技术资料

Immunostimulatory agents such as agonistic anti-CD137 and interleukin (IL)-2 generate effective anti-tumor immunity but also elicit serious toxicities,hampering their clinical application. Here we show that combination therapy with anti-CD137 and an IL-2-Fc fusion achieves significant initial anti-tumor activity,but also lethal immunotoxicity deriving from stimulation of circulating leukocytes. To overcome this toxicity,we demonstrate that anchoring IL-2 and anti-CD137 on the surface of liposomes allows these immune agonists to rapidly accumulate in tumors while lowering systemic exposure. In multiple tumor models,immunoliposome delivery achieves anti-tumor activity equivalent to free IL-2/anti-CD137 but with the complete absence of systemic toxicity. Immunoliposomes stimulated tumor infiltration by cytotoxic lymphocytes,cytokine production,and granzyme expression,demonstrating equivalent immunostimulatory effects to the free drugs in the local tumor microenvironment. Thus,surface-anchored particle delivery may provide a general approach to exploit the potent stimulatory activity of immune agonists without debilitating systemic toxicities. View Publication

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5,originally isolated from a NOD mouse,has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy,leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge,this work is the first to use a hybrid insulin peptide,or any neoepitope,to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients. View Publication

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here,we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7),a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2,a receptor that recognizes NAP-2,abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. View Publication

Regulation of gene expression in immune cells is known to be under genetic control,and likely contributes to susceptibility to autoimmune diseases such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice,more accurately reflecting genetically diverse human populations,we provide an extensive characterization of the genetic regulation of gene expression in five different naive immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control,exhibiting diverse patterns: global,cell-specific and sex-specific. Bioinformatic analysis of the genetically controlled transcript networks reveals reduced cell type specificity and inflammatory activity in wild-derived PWD/PhJ mice,compared with the conventional laboratory strain C57BL/6J. Additionally,candidate MS-GWAS (genome-wide association study candidate genes for MS susceptibility) genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared with PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis,the principal model of MS,and skewing of the encephalitogenic T-cell responses. Taken together,our results provide functional insights into the genetic regulation of the immune transcriptome,and shed light on how this in turn contributes to susceptibility to autoimmune disease.Genes and Immunity advance online publication,22 September 2016; doi:10.1038/gene.2016.37. View Publication

Because lymphoid progenitors can give rise to natural killer (NK) cells,NK ontogeny has been considered to be exclusively lymphoid. Here,we show that rare human CD34(+) hematopoietic progenitors develop into NK cells in vitro in the presence of cytokines (interleukin-7,interleukin-15,stem cell factor,and fms-like tyrosine kinase-3 ligand). Adding hydrocortisone and stromal cells greatly increases the frequency of progenitor cells that give rise to NK cells through the recruitment of myeloid precursors,including common myeloid progenitors and granulocytic-monocytic precursors to the NK-cell lineage. WNT signaling was involved in this effect. Cells at more advanced stages of myeloid differentiation (with increasing expression of CD13 and macrophage colony-stimulating factor receptor [M-CSFR]) could also differentiate into NK cells in the presence of cytokines,stroma,and hydrocortisone. NK cells derived from myeloid precursors (CD56(-)CD117(+)M-CSFR(+)) showed more expression of killer immunoglobulin-like receptors,a fraction of killer immunoglobulin-like receptor-positive-expressing cells that lacked NKG2A,a higher cytotoxicity compared with CD56(-)CD117(+)M-CSFR(-) precursor-derived NK cells and thus resemble the CD56(dim) subset of NK cells. Collectively,these studies show that NK cells can be derived from the myeloid lineage. View Publication

Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity,and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs,but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA,human pDCs upregulate the mannose receptor (MR,CD206). To examine the functional outcome of this upregulation,inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha,viability,and upregulation of several pDC-activation markers: CD40,CD86,HLA-DR,CCR7,and PD-L1. The level of activation negatively correlated with degree of mannosylation,however,subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore,two of the infectious HIV-1 strains induced profound necrosis in pDCs,also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs. View Publication

Mixed lineage kinase domain-like (MLKL)-dependent necroptosis is thought to be implicated in the death of mycobacteria-infected macrophages,reportedly allowing escape and dissemination of the microorganism. Given the consequent interest in developing inhibitors of necroptosis to treat Mycobacterium tuberculosis (Mtb) infection,we used human pharmacologic and murine genetic models to definitively establish the pathophysiological role of necroptosis in Mtb infection. We observed that Mtb infection of macrophages remodeled the intracellular signaling landscape by upregulating MLKL,TNFR1,and ZBP1,whilst downregulating cIAP1,thereby establishing a strong pro-necroptotic milieu. However,blocking necroptosis either by deleting Mlkl or inhibiting RIPK1 had no effect on the survival of infected human or murine macrophages. Consistent with this,MLKL-deficiency or treatment of humanized mice with the RIPK1 inhibitor Nec-1s did not impact on disease outcomes in vivo,with mice displaying lung histopathology and bacterial burdens indistinguishable from controls. Therefore,although the necroptotic pathway is primed by Mtb infection,macrophage necroptosis is ultimately restricted to mitigate disease pathogenesis. We identified cFLIP upregulation that may promote caspase 8-mediated degradation of CYLD,and other necrosome components,as a possible mechanism abrogating Mtb's capacity to coopt necroptotic signaling. Variability in the capacity of these mechanisms to interfere with necroptosis may influence disease severity and could explain the heterogeneity of Mtb infection and disease. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication

Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine,we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly,neoadjuvant vaccination was far superior and provided approximately 100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. View Publication

The maintenance of peripheral naive T lymphocytes in humans is dependent on their homeostatic division,not continuing emigration from the thymus,which undergoes involution with age. However,postthymic maintenance of naive T cells is still poorly understood. Previously we reported that recent thymic emigrants (RTEs) are contained in CD31+CD25- naive T cells as defined by their levels of signal joint T cell receptor rearrangement excision circles (sjTRECs). Here,by differential gene expression analysis followed by protein expression and functional studies,we define that the naive T cells having divided the least since thymic emigration express complement receptors (CR1 and CR2) known to bind complement C3b- and C3d-decorated microbial products and,following activation,produce IL-8 (CXCL8),a major chemoattractant for neutrophils in bacterial defense. We also observed an IL-8-producing memory T cell subpopulation coexpressing CR1 and CR2 and with a gene expression signature resembling that of RTEs. The functions of CR1 and CR2 on T cells remain to be determined,but we note that CR2 is the receptor for Epstein-Barr virus,which is a cause of T cell lymphomas and a candidate environmental factor in autoimmune disease. View Publication