技术资料

PURPOSE Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors,such as prostate cancers. However,targeting MDSCs proved challenging due to their phenotypic heterogeneity. EXPERIMENTAL DESIGN Myeloid cell populations were evaluated using flow cytometry on blood samples,functional assays,and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects,localized and metastatic castration-resistant prostate cancer patients. RESULTS Here,we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3,which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA,thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1,a downstream STAT3 target gene and a potent T-cell inhibitor. CONCLUSIONS Overall,we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression. View Publication

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live,but not killed,virulent MTB. Additionally,we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live,virulent MTB,and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases,and by knockdown of crucial transcriptional regulators,we demonstrate that protein kinase-A (PKA),CREB,and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis,we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live,virulent MTB to subvert the immune system to survive and replicate in the host. View Publication

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A,POLR3B,and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report,we describe three individuals with biallelic variants in POLR3GL,a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual,we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis,oligodontia,short stature,and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A,POLR3B,POLR1C,and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected. View Publication

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication

Neural stem cells (NSCs) possess immunosuppressive characteristics,but effects of NSCs on human dendritic cells (DCs),the most important antigen presenting cells,are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14+ monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line,as well as human bone marrow derived mesenchymal stem cells (MSCs),were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14+ monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a,whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However,when effects on the function of derived DCs were investigated,NSCs suppressed the elevation of the DC maturation marker CD83,although not the up-regulation of costimulatory molecules CD80,CD86 and CD40,and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs,these stem cells can still affect the function of DCs,ultimately regulating specific immune responses. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus,it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression-based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide,we identified AR-42 (OSU-HDAC42),a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate,but with improved activity at submicromolar concentrations. Here,we report that AR-42 induces NF-κB inhibition,disrupts the ability of Hsp90 to stabilize its oncogenic clients,and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide,the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2-driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials,we expect that our results can be extended to the clinic. View Publication