was characterized by RNA interference and overexpression studies, and the anti-apoptotic function of Daxx during DENV infection was identified. Furthermore, the viral component, DENV capsid protein (DENV C), interacted with Daxx to disrupt interaction between Daxx and NF-κB. The liberated NF-κB activated the promoter of CD137, which is a member of the TNF family, and is previously shown to induce apoptosis during DENV infection. In summary, DENV C disrupts Daxx and NF-κB interaction to induce CD137-mediated apoptosis during DENV infection.

Promising immunotherapeutic approaches for primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a large B-cell neoplasm usually presenting as a serious effusion in body cavities without detectable tumor masses. It is an AIDS-related non-Hodgkin's lymphoma (HL) with human herpes virus 8 (HHV8)/Kaposi sarcoma-associated herpes virus (KSHV) infection. A combination antiretroviral therapy (cART) prolongs the lifespan of AIDS and AIDS-related malignant lymphoma patients, but PEL continues to have a dismal prognosis. PEL showed disappointing outcomes with standard chemotherapy such as CHOP or CHOP-like regimens. A PEL status highlights the urgent need for new therapeutic approaches and treatment strategies and improve clinical outcomes. This review discusses the current knowledge and some recent clinical trials for PEL in the platform of immunotherapy as well as promising future immunotherapeutic approaches for PEL.

Interleukin-1ß: Friend or foe for gastrointestinal cancers.

Gastrointestinal (GI) cancer is a malignancy arising in the digestive system and accounts for approximately a third of increasing global cancer-related mortality, especially in the colorectum, esophagus, stomach, and liver. Interleukin-1ß (IL-1ß) is a leukocytic pyrogen recognized as a tumor progression-related cytokine. IL-1ß secretion and maturation in inflammatory responses could be regulated by nuclear factor-kappaB-dependent expression of NLR family pyrin domain containing 3, inflammasome formation, and activation of IL-1 converting enzyme. Several studies have documented the pro-tumorigenic effects of IL-1ß in tumor microenvironments, promoting proliferation and metastatic potential of cancer cells in vitro and tumorigenesis in vivo. The application of IL-1ß inhibitors is also promising for targeted therapy development in some cancer types. However, as a leukocytic pro-inflammatory cytokine, IL-1ß may also possess anti-tumorigenic effects and be type-specific in different cancers. This editorial discusses the up-to-date roles of IL-1ß in GI cancers, including underlying mechanisms and downstream signaling pathways. Understanding and clarifying the roles of IL-1ß would significantly benefit future therapeutic targeting and help improve therapeutic outcomes in patients suffering from GI cancer.

Trastuzumab, a monoclonal anti-HER2 antibody modulates cytotoxicity against cholangiocarcinoma via multiple mechanisms.

Cholangiocarcinoma (CCA) is an aggressive and fatal cancer. The prognosis is very poor and no optimal chemotherapy has been established. Human epidermal growth factor receptor 2 (HER2, neu, and erbB2) is highly-expressed in breast cancer and is expressed in many other tumors but poorly expressed in CCA. The anti-HER2 antibody, trastuzumab, has been used for the treatment of HER2-positive breast and gastric cancer. In this study, we examined the surface expression of HER2 on seven Thai liver-fluke-associated CCA cell lines by flow cytometry, and found all of these CCA cells were weakly positive for HER2. MTT assay revealed that trastuzumab directly suppressed the growth of CCA. By using FcR-bearing recombinant Jurkat T-cell-expressing firefly luciferase gene under the control of NFAT response elements, we defined the activities of antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). ADCC was confirmed by using expanded NK cells. ADCP was confirmed by using mouse peritoneal macrophages and human monocyte-derived macrophages as effector cells. Rabbit serum was administered to test the complement-dependent cytotoxicity (CDC) activity of trastuzumab. Finally, we evaluated the efficacy of trastuzumab in in vivo patient-derived cell xenograft and patient-derived xenograft (PDX) models. Our results showed that a distinct population of CCA (liver-fluke-associated CCA) expressed HER2. Trastuzumab demonstrated a potent inhibitory effect on even HER2 weakly positive CCA both in vitro and in vivo via multiple mechanisms. Thus, HER2 is a promising target in anti-CCA therapy, and trastuzumab can be considered a promising antibody immunotherapy agent for the treatment of CCA.

Magnetite nanoparticles: an emerging adjunctive tool for the improvement of cancer immunotherapy.

Cancer immunotherapy has emerged as a groundbreaking field, offering promising and transformative tools for oncological research and treatment. However, it faces several limitations, including variations in cancer types, dependence on the tumor microenvironments (TMEs), immune cell exhaustion, and adverse reactions. Magnetic nanoparticles, particularly magnetite nanoparticles (MNPs), with established pharmacodynamics and pharmacokinetics for clinical use, hold great promise in this context and are now being explored for therapeutic aims. Numerous preclinical studies have illustrated their efficacy in enhancing immunotherapy through various strategies, such as modulating leukocyte functions, creating favorable TMEs for cytotoxic T lymphocytes, combining with monoclonal antibodies, and stimulating the immune response via magnetic hyperthermia (MHT) treatment (Front Immunol. 2021;12:701485. doi: 10.3389/fimmu.2021.701485). However, the current clinical trials of MNPs are mostly for diagnostic aims and as a tool for generating hyperthermia for tumor ablation. With concerns about the adverse effects of MNPs in the in vivo systems, clinical translation and clinical study of MNP-boosted immunotherapy remains limited. The lack of extensive clinical investigations poses a current barrier to patient application. Urgent efforts are needed to ascertain both the efficacy of MNP-enhanced immunotherapy and its safety profile in combination therapy. This article reviews the roles, potential, and challenges of using MNPs in advancing cancer immunotherapy. The application of MNPs in boosting immunotherapy, and its perspective role in research and development is also discussed.

Involvement of interleukin-1ß in high glucose-activated proliferation of cholangiocarcinoma.

Background: Diabetes mellitus (DM) is associated with the increased risk of development and the advancement of cholangiocarcinoma (CCA). High glucose levels were previously shown for upregulating interleukin-1ß (IL-1ß) in CCA cells with unclear functions. The present study, thus, aimed to investigate molecular mechanisms linking DM to CCA progression, with IL-1ß hypothesized as a communicating cytokine. Methods: CCA cells were cultured in media with normal (5.6 mM) or high (25 mM) glucose, resembling euglycemia and hyperglycemia, respectively. Expressions of IL-1ß and IL-1 receptor (IL-1R) in CCA tissues from patients with and without DM were examined using immunohistochemistry. Functional analyses of IL-1ß were performed using siRNA and recombinant human IL-1R antagonist (rhIL-1RA), in which Western blots investigated the knockdown efficacy. BALB/c Rag-2-/- Jak3-/- (BRJ) mice were implanted with CCA xenografts to investigate hyperglycemia's effects on CCA growth and the anti-tumor effects of IL-1RA. Results: CCA tumors from patients with hyperglycemia showed significantly higher IL-1ß expression than those from non-DM patients, while IL-1ß was positively correlated with fasting blood glucose (FBG) levels. CCA cells cultured in high glucose showed increased IL-1ß expression, resulting in increased proliferation rates. Suppressing IL-1ß signaling by si-IL-1ß or rhIL-1RA significantly reduced CCA cell proliferation in vitro. Anakinra, a synthetic IL-1RA, also exerted significant anti-tumor effects in vivo and significantly reversed the effects of hyperglycemia-induced growth in CCA xenografts. Conclusions: IL-1ß plays a crucial role in CCA progression in a high-glucose environment. Targeting IL-1ß might, then, help improve therapeutic outcomes of CCA in patients with DM and hyperglycemia.

Precision immunotherapy for cholangiocarcinoma: Pioneering the use of human-derived anti-cMET single chain variable fragment in anti-cMET chimeric antigen receptor (CAR) NK cells.

Cholangiocarcinoma (CCA) presents a significant clinical challenge which is often identified in advanced stages, therby restricting the effectiveness of surgical interventions for most patients. The high incidence of cancer recurrence and resistance to chemotherapy further contribute to a bleak prognosis and low survival rates. To address this pressing need for effective therapeutic strategies, our study focuses on the development of an innovative cellular immunotherapy, specifically utilizing chimeric antigen receptor (CAR)-engineered natural killer (NK) cells designed to target the cMET receptor tyrosine kinase. In this investigation, we initiated the screening of a phage library displaying human single-chain variable fragment (ScFv) to identify novel ScFv molecules with specificity for cMET. Remarkably, ScFv11, ScFv72, and ScFv114 demonstrated exceptional binding affinity, confirmed by molecular docking analysis. These selected ScFvs, in addition to the well-established anti-cMET ScFvA, were integrated into a CAR cassette harboring CD28 transmembrane region-41BB-CD3ζ domains. The resulting anti-cMET CAR constructs were transduced into NK-92 cells, generating potent anti-cMET CAR-NK-92 cells. To assess the specificity and efficacy of these engineered cells, we employed KKU213A cells with high cMET expression and KKU055 cells with low cMET levels. Notably, co-culture of anti-cMET CAR-NK-92 cells with KKU213A cells resulted in significantly increased cell death, whereas no such effect was observed with KKU055 cells. In summary, our study identified cMET as a promising therapeutic target for CCA. The NK-92 cells, armed with the anti-cMET CAR molecule, have shown strong ability to kill cancer cells specifically, indicating their potential as a promising treatment for CCA in the future.

Establishment and characterization of TK-ALCL1: a novel NPM-ALK-positive anaplastic large-cell lymphoma cell line.

TK-ALCL1, a novel anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) cell line, was established from the primary tumor site of a 59-year-old Japanese male patient. The immune profile of TK-ALCL1 corresponds to that seen typically in primary ALCL cells, i.e., positive for ALK, CD30, EMA, and CD4, but negative for CD2, CD3, CD5, CD8a, and EBV-related antigens. The rearrangement of the T cell receptor-gamma locus shows that TK-ALCL1 is clonally derived from T-lineage lymphoid cells. FISH and RT-PCR analysis revealed that TK-ALCL1 has the nucleophosmin (NPM)-ALK fusion transcript, which is typical for ALK+ ALCL cell lines. When TK-ALCL1 was subcutaneously inoculated into 6-week-old BALB/c Rag2-/-/Jak3-/- (BRJ) mice, it formed tumor masses within 4-6 weeks. Morphological, immunohistochemical, and molecular genetic investigations confirmed that the xenograft and the original ALCL tumor were identical. The ALK inhibitors Alectinib and Lorlatinib suppressed proliferation in a dose-dependent manner. Thus, TK-ALCL1 provides a useful in vitro and in vivo model for investigation of the biology of ALK+ ALCL and of novel therapeutic approaches targeting ALK.

Role of cathepsin B in dengue virus-mediated apoptosis.

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.

Compound A, a dissociated glucocorticoid receptor modulator, reduces dengue virus-induced cytokine secretion and dengue virus production.

Dengue Virus (DENV) infection is an important mosquito-borne viral disease and its clinical symptoms range from a predominantly febrile disease, dengue fever (DF), to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased levels of cytokines - the so-called 'cytokine storm', contribute to the pathogenesis of DHF/DSS. In this study, we compared the expression of cytokine genes between mock-infected and DENV-infected HepG2 cells using a real-time PCR array and revealed several up-regulated chemokines and cytokines, including CXCL10 and TNF-α. Compound A (CpdA), a plant-derived phenyl aziridine precursor containing anti-inflammatory action and acting as a dissociated nonsteroidal glucocorticoid receptor modulator, was selected as a candidate agent to modulate secretion of DENV-induced cytokines. CpdA is not a glucocorticoid but has an anti-inflammatory effect with no metabolic side effects as steroidal ligands. CpdA significantly reduced DENV-induced CXCL10 and TNF-α secretion and decreased leukocyte migration indicating for the first time the therapeutic potential of CpdA in decreasing massive immune activation during DENV infection.

Metformin as a booster of cancer immunotherapy.

Metformin, a biguanide antidiabetic, has been studied for its repurposing effects in oncology. Although a modest effect was observed in a single-agent regimen, metformin can synergize the anti-tumor effects of other modalities. The promising combination for cancer treatment is with immunotherapy. Despite high efficacy for some cancers, immunotherapy could be limited by modulation of the tumor immune microenvironment and the immune exhaustion of cytotoxic immune cells. Combining immunotherapy with metformin, thus, exerted a rescuing effect of immunotherapy and potentiated the anti-tumor effects of each other. Although not fully understood, metformin shows promoting effects of immunotherapy by several mechanisms. Those proposed mechanisms have been partially proven and are suggested for possible therapeutic strategies for cancer treatment. In this review, a state-of-the-art of metformin's boosting effects on immunotherapy is reviewed and discussed. The future directions for metformin research in preclinical and clinical immunotherapy are also suggested.

Dihydroartemisinin Induced Apoptosis and Synergized With Chemotherapy in Pleural Effusion Lymphoma Cells.

BACKGROUND/AIM: Primary effusion lymphoma (PEL) is a rare aggressive B-cell lymphoma associated with HHV-8. With a median survival of fewer than six months, the prognosis of the disease with current standard therapies is usually dismal. Dihydroartemisinin (DHA) is a derivative of artemisinin, originally designed as an antimalarial drug. Several studies have shown that this compound also demonstrates anti-cancer activity in various types of cancer, including hematologic malignancies. MATERIALS AND METHODS: Anti-proliferation activity of DHA on 5 PEL cell lines was assessed by MTT assay. Cell cycle arrest was determined by propidium iodide staining and flow cytometry analysis. DHA-induced PEL apoptosis was shown by annexin V/PI staining and western blotting for cleaved caspases 3, 8, and 9. An inhibitory effect on PEL growth was evaluated in a PEL-xenograft mouse model. A synergistic effect of DHA and doxorubicin combination treatment was shown in vitro. RESULTS: DHA showed anti-proliferative activity on PEL and induced caspase-dependent apoptosis in a time- and dose-dependent manner. DHA-induced cell death appeared to be triggered by increased levels of reactive oxygen species (ROS). N-acetylcysteine treatment inhibited DHA-induced ROS elevation and suppressed expression of cleaved caspases leading to significantly reduced PEL apoptosis. DHA treatment also demonstrated an inhibitory effect on PEL cell growth in an in-vivo xenograft model. Moreover, we found that a combination treatment of DHA and doxorubicin, the standard chemotherapy drug for PEL, demonstrated a synergistic effect on PEL cell lines. CONCLUSION: DHA is a potentially effective candidate drug for PEL treatment.

Cepharanthine inhibits dengue virus production and cytokine secretion.

Dengue virus (DENV) infection is a public health problem in tropical and subtropical regions. It can cause a spectrum of clinical manifestations ranging from mild dengue fever (DF) to severe dengue haemorrhagic fever (DHF) and potentially life-threatening disease including dengue shock syndrome (DSS). Severe DENV infection is caused by high viral load and cytokine storm in dengue-infected patients. Currently, there is no specific antiviral drug for DENV infection. An anti-DENV agent that demonstrates inhibitory effects on both DENV replication and cytokine secretion is urgently needed. In this study, cepharanthine (CEP), which is an anti-inflammatory, anti-HIV, and anti-tumor compound isolated from Stephania cepharantha Hayata, was tested for inhibition of DENV infection. We investigated the efficacy of CEP to inhibit DENV infection, replication, and cytokine production. The inhibitory effect of CEP treatment was studied in DENV-infected human chronic myeloid leukemia (K562) cells. The levels of DENV E protein and DENV production were determined by flow cytometry and FFU assay, respectively. CEP treatment significantly reduced viral E protein and viral production in all DENV-1, 2, 3, 4 serotypes. In addition, CEP treatment reduced the IL-6 proinflammatory cytokine production in DENV-infected A549 cells. Taken together, CEP has inhibitory effects on DENV infection specifically at the initial viral replication states and proinflammatory cytokine secretion, and is a promising candidate for further development as an anti-DENV treatment.

Establishment of Nude Mice Lacking NK Cells and Their Application for Human Tumor Xenografts.

OBJECTIVE: Nude mice are used as a recipient for human tumor cell xenografts. However, the success rate of xenotransplantation is unsatisfactory due to high natural killer (NK) activity. To overcome this limitation, we established nude mice with no NK cells, and compared the transplantation efficiency with that in nude mice. METHODS: BALB/c Nude Jak3-/- (Nude-J) mice were established by crossing BALB/c Nude mice and BALB/c Jak-3-/- mice. Hematopoietic malignant cell lines (BCBL-1 and Z138) were implanted subcutaneously to compare the tumorigenicity in Nude-J mice with Nude Rag-2/Jak3 double deficient (Nude RJ) mice and nude mice. RESULTS: Nude-J mice showed complete loss of NK and T lymphocytes, whereas B lymphocytes remained. Both BCBL-1 and Z138 human lymphoid malignant cell lines formed almost the same sizes of subcutaneous tumors in Nude-J and Nude RJ mice, whereas they formed no or only small tumors in nude mice. Splenocytes from Nude-J mice showed no cytotoxic activity in vitro. CONCLUSION: Nude-J mice can be a valuable tool for human tumor cell transplantation studies.
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Establishment of bone marrow-derived M-CSF receptor-dependent self-renewing macrophages.

Recent studies have revealed that tissue macrophages are derived from yolk sac precursors or fetal liver monocytes, in addition to bone marrow monocytes. The relative contribution of these cells to the tissue macrophage pool is not fully understood, but embryo-derived cells are supposed to be more important because of their capacity to self-renew. Here, we show the presence of adult bone marrow-derived macrophages that retain self-renewing capacity. The self-renewing macrophages were readily obtained by long-term culture of mouse bone marrow cells with macrophage colony-stimulating factor (M-CSF), a key cytokine for macrophage development. They were non-tumorigenic and proliferated in the presence of M-CSF in unlimited numbers. Despite several differences from non-proliferating macrophages, they retained many features of cells of the monocytic lineage, including the differentiation into dendritic cells or osteoclasts. Among the transcription factors involved in the self-renewal of embryonic stem cells, Krüppel-like factor 2 (KLF2) was strongly upregulated upon M-CSF stimulation in the self-renewing macrophages, which was accompanied by the downregulation of MafB, a transcription factor that suppresses KLF2 expression. Indeed, knockdown of KLF2 led to cell cycle arrest and diminished cell proliferation in the self-renewing macrophages. Our new cell model would be useful to unravel differences in phenotype, function, and molecular mechanism of proliferation among self-renewing macrophages with different origins.