Sex and Gender Differences in Overlap Syndrome of Functional Gastrointestinal Disorder and Effect of Genetic Polymorphisms in South Korea: A Long-term Follow-up Study.

BACKGROUND/AIMS: Overlap functional gastrointestinal disorder (FGID) is associated with more severe gastrointestinal symptoms and lower quality of life. The aim of this study is to evaluate clinical features of non-erosive reflux disease (NERD), functional dyspepsia, irritable bowel syndrome, their overlap in terms of sex and gender, and to assess the risk factors, including genetic polymorphisms. METHODS: A total of 494 FGIDs and 239 controls were prospectively enrolled between 2004 and 2020. FGIDs were diagnosed based on the Rome III criteria and symptoms were evaluated using a questionnaire. Follow-up questionnaires were conducted to determine the change of symptoms during the 75.8-month mean observation period. Risk factors including genetic polymorphisms in neurotransmitter receptor (SLC6A4 5-HTTLPR, GNB3, ADRA2A, CCKAR, and TRPV1) and cytokine (TNFA and IL10) genes. RESULTS: NERD was more prevalent in men, and functional dyspepsia in women. Overlap FGIDs (n = 239) were more prevalent than nonoverlap FGIDs (n = 255) in women (P = 0.019). Anxiety and depression scores were higher in the overlaps (P = 0.012 and P < 0.001, respectively). Symptoms were more frequent and severe in the overlap FGIDs than in the non-overlaps (P < 0.001). During followup, symptoms progressed more frequently in the overlap FGIDs, especially in patients with the L/S genotype of SLC6A4 5-HTTLPR and anxiety/depression. CONCLUSIONS: Overlap FGID patients need attention given their association with anxiety/depression and more severe symptoms, especially in women. Genetic polymorphisms also may be associated with certain symptoms of overlap FGIDs.

A phase I study of IMC-001, a PD-L1 blocker, in patients with metastatic or locally advanced solid tumors.

Introduction IMC-001 is a fully human IgG1 monoclonal antibody that binds to human PD-L1 (programmed death-ligand 1). This study evaluated the safety, pharmacokinetics, and pharmacodynamics of IMC-001 in patients with advanced solid tumors. Materials and Methods This open-labeled phase I study used a standard 3 + 3 dose-escalation design, with doses ranging from 2 to 20 mg/kg. IMC-001 was administered intravenously every 2 weeks until disease progression or unacceptable toxicity. The dose-limiting toxicity window was defined as 21 days from the first dose. Results Fifteen subjects were included in 5 dose-escalation cohorts. No dose-limiting toxicity was observed, and the maximum tolerated dose was not reached. The most common adverse events (AEs) were general weakness, decreased appetite, fever, and cough. No grade 4 or 5 treatment emergent AEs were reported during the study. One subject in the 2 mg/kg cohort showed grade 2 immune-induced thyroiditis and diabetes mellitus suspected to be related to IMC-001. Over the dose range of 2-20 mg/kg IMC-001, the AUC0-14d, AUC0-∞, and Cmax generally increased in a dose-proportional manner for each step of dose escalation. Of the 15 enrolled patients, 1 subject with rectal cancer showed a partial response, and the disease control rate was 33.3%. Conclusions IMC-001 demonstrated a favorable safety profile up to 20 mg/kg administered intravenously every 2 weeks and showed preliminary efficacy in patients with advanced solid tumors. Based on pharmacokinetic and pharmacodynamic data, 20 mg/kg was selected as the recommended phase II dose. Clinical trial identification NCT03644056 (date of registration: August 23, 2018).

Circulating TNF-like protein 1A (TL1A) is elevated early in rheumatoid arthritis and depends on TNF.

BACKGROUND: The tumor necrosis factor (TNF) superfamily cytokine TNF-like protein 1A (TL1A) and its receptor DR3 are essential for diverse animal models of autoimmune disease and may be pathogenic in rheumatoid arthritis (RA). However, the relationship of TL1A to disease duration, activity, and response to anti-TNF and other therapies in RA is not clear. METHODS: We measured soluble TL1A in synovial fluid (SF), serum, or plasma from RA first-degree relatives (FDRs) and in early RA and established disease. We measured the effects of anti-TNF and methotrexate (MTX) therapy on circulating TL1A from multiple independent RA treatment trials. We also determined the ability of a blocking anti-TL1A antibody to inhibit clinical disease and articular bone destruction in the murine collagen-induced arthritis (CIA) model of human RA. RESULTS: Soluble TL1A was specifically elevated in the blood and SF of patients with RA compared to patients with other diseases and was elevated early in disease and in at-risk anti-cyclic citrullinated peptide (CCP) (+) first-degree relatives (FDRs). Therapeutic TNF inhibition reduced serum TL1A in both responders and non-responders, whereas TL1A declined following MTX treatment only in responders. In murine CIA, TL1A blockade was clinically efficacious and reduced bone erosions. CONCLUSIONS: TL1A is specifically elevated in RA from early in the disease course and in at-risk FDRs. The decline in TL1A after TNF blockade suggests that TL1A levels may be a useful biomarker for TNF activity in RA. These results support the further investigation of the relationship between TL1A and TNF and TL1A blockade as a potential therapeutic strategy in RA.

Receptor Binding Affinities of Synthetic Cannabinoids Determined by Non-Isotopic Receptor Binding Assay.

A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor (CB1) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified CB1 were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid Δ9-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to CB1 were determined to be (from highest to lowest) 9.52 × 10-13 M (JWH-210), 6.54 × 10-12 M (JWH-250), 1.56 × 10-11 M (Δ9-tetrahydrocannabinol), 2.75 × 10-11 M (RCS-4), and 6.80 ×10-11 M (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.

A synthetic cannabinoid JWH-210 reduces lymphoid organ weights and T-cell activator levels in mice via CB2 receptors.

The problem of new psychoactive substances (NPS) is emerging globally. However, the immunotoxicity of synthetic cannabinoids is not evaluated extensively yet. The purpose of the present study was to investigate whether synthetic cannabinoids (JWH-210 and JWH-030) induce adverse effects on lymphoid organs, viability of splenocytes and thymocytes, and immune cell activator and cytokines in mice. JWH-210 (10 mg/kg, 3 days, i.p.) is more likely to have cytotoxicity and reduce lymphoid organ weight than JWH-030 of ICR mice in vivo. We also demonstrated that JWH-210 administration resulted in the decrease of expression levels of T-cell activator including Cd3e, Cd3g, Cd74p31, and Cd74p41, while JWH-030 increased Cd3g levels. In addition, JWH-210 reduced expression levels of cytokines, such as interleukin-3, interleukin-5, and interleukin-6. Furthermore, we demonstrated that a CB2 receptor antagonist, AM630 inhibited JWH-210-induced cytotoxicity, whereas a CB1 receptor antagonist, rimonabant did not in primary cultured splenocytes. These results suggest that JWH-210 has a cytotoxicity via CB2 receptor action and results in decrement of lymphoid organ weights, T-cell activator, and cytokine mRNA expression levels.

Synthetic Cannabinoid-Induced Immunosuppression Augments Cerebellar Dysfunction in Tetanus-Toxin Treated Mice.

Synthetic cannabinoids are one of most abused new psychoactive substances. The recreational use of abused drug has aroused serious concerns about the consequences of these drugs on infection. However, the effects of synthetic cannabinoid on resistance to tetanus toxin are not fully understood yet. In the present study, we aimed to determine if the administration of synthetic cannabinoids increase the susceptibility to tetanus toxin-induced motor behavioral deficit and functional changes in cerebellar neurons in mice. Furthermore, we measured T lymphocytes marker levels, such as CD8 and CD4 which against tetanus toxin. JWH-210 administration decreased expression levels of T cell activators including cluster of differentiation (CD) 3ε, CD3γ, CD74p31, and CD74p41. In addition, we demonstrated that JWH-210 induced motor impairment and decrement of vesicle-associated membrane proteins 2 levels in the cerebellum of mice treated with tetanus toxin. Furthermore, cerebellar glutamatergic neuronal homeostasis was hampered by JWH-210 administration, as evidenced by increased glutamate concentration levels in the cerebellum. These results suggest that JWH-210 may increase the vulnerability to tetanus toxin via the regulation of immune function.

Synthetic cannabinoid, JWH-030, induces QT prolongation through hERG channel inhibition.

The problem of new psychoactive substance (NPS) abuse, which includes synthetic cannabinoids, is emerging globally, and the cardiotoxicity of these synthetic cannabinoids has not yet been evaluated extensively. In the present study, we investigated the effects of synthetic cannabinoids on the cytotoxicity, human Ether-à-go-go-related gene (hERG) channel, action potential duration (APD), and QT interval. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that JWH-030 was more cytotoxic than JWH-210, JWH-250, and RCS4 in H9c2 cells at 0.1 µM. In addition, the cytotoxicity was associated with its pro-apoptotic effects as evidenced by the increase in caspase-3 levels. We demonstrated that a cannabinoid receptor type 2 (CB2) antagonist, AM630, inhibited JWH-030-induced cytotoxicity, whereas a CB1 antagonist, rimonabant, did not. Furthermore, fluorescence polarization assay showed JWH-030 to block the hERG channel (half-maximal inhibitory concentration, IC50 was 88.36 µM). JWH-030 significantly reduced the APD at 90% repolarization (APD90) in rabbit Purkinje fibers and decreased the left ventricular end diastolic pressure (LVEDP) in Langendorff-perfused Sprague-Dawley (SD) rat hearts at 30 µM. In addition, the electrocardiogram (ECG) measurement revealed that the intravenous injection of JWH-030 (0.5 mg kg-1) prolonged the QT interval in SD rats. These results suggest that JWH-030 is cytotoxic and its cytotoxicity is mediated by its action on the CB2 receptor; it prolongs the QT interval by regulating ion current channels and APD.

Rapid expansion and auto-grafting efficiency of porcine full skin expanded by a skin bioreactor ex vivo.

Full skin auto-grafts are required for reconstruction of skin burns and trauma scars. However, currently available clinical approaches such as sheet skin graft, mesh skin grafts, artificial skin graft, and in vivo skin expansion have limitations due to their potential danger for secondary damage and scar formation at the donor site, and discomfort during skin expansion. We developed an advanced bioreactor system and evaluated its function in skin expansion using porcine full skin. The reactor was designed as a pneumatic cylinder type, was programmed to adjust the pressure and the operating time. The system was composed of culture chamber unit, environmental control unit, and monitoring unit. Skins were expanded at 200 kPa pneumatic force and the expanded skins were analyzed by immunohistochemistry and histology. Furthermore we carried out auto-grafting experiment of the expanded skins in vivo using Yucatan pigs and skins were harvested and histologically analyzed after 8 weeks. The results showed that the bioreactor expanded skins to 160% in 4 hours. Histological analysis of the expanded skins revealed that epidermal cells and dermal fibroblasts were viable and remained integrity. The results of auto-grafting experiment indicated that fibrosis and scars were not detected in the grafted skins. This study demonstrates that the newly developed skin bioreactor enabled to obtain large sized full skin rapidly and successful grating.

Cardiovascular Safety Pharmacology of Sibutramine.

Sibutramine is an anorectic that has been banned since 2010 due to cardiovascular safety issues. However, counterfeit drugs or slimming products that include sibutramine are still available in the market. It has been reported that illegal sibutramine-contained pharmaceutical products induce cardiovascular crisis. However, the mechanism underlying sibutramine-induced cardiovascular adverse effect has not been fully evaluated yet. In this study, we performed cardiovascular safety pharmacology studies of sibutramine systemically using by hERG channel inhibition, action potential duration, and telemetry assays. Sibutramine inhibited hERG channel current of HEK293 cells with an IC50 of 3.92 µM in patch clamp assay and increased the heart rate and blood pressure (76 Δbpm in heart rate and 51 ΔmmHg in blood pressure) in beagle dogs at a dose of 30 mg/kg (per oral), while it shortened action potential duration (at 10 µM and 30 µM, resulted in 15% and 29% decreases in APD50, and 9% and 17% decreases in APD90, respectively) in the Purkinje fibers of rabbits and had no effects on the QTc interval in beagle dogs. These results suggest that sibutramine has a considerable adverse effect on the cardiovascular system and may contribute to accurate drug safety regulation.

USP8 modulates ubiquitination of LRIG1 for Met degradation.

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301 employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301.

A new anti-c-Met antibody selected by a mechanism-based dual-screening method: therapeutic potential in cancer.

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

New insights into T cell biology and T cell-directed therapy for autoimmunity, inflammation, and immunosuppression.

T cell-directed therapies have become mainstays in the management of various autoimmune diseases and organ transplantation. The understanding of T cell biology has expanded greatly since the development of most agents currently in use. Here we discuss important recent discoveries pertaining to T helper cell differentiation, lineage commitment, and function. Within this context, we examine existing T cell-directed therapies, including new agents being evaluated in clinical and preclinical studies. We also use recent findings to speculate on novel targets.

A dual regulatory role of apurinic/apyrimidinic endonuclease 1/redox factor-1 in HMGB1-induced inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/Redox factor-1 (APE1) is a multifunctional protein involved in reduction-oxidation regulation. High-mobility group box 1 (HMGB1) is released by necrotic cells and various inflammatory stimuli, acting as an inflammatory marker in sepsis and autoimmune diseases. Here, we report the dual regulatory role of APE1 in inflammatory signaling to extracellular HMGB1 or in the release of endogenous HMGB1 in human monocytes/macrophages. Forced cytoplasmic overexpression of APE1 profoundly attenuated the upregulation of HMGB1-mediated reactive oxygen species generation, cytokine secretion, and cyclooxygenase-2 expression by primary monocytes and macrophage-like THP-1 cell lines. In addition, HMGB1-induced activation of p38 and c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2, was strongly abrogated by the overexpression of APE1. The activation of apoptosis signal-regulating kinase 1 was required for both the p38 and JNK activation challenge with HMGB1. The extracellular release of HMGB1 by activated macrophages was inhibited by APE1 transfection. Small interfering RNA (siRNA) knockdown of endogenous APE1 impaired HMGB1-mediated cytokine expression and MAPK activation in THP-1 cells. HMGB1 stimulation induced the translocation of APE1 to the nucleus of the cell. In addition, APE1 silencing via siRNA transfection inhibited both the nuclear and cytoplasmic expression of APE1. These data identify APE1 as a novel dual regulator of inflammatory signaling to HMGB1 by human monocytes/macrophages. The modulation of cytosolic APE1 expression might be useful as a potential therapeutic modality for the treatment of inflammatory or autoimmune diseases.

Alteration of p66shc is associated with endothelial dysfunction in the abdominal aortic coarctation of rats.

To examine the role of p66shc in endothelial dysfunction, we investigated the endothelium-dependent relaxation, protein expression and superoxide production in abdominal aortic coarctation rats. Endothelium-dependent relaxation to acetylcholine was impaired only in the aortic segments above the aortic coarctation (35.0+/-7.1% vs. 86.6+/-6.0% for sham control at 1 microM Ach). The aortic segments exposed to increased blood pressure showed a decreased phosphorylation of endothelial nitric oxide synthase, an increased phosphorylation of p66shc, and an increased superoxide production. Angiotensin II elicited a significantly increased phosphorylation of p66shc in the endothelial cells. Taken together, these findings suggest that the increased phosphorylation of p66shc is one of the important mediators in the impaired endothelium-dependent relaxation of aortic coarctation rats.

HLA alleles determine differences in human natural killer cell responsiveness and potency.

Epidemiological studies have associated certain human disease outcomes with particular killer cell Ig-like receptor (KIR) and HLA genotypes. However, the functional explanation for these associations is poorly understood, because the KIRs were initially described as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. Yet resolution of infections is often associated with genotypic pairing of inhibitory KIRs with their cognate HLA ligands. Recent studies in mice indicate a second role for MHC-specific inhibitory receptors, i.e., self-MHC recognition confers functional competence on the NK cell to be triggered through their activation receptors, a process termed licensing. As a result, licensed NK cells with self-MHC-specific receptors are more readily activated as compared with unlicensed NK cells without self-MHC-specific receptors. Such results predict that human NK cells may undergo a similar process. Here, we examined the human NK cell subset expressing KIR3DL1, the only known KIR specific for HLA-Bw4 alleles. The KIR3DL1(+) subset in normal donors with two HLA-B-Bw4 genes displayed increased responsiveness to tumor stimulation compared with the KIR3DL1(+) subset from individuals with only one or no Bw4 genes. By contrast, NK cells lacking KIR3DL1 showed no differences. Therefore, these data indicate that particular KIR and HLA alleles are associated with more responsive NK cells, strongly suggesting that human NK cells are also subjected to NK cell licensing, and providing a potential functional explanation for the influence of KIR and HLA genes in disease as well as interindividual differences in NK cell potency.

Tat-APE1/ref-1 protein inhibits TNF-alpha-induced endothelial cell activation.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-alpha-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-alpha-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.

Alteration of APE1/ref-1 expression in non-small cell lung cancer: the implications of impaired extracellular superoxide dismutase and catalase antioxidant systems.

PURPOSE: Apurinic/apyrimidinic endonuclease1/ref-1(APE1/ref-1) is a key enzyme in the base excision repair and in transcriptional modulation against oxidative stress. We investigated the altered expression of APE1/ref-1 and antioxidant systems in lung cancer. PATIENTS AND METHODS: Tumor specimens from 48 patients with operable non-small cell lung cancer were obtained from 2004 to 2006. Immunohistochemistry, Western blot, lipid peroxidation and superoxide production were performed on the tumor samples and a cultured H460 cell line. RESULTS: APE1/ref-1 was mainly localized to the nucleus in the non-tumor regions of the lung cancer tissue specimens. However, nuclear and cytoplasmic expressions of APE1/ref-1 in the lung cancers were markedly up-regulated in the non-small cell lung cancer (NSCLC) specimens including squamous cell and adenocarcinoma specimens. Extracellular superoxide dismutase (ECSOD) and catalase were down-regulated and manganese superoxide dismutase (MnSOD) was up-regulated in the tumor regions of the NSCLC. Tumor regions of the NSCLC showed higher superoxide production and lipid peroxidation levels than non-tumor regions. In the lung adenocarcinoma cell line, H460, treatment with hydrogen peroxide in the presence of a catalase inhibitor, aminotriazole, increased APE1/ref-1 expression, suggesting oxidative stress might have contributed to the induction of APE1/ref-1. CONCLUSION: The results of this study suggest that APE1/ref-1 is up-regulated in the tumor regions of NSCLC. Altered expression of antioxidant systems lead to enhanced production of superoxide production and lipid peroxidation, which can induce APE1/ref-1 in the tumor regions of NSCLS.

Chronic lymphocytosis of functionally immature natural killer cells.

BACKGROUND: The development of natural killer (NK) cells in the bone marrow is not well characterized. We recently described a mouse (referred to as an NK cell-deficient [NKD] mouse) with a selective deficiency in NK cells caused by the insertion of a transgene construct into the genetic locus for the basic leucine zipper transcription factor ATF-2. NK cells in this mouse were both phenotypically and functionally immature and accumulated in the bone marrow at a stage at which constitutive NK cell proliferation occurs in wild-type mice. OBJECTIVE: We hypothesized that excess IL-15 could potentially overcome this developmental block, allowing normal emigration of mature NK cells from the bone marrow to the periphery. METHODS: Double-transgenic mice were generated by crossing the NKD mice with transgenic mice overexpressing IL-15. RESULTS: The double-transgenic mice had a dramatic accumulation of phenotypically immature NK cells in the bone marrow and subsequently in the blood, liver, and spleen. NK cells from these double-transgenic mice manifested functional deficits similar to those observed in NK cells from NKD mice, as assessed by decreased cytokine production and cytotoxicity. CONCLUSION: Rather than bypass the observed developmental defect in NKD mice, excess IL-15 drove a massive accumulation of phenotypically and functionally immature NK cells in the bone marrow and periphery. CLINICAL IMPLICATIONS: We propose that these double-transgenic mice will serve as a murine model of chronic NK cell lymphocytosis in human patients.

Arrested natural killer cell development associated with transgene insertion into the Atf2 locus.

Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1-specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.

Licensing of natural killer cells by host major histocompatibility complex class I molecules.

Self versus non-self discrimination is a central theme in biology from plants to vertebrates, and is particularly relevant for lymphocytes that express receptors capable of recognizing self-tissues and foreign invaders. Comprising the third largest lymphocyte population, natural killer (NK) cells recognize and kill cellular targets and produce pro-inflammatory cytokines. These potentially self-destructive effector functions can be controlled by inhibitory receptors for the polymorphic major histocompatibility complex (MHC) class I molecules that are ubiquitously expressed on target cells. However, inhibitory receptors are not uniformly expressed on NK cells, and are germline-encoded by a set of polymorphic genes that segregate independently from MHC genes. Therefore, how NK-cell self-tolerance arises in vivo is poorly understood. Here we demonstrate that NK cells acquire functional competence through 'licensing' by self-MHC molecules. Licensing involves a positive role for MHC-specific inhibitory receptors and requires the cytoplasmic inhibitory motif originally identified in effector responses. This process results in two types of self-tolerant NK cells--licensed or unlicensed--and may provide new insights for exploiting NK cells in immunotherapy. This self-tolerance mechanism may be more broadly applicable within the vertebrate immune system because related germline-encoded inhibitory receptors are widely expressed on other immune cells.