技术资料

Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore,bone marrow samples from 100 children with acute lymphoblastic leukemia (ALL) were cultured in the cytokines TNF-alpha,GM-CSF,c-kit ligand,and fetal liver tyrosine kinase 3 ligand,with or without IL-3 and -4 and after administration of HdI valproic acid (VAL),suberoylanilide hydroxamic acid (SAHA),isobutyramid,or trichostatin A. Among the tested samples,25 were positive for the chromosomal translocation t(12;21),encoding the fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 (TEL/AML1). SAHA increased CD83 expression of TEL/AML1-positive blasts in conditions without ILs,and SAHA and VAL increased the number of CD86(+)80(-) cells in the presence of ILs. VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive,leukemic DC; VAL and SAHA reduced those of TEL/AML1-negative DC. Cytotoxic T cells sensitized with leukemic DC produced more IFN-gamma and TNF-alpha upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of nondifferentiated blasts,which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. Therefore,the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended. View Publication

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases,their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients,but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study,we further showed elevated Th17 cell populations in the TILs obtained from melanoma and breast and colon cancers,suggesting that development of tumor-infiltrating CD4(+) Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition to their recruitment,we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and nucleotide oligomerization binding domain 2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs,demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments,and provide new insights relevant for the development of novel cancer immunotherapeutic approaches. View Publication

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study,we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First,CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes,including macrophages,CD4+ T cells,and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40,systemic cryptococcal dissemination was increased,and mice died of central nervous system infection. Second,CD40 promoted pathological changes in the airways,including intraluminal mucus production and subepithelial collagen deposition,but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively,these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some,but not all,elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung. View Publication

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication,their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly,both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis,antibody-dependent cellular cytotoxicity,and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS. View Publication

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process,cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes,driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire,it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ,and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly,postrevision cells do not proliferate in response to the tolerizing superantigen,implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host. View Publication

Lipid rafts accumulate in the immunological synapse formed by an organized assembly of the TCR/CD3,LFA-1,and signaling molecules. However,the precise role of lipid rafts in the formation of the immunological synapse is unclear. In this study,we show that LFA-1 on CTL is constitutively active and mediates Ag-independent binding of CTL to target cells expressing its ligands. LFA-1 and CD3 on CTL,but not resting T cells,colocalize in lipid rafts. Binding of LFA-1 on CTL to targets initiates the formation of the immunological synapse,which is formed by LFA-1,CD3,and ganglioside GM1 distributed in the periphery of the cell contact site and cholesterol is more widely distributed. The formation of this synapse is Ag independent,but the recognition of Ag by the TCR induces accumulation of tyrosine phosphorylated proteins in the synapse as well as redistribution of the microtubule organization center toward the cell contact site. Our results suggest that LFA-1 recruits lipid rafts and the TCR/CD3 to the synapse,and facilitates efficient and rapid activation of CTL. View Publication

In a murine model of autoimmunity targeted against the epidermal cell Ags,Skn,adoptive transfer of Skn-immune T cells to immunosuppressed recipients elicits skin lesions in areas of mild epidermal trauma. In this study,we examined peripheral regulation of Skn-induced autoreactivity disrupted by rendering the mice immunoincompetent. We found that regulation of Skn-directed autoimmunity was restored by cotransfer of normal syngeneic spleen cells at twice the concentration of Skn-immune cells and was evidenced by significantly reduced lesion severity by days 5-7 post-cotransfer compared with animals given injections of Skn-immune cells alone. Enrichment and depletion of normal CD4(+) or CD8(+) spleen cells and RT-PCR analysis of selected cytokines identified CD4(+) cells as the regulatory cells in the cotransfer inoculum; however,significant reduction in lesion severity was observed only when there was a concomitant increase in levels of IL-7. The role of IL-7 was further supported in that mice cotransferred with Skn-immune cells plus normal spleen cells,but also treated with anti-IL-7 Ab,no longer exhibited reduced lesion severity. To determine whether IL-7 expression without normal spleen cell cotransfer could modulate lesion development,an IL-7-encoding plasmid (pCMV-Tag1-IL-7) was topically delivered to sites flanking the stressed skin site in Skn-induced autoimmune mice. Daily application of 15 mug of pCMV-Tag1-IL-7 significantly suppressed lesion severity. Our results support a mechanism for CD4(+) T cells and IL-7 in contributing to the control of autoreactivity. View Publication

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However,less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved,we show that absence of MyD88,a key innate Toll like receptor signal adaptor,abrogates this resistance and facilitates inducible allograft acceptance. In our model,absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+ CD25+ regulatory T cells. Therefore,this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential,clinically relevant strategy to facilitate transplantation tolerance. View Publication

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. View Publication

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision,RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study,we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing,revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision,thereby ensuring the utility of the postrevision repertoire. View Publication

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs,substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples,39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However,33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients,but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction,and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function,and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies. View Publication

Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific,as they reduce infection rates in elective surgery,but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably,AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery,consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly,AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells,which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms. View Publication