Replication Stress in Activated Human NK Cells Induces Sensitivity to Apoptosis.

NK cells are innate immune effectors that kill virally infected or malignant cells. NK cell deficiency (NKD) occurs when NK cell development or function is impaired and variants in MCM4, GINS1, MCM10, and GINS4 result in NKD. Although NK cells are strongly impacted by mutational deficiencies in helicase proteins, the mechanisms underlying this specific susceptibility are poorly understood. In this study, we induced replication stress in activated NK cells or T cells by chemical and genetic methods. We found that the CD56bright subset of NK cells accumulates more DNA damage and replication stress during activation than do CD56dim NK cells or T cells. Aphidicolin treatment increases apoptosis of CD56bright NK cells through increased pan-caspase expression and decreases perforin expression in surviving cells. These findings show that sensitivity to replication stress affects NK cell survival and function and contributes to NKD.

A subset of virus-specific CD161+ T cells selectively express the multidrug transporter MDR1 and are resistant to chemotherapy in AML.

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.

NK92-CD16 cells are cytotoxic to non-small cell lung cancer cell lines that have acquired resistance to tyrosine kinase inhibitors.

BACKGROUND: Treatment with tyrosine kinase inhibitors (TKIs) has improved the outcomes for patients with non-small cell lung cancer (NSCLC) harboring targetable driver mutations. However, acquired resistance to TKIs invariably develops within approximately 1 year of treatment by various mechanisms, including gatekeeper mutations, alternative pathway activation and histological transformations. Because immunotherapy is an option for patients with drug-resistant cancers, we generated several TKI-resistant NSCLC cell lines in vitro, and then evaluated the cytotoxicity of NK92-CD16 cells to these resistant cells. MATERIALS AND METHODS: TKI-resistant NSCLC cells (H3122CR1, H3122LR1, H3122CR1LR1, PC-9GR, PC-9ER, EBC-CR1 and EBC-CR2) were established from NCI-H3122 (EML4-ALK fusion), PC-9 (EGFR exon19 deletion) and EBC-1 (MET amplification) after continuous exposure to crizotinib, ceritinib, gefitinib, erlotinib and capmatinib. Expression of ligands for natural killer (NK) cell receptors and total EGFR were analyzed using flow cytometry. NK cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) using anti-EGFR monoclonal antibody (mAb) cetuximab were measured using NK92-CD16 as effectors and detected using the 51Chromium-release assay. RESULTS: We found that NK92-CD16 cells preferentially killed TKI-resistant NSCLC cells when compared with their parental NSCLC cells. Mechanistically, intracellular adhesion molecule 1 (ICAM-1) was up-regulated in the TKI-resistant NSCLC cells and patients' tumors, and the ICAM-1 up-regulated cancer cells lines were less susceptible to NK cytotoxicity by blocking ICAM-1. Moreover, NK92-CD16 cell-induced cytotoxicity toward TKI-resistant NSCLC cells was enhanced in the presence of cetuximab, an EGFR-targeting mAb. CONCLUSION: These data suggest that combinational treatment with NK cell-based immunotherapy and cetuximab may be promising for patients with TKI-resistant NSCLC.

CD21-independent Epstein-Barr virus entry into NK cells.

Extranodal natural killer (NK)/T-cell lymphoma is an aggressive malignant disease that is associated with Epstein-Barr viral (EBV) infection. To date, the mechanism of viral entry into NK cells remains uncertain. Here, we investigated this mechanism using human NK cells in vitro. CD21 mRNA expression, an EBV-entry receptor, was transiently detected in NK cells after exosome treatment, and levels decreased after further culture. CD21 protein expression was also transiently transferred to NK cells after co-culture with an EBV-positive Burkitt lymphoma cell line (Raji) via trogocytosis. However, EBV did not infect NK cells through CD21-mediated trogocytosis. Unexpectedly, when NK cell leukemia cells, as well as primary NK cells, were treated with viral supernatant, EBV genes, but not RNA, were detected in the NK cells, at latency stage 0. Therefore, these results suggest that EBV-NK cell infection results from the direct transfer of viral episomes, independent of EBV-positive B cells.

Novel JAK3-Activating Mutations in Extranodal NK/T-Cell Lymphoma, Nasal Type.

Inhibition of the Janus kinase (JAK)-STAT pathway has been implicated as a treatment option for extranodal natural killer/T-cell lymphoma, nasal type (NTCL). However, JAK-STAT pathway alterations in NTCL are variable, and the efficacy of JAK-STAT pathway inhibition has been poorly evaluated. JAK3 mutation and STAT3 genetic alterations were investigated by direct sequencing and immunohistochemistry in 84 patients with newly diagnosed NTCL. Five of 71 patients with NTCL (7.0%) had JAK3 mutations in the pseudokinase domain: two JAK3A573V, two JAK3H583Y, and one JAK3G589D mutation. Proliferation of Ba/F3 cells transduced with novel JAK3 mutations (JAK3H583Y and JAK3G589D) was independent of IL-3 and was inhibited by the JAK3 inhibitor tofacitinib (means ± SD drug concentration causing a 50% inhibition of the desired activity, 85 ± 10 nmol/L and 54 ± 9 nmol/L). Ribbon diagrams revealed that these JAK3 pseudokinase domain mutations were located at the pseudokinase-kinase domain interface. Although phosphorylated STAT3 was overexpressed in 35 of 68 patients with NTCL (51.4%), a STAT3 mutation (p.Tyr640Phe; STAT3Y640F) at the SRC homology 2 domain was detected in 1 of the 63 patients (1.5%). A STAT3 inhibitor was active against STAT3-mutant SNK-6 and YT cells. Novel JAK3 mutations are oncogenic and druggable in NTCL. The JAK3 or STAT3 signal was altered in NTCL, and pathway inhibition might be a therapeutic option for patients with JAK3- or STAT3-mutant NTCL.

Human group3 innate lymphoid cells express DR3 and respond to TL1A with enhanced IL-22 production and IL-2-dependent proliferation.

Death receptor 3 (DR3, TNFRSF25) is expressed by activated lymphocytes and signaling by its ligand, TL1A, enhances cytokine expression and proliferation. Recent studies show that DR3 is also present on murine type 2 innate lymphoid cells (ILC2s). Here, we show that DR3 is expressed by IL-22-producing human group 3 innate lymphoid cells (ILC3s). Stimulation of ILC3s with exogenous TL1A alone had no impact on cytokine production or proliferation. Addition of TL1A to IL-1ß + IL-23 significantly enhanced the amount IL-22 produced by ILC3s as well as the percentage IL-22- and IL-8-producing cells. Addition of TL1A to IL-1ß + IL-23 also augmented ILC3 proliferation. Mechanistically, this occurred through the upregulation of CD25 and responsiveness to IL-2 stimulation. The combination of TL1A, IL-1ß+ IL-23, and IL-2 expanded ILC3s while IL-1ß+ IL-23 did not increase proliferation above controls. After 2 weeks of expansion, ILC3s maintained their phenotype, transcription factor expression, and function (IL-22 production). These findings identify DR3 as a costimulatory molecule on ILC3s that could be exploited for ex vivo expansion and clinical use.

The effects of 1,25-dihydroxyvitamin D3 on in vitro human NK cell development from hematopoietic stem cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is the biologically active form of vitamin D and is immunoregulatory. 1,25(OH)2D3 binds the vitamin D receptor complex present in many immune populations and can illicit transcriptional responses that vary among different immune subsets. The effects of 1,25(OH)2D3 on mature and developing human NK cells are not well characterized. In the present study, we examined the influence of 1,25(OH)2D3 using an established NK cell differentiation system. Briefly, umbilical cord blood CD34(+) cells were isolated and cultured in conditions optimal for NK cell differentiation, and varying concentrations of 1,25(OH)2D3 were administered. At physiological concentrations (10 nM), 1,25(OH)2D3 impaired NK cell development. Moreover, the NK cells that did develop under the influence of 1,25(OH)2D3 showed a significant reduction in function (cytotoxicity and cytokine production). Conversely, 1,25(OH)2D3 strongly induced hematopoietic stem cells to differentiate along a myeloid pathway, giving rise to CD14(+) cells. Mechanistically, 1,25(OH)2D3 drives hematopoietic progenitor cells to rapidly upregulate monocyte genes (i.e., C/EBP-α and CD14). There were no effects of 1,25(OH)2D3 on mature NK cytotoxicity or cytokine production. Collectively, these studies provide novel data showing the negative regulatory effect of 1,25(OH)2D3 on NK cell development.

Lineage relationships of human interleukin-22-producing CD56+ RORγt+ innate lymphoid cells and conventional natural killer cells.

Human interleukin (IL)-22-producing RORγt(+) innate lymphoid cells (ILC22) and conventional natural killer (cNK) cells are present in secondary lymphoid tissues. Both have an immunophenotype corresponding to stage III NK progenitors (CD56(+/-)CD117(high)CD94(-)). Using an in vitro differentiation and primary human tissues, we investigated their developmental relationships. cNK cells showed a CD56(+)CD117(+)CD7(+/-)LFA-1(high) phenotype and expressed surface receptors, cytokines, and transcription factors found on mature cNK cells. In contrast, ILC22 cells were contained within the CD56(+)CD117(high)CD94(-)CD7(-)LFA-1(-) fraction and produced IL-22, IL-8, and granulocyte macrophage colony stimulating factor. Although ILC22 cells expressed NKp44 and CD161, they lacked most other NK receptors and NK-associated transcription factors (T-bet and Eomes) and were incapable of interferon-γ production or cytotoxic responses. Most purified CD56(+)CD117(+)CD7(+/-)LFA-1(-) remained as ILC22 cells and never became cNK cells. In the absence of IL-15, CD34(+) cells showed a complete block in cNK differentiation and instead gave rise to a CD56(+) population of ILC22 cells. Conversely, in the absence of IL-7 and stem cell factor, cNK cells were generated but ILC22 cells showed minimal differentiation. Although human ILC22 cells and cNK progenitors have a phenotype that overlaps with stage III NK progenitors, they have unique cytokine requirements and can be distinguished by LFA-1 expression.

Development of IL-22-producing NK lineage cells from umbilical cord blood hematopoietic stem cells in the absence of secondary lymphoid tissue.

Human secondary lymphoid tissues (SLTs) contain interleukin-22 (IL-22)-producing cells with an immature NK phenotype. Given their location, these cells are difficult to study. We have generated large numbers of NK22 cells from hematopoietic stem cells. HSC-derived NK22 cells show a CD56(+)CD117(high)CD94(-) phenotype, consistent with stage III NK progenitors. Like freshly isolated SLT stage III cells, HSC-derived NK22 cells express NKp44, CD161, CCR6, IL1 receptor, AHR, and ROR-γτ. IL-1ß and IL-23 stimulation results in significant IL-22 but not interferon-γ production. Supernatant from these cells increases CD54 expression on mesenchymal stem cells. Thus, IL-22-producing NK cells can be generated in the absence of SLT. HSC-derived NK22 cells will be valuable in understanding this rare NK subset and create the opportunity for human translational clinical trials.

Ex vivo activation and expansion of natural killer cells from patients with advanced cancer with feeder cells from healthy volunteers.

BACKGROUND AIMS: Culturing natural killer (NK) cells from patients with advanced cancer is difficult and has restricted the generation of sufficient cell numbers for autologous adoptive NK-cell therapy. The aim of this study was to establish a novel method for ex vivo NK-cell expansion from patients with cancer. METHODS: NK cells (CD3(-)CD56(+)) were isolated from peripheral blood mononuclear cells from healthy volunteers and cancer patients, and NK(-) fractions were used as feeder cells. Purified NK cells were co-cultured with feeder cells in AIM-V medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% human serum and 1000 units/mL human interleukin-2. RESULTS: NK cells co-cultured with feeder cells from healthy volunteers (feeder-HV) expanded more than NK cells co-cultured with feeder cells from cancer patients (feeder-CP). During the 14-day culture period, NK cells from patients with advanced cancer co-cultivated with feeder-HV expanded on average 300-fold. NK cells co-cultivated with feeder-CP expanded on average 169.4-fold. Cultures grown in the presence of feeder-HV contained 93.8 ± 7.0% (mean ± standard deviation; n = 6) CD3(-)CD56(+) NK cells, and cultures grown in the presence of feeder-CP contained 83.6 ± 15.9% CD3(-)CD56(+) NK cells. Feeder-HV caused a relative increase in CD3(+)CD4(+) T cells, whereas feeder-CP did not induce changes. Interleukin-15, a cytokine that induces NK-cell proliferation, was detected in the culture supernatants of feeder-HV but not in those of feeder-CP. CONCLUSIONS: Feeder cells obtained from healthy volunteers have the potential to expand and activate NK cells from patients with advanced cancer. The novel NK-cell expansion method described here provides a technique for acquiring the large numbers of highly active NK cells from patients with cancer for autologous adoptive immunotherapy.

iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment.

Cell proliferation is a ubiquitous process required for organismal development and homeostasis. However, individuals with partial loss-of-function variants in DNA replicative helicase components often present with immunodeficiency due to specific loss of natural killer (NK) cells. Such lineage-specific disease phenotypes raise questions on how the proliferation is regulated in cell type-specific manner. We aimed to understand NK cell-specific proliferative dynamics and vulnerability to impaired helicase function using iPSCs from individuals with NK cell deficiency (NKD) due to hereditary compound heterozygous GINS4 variants. We observed and characterized heterogeneous cell populations that arise during the iPSC differentiation along with NK cells. While overall cell proliferation decreased with differentiation, early NK cell precursors showed a short burst of cell proliferation. GINS4 deficiency induced replication stress in these early NK cell precursors, which are poised for apoptosis, and ultimately recapitulate the NKD phenotype.

Influence of chemotherapy on nitric oxide synthase, indole-amine-2,3-dioxygenase and CD124 expression in granulocytes and monocytes of non-small cell lung cancer.

There is no specific marker to evaluate the immuno-suppressive status of cancer patients. Several markers, such as CD124, latency-associated peptide (LAP), arginase I, indole-amine-2,3-dioxygenase (IDO) and inducible nitric oxide synthase (iNOS), are known to be associated with immune suppression. However, there is little research regarding the change in these parameters after chemotherapy. The present study enrolled 23 chemo-naïve non-small cell lung cancer (NSCLC) patients and 19 healthy donors. From the 23 NSCLC patients, 11 post-chemotherapy samples were collected. Surface and functional markers were analyzed by flow-cytometry. The mean fluorescence intensities (MFI) of iNOS were higher and the MFI of LAP were lower in NSCLC patient than in healthy donors (P < 0.05). In a comparison of pre-chemotherapy and post-chemotherapy groups with NSCLC, the MFI of iNOS on granulocytes and monocytes and IDO on monocytes were significantly lower in the post-chemotherapy group than in the pre-chemotherapy group (P < 0.05). In a serial analysis with 10 patients who had paired samples and who showed clinical benefits from chemotherapy, the MFI of iNOS for both cell types, and of IDO and CD124 for monocytes decreased significantly after chemotherapy, compared with those before chemotherapy (iNOS, 4.79 ± 1.75 vs 2.83 ± 0.77, P = 0.005, for granulocytes and 6.15 ± 2.94 vs 2.76 ± 1.05, P = 0.005 for monocytes; IDO, 6.81 ± 3.43 vs 4.64 ± 1.55, P = 0.012 for monocytes; CD124, 2.31 ± 0.39 vs 1.94 ± 0.43, P = 0.008 for monocytes). The changes in arginase I and LAP expression were not significant. The changes in iNOS, IDO and CD124 expression were significant after chemotherapy in NSCLC. Further evaluation of the possibility of immune status monitoring using these parameters is needed.

Natural killer cell differentiation from hematopoietic stem cells: a comparative analysis of heparin- and stromal cell-supported methods.

Natural killer (NK) cells differentiated from hematopoietic stem cells (HSCs) may have significant clinical benefits over NK cells from adult donors, including the ability to choose alloreactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study the differentiation of NK cells from HSCs. Stroma and cytokines support NK cell differentiation, but may face considerable regulatory hurdles. A recently reported clinical-grade, heparin-based method could serve as an alternative. How the stromal-based and heparin-based approaches compare in terms of NK cell generating efficiency or function is unknown. We show that compared with heparin-based cultures, stroma significantly increases the yield of HSC-derived NK cells by differentiating less-committed progenitors into the NK lineage. NK cells generated by both approaches were similar for most NK-activating and -inhibiting receptors. Although both approaches resulted in a phenotype consistent with CD56(bright) stage IV NK cells, heparin-based cultures favored the development of CD56(+)CD16(+) cells, whereas stroma produced more NK cell immunoglobulin-like receptor-expressing NK cells, both of which are markers of terminal maturation. At day 21, stromal-based cultures demonstrated significantly more IL-22 production, and both methods yielded similar amounts of IFN-γ production and cytotoxicity by day 35. These findings suggest that heparin-based cultures are an effective replacement for stroma and may facilitate clinical trials testing HSC-derived NK cells.

CD15+/CD16low human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function.

Myeloid-derived suppressor cells (MDSCs) are a subpopulation of myeloid cells with immunosuppressive function whose numbers are increased in conditions such as chronic infection, trauma, and cancer. Unlike murine MDSCs defined as CD11b(+)/Gr-1(+), there are no specific markers for human MDSCs. The goal of this study was to delineate a specific human MDSCs subpopulation in granulocytes from terminal cancer patients and investigate its clinical implications. Here, we show that the CD15(+)/CD16(low) subset was increased in terminal cancer patients compared with healthy donors (P = 0.009). Phorbol 12-myristate 13-acetate-activated granulocytes (CD16(low)/CD66b(++)/CD15(+)) that have a phenotype similar to MDSCs from cancer patients, effectively suppressed both proliferation and cytotoxicity of normal T cells. Among cancer patients, T-cell proliferation was highly suppressed by granulocytes isolated from terminal cancer patients with a high proportion of CD15(+)/CD16(low) cells. Patients with low peripheral blood levels of CD15(+)/CD16(low) cells had significantly longer survival than those with high levels (P = 0.0011). Patients with higher levels of CD15(+)/CD16(low) also tended to have poor performance status (P = 0.05). These data suggest that CD15(+)/CD16(low) granulocytes found in terminal cancer patients may play a role in the progression of cancer by inhibiting tumor immunity.

Heterogeneous genetic landscape of congenital neutropenia in Korean patients revealed by whole exome sequencing: genetic, phenotypic and histologic correlations.

Congenital neutropenia (CN) is a hematological disease heterogeneous in its genetic, phenotypic and histologic aspects. We aimed to identify the genetic etiology of Korean CN patients in the context of bone marrow (BM) histology and clinical phenotype. Whole-exome sequencing (WES) or targeted sequencing was performed on the BM or peripheral blood specimens of 16 patients diagnosed with CN based on BM exam from 2009 to 2018. Absolute count of myeloperoxidase (MPO)-positive cells was calculated using ImageJ software. Semi-quantitation of MPO-positive cells in BM sections was performed by MPO grading (grades 0-3). Comprehensive retrospective review on real-world data of 345 pediatric patients with neutropenia including 16 patients in this study during the same period was performed. Seven disease-causing variants were identified in ELANE, G6PC3 and CXCR4 in 7 patients. A novel homozygous G6PC3 variant (K72fs) of which the mechanism was copy-neutral loss of heterozygosity was detected in two brothers. A low myeloid-to-erythroid ratio (0.5-1.5) was consistently observed in patients with ELANE mutations, while MPO-positive cells (40%-50%) with MPO grade 1 or 2 were detected in myelokathexis caused by G6PC3 and CXCR4 mutations. Meanwhile, disease-causing variants were detected in ELANE, TAZ and SLC37A4 in 5 patients by retrospective review of medical records. Our results suggest that following the immunological study and BM exam, WES or an expanded next generation sequencing panel that covers genes related to immunodeficiency and other inherited bone marrow failures as well as CN is recommended for neutropenia patient diagnosis.

Natural Killer Cell Expansion and Cytotoxicity Differ Depending on the Culture Medium Used.

Background: Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality. Methods: UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. Results: The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity. Conclusions: Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.

Presence of membrane-bound TGF-beta1 and its regulation by IL-2-activated immune cell-derived IFN-gamma in head and neck squamous cell carcinoma cell lines.

The presence of membrane-bound TGF-beta1 (mTGF-beta1) has been recently observed in regulatory T cells, but only a few studies have reported the same phenomenon in cancer cells. In this study, we investigate the regulation of mTGF-beta1 expression in five head and neck squamous cell carcinoma cell lines using FACS analysis. Through blocking Ab and exogenous cytokine treatment experiments, we found that expression of mTGF-beta1 is significantly induced by the activated immune cell-derived factor IFN-gamma. In addition, IFN-gamma and TNF-alpha are shown to have a synergistic effect on mTGF-beta1 expression. Moreover, we found that exogenous TNF-alpha induces endogenous TNF-alpha mRNA expression in an autocrine loop. In contrast to previous reports, we confirm that, in this model, mTGF-beta1 is neither a rebound form of once-secreted TGF-beta1 nor an activated form of its precursor membrane latency-associated peptide. Inhibitors of transcription (actinomycin D), translation (cycloheximide), or membrane translocation (brefeldin A) effectively block the induction of mTGF-beta1, which suggests that induction of mTGF-beta1 by IFN-gamma and/or TNF-alpha occurs through de novo synthesis. These findings suggest that some cancer cells can detect immune activating cytokines, such as IFN-gamma and TNF-alpha, and actively block antitumor immunity by induction of mTGF-beta1.

A possible mechanism of impaired NK cytotoxicity in cancer patients: down-regulation of DAP10 by TGF-beta1.

AIMS AND BACKGROUND: Elevated TGF-BETA1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. However, the molecular mechanism of immunosuppression by TGF-BETA1 is not yet clarified. METHODS: IL-2-activated human NK cells were cultured with TGF-BETA1. Protein levels of NKG2D and DAP10 were examined by FACS or immunoblot analyses. Real-time RTPCR was performed to quantify the transcription levels. MAPK inhibitors were used to investigate intracellular signaling. RESULTS: TGF-BETA1 down-regulated total and surface NKG2D, which was partially dependent on transcriptional regulation. TGF-BETA1 treatment of human NK cells resulted in significant changes in both transcriptional and translational levels of DAP10. Moreover, treatment with bafilomycin A1 or folimycin restored total NKG2D levels in TGF-BETA1-treated NK cells. The impaired NKG2D down-modulation by TGF-BETA1 was not associated with activation of the MAPK signaling pathway. CONCLUSIONS: TGF-BETA1 down-modulates surface NKG2D expression by controlling the transcriptional and translational levels of DAP10.

Prognostic value of integrated cytogenetic, somatic variation, and copy number variation analyses in Korean patients with newly diagnosed multiple myeloma.

BACKGROUND: To investigate the prognostic value of gene variants and copy number variations (CNVs) in patients with newly diagnosed multiple myeloma (NDMM), an integrative genomic analysis was performed. METHODS: Sixty-seven patients with NDMM exhibiting more than 60% plasma cells in the bone marrow aspirate were enrolled in the study. Whole-exome sequencing was conducted on bone marrow nucleated cells. Mutation and CNV analyses were performed using the CNVkit and Nexus Copy Number software. In addition, karyotype and fluorescent in situ hybridization were utilized for the integrated analysis. RESULTS: Eighty-three driver gene mutations were detected in 63 patients with NDMM. The median number of mutations per patient was 2.0 (95% confidence interval [CI] = 2.0-3.0, range = 0-8). MAML2 and BHLHE41 mutations were associated with decreased survival. CNVs were detected in 56 patients (72.7%; 56/67). The median number of CNVs per patient was 6.0 (95% CI = 5.7-7.0; range = 0-16). Among the CNVs, 1q gain, 6p gain, 6q loss, 8p loss, and 13q loss were associated with decreased survival. Additionally, 1q gain and 6p gain were independent adverse prognostic factors. Increased numbers of CNVs and driver gene mutations were associated with poor clinical outcomes. Cluster analysis revealed that patients with the highest number of driver mutations along with 1q gain, 6p gain, and 13q loss exhibited the poorest prognosis. CONCLUSIONS: In addition to the known prognostic factors, the integrated analysis of genetic variations and CNVs could contribute to prognostic stratification of patients with NDMM.