技术资料

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif),which promotes degradation of A3G. We report that viral protein R (Vpr),which interacts with a uracil glycosylase,also counteracted A3G by diminishing the incorporation of uridine. However,this process resulted in activation of the DNA-damage–response pathway and the expression of natural killer (NK) cell–activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched,full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell,HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units,CD56+CD3- placenta-derived NK cells,termed pNK cells,were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were textgreater80% CD56+CD3-,comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D,NKp46,and NKp44 (p textless 0.001,p textless 0.001,and p textless 0.05,respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile,immunophenotype,and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development,pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options. View Publication

Natural killer (NK) cells have been originally defined by their naturally occurring" effector function. However� View Publication

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. View Publication

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice,the development of granulocytes is favored at the expense of monocytes. However,Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras,we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils,Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα,C/EBPβ,and PU.1,depends on Btk. In addition,expression of several granule proteins,including myeloperoxidase,neutrophilic granule protein,gelatinase and neutrophil elastase,is Btk-dependent. In the Arthus reaction,an acute inflammatory response,neutrophil migration into tissues,edema formation,and hemorrhage are significantly reduced in Btk-deficient animals. Together,our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo. View Publication

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here,we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model,HMBA did not reactivate HIV-1,yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However,HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells,hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside,HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall,our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells,providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug. View Publication

BACKGROUND The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here,we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS EXID's clinical and immunological characteristics were compared to immunological responders (IRs),immunological nonresponders (INRs),healthy controls (HCs),and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/mul,compared with CD4+ increases of 193 cells/mul and 427 cells/mul in INR and IR,respectively. EXID had reduced naive CD4+ T cells,but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR,but the IL-7 axis was profoundly perturbed compared with HC,IR,INR,and ICL. Genes involved in T cell and monocyte/macrophage function,autophagy,and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC,while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS EXID is a distinct immunological outcome compared with previously described INR. Anti-CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID. View Publication