技术资料

In herpesvirus infections,the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection,we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist,we were able to dissect the changes in the antiviral CD8(+) T-cell response that are induced by virus persistence. Surprisingly,persistently infected mice controlled a secondary challenge infection more rapidly than nonpersistently infected mice,indicating enhanced rather than diminished effector functions. Consistent with this,virus-specific CD8 T cells from these mice exhibited faster upregulation of the cytotoxic mediator granzyme B. Another unexpected finding was that CD8(+) T cells from neither infection responded efficiently to homeostatic cytokines. The unresponsiveness of the memory cells from the nonpersistently infected mice appears to be linked to the prolonged replication of virus within the lungs. Other changes seen in different chronic infection models were also observed,such as changes in Bcl-2 levels,interleukin-2 production,and the immunodominance hierarchy. These data show persistence of gammaherpesvirus type 68 alters the properties of CD8(+) T cells and illustrates that immune surveillance does not require CD8 T cells with the same attributes as classical" memory CD8(+) T cells." View Publication

While during the first trimester of pregnancy natural killer (NK) cells represent the most abundant lymphocyte population in the decidua,their actual function at this site is still debated. In this study we analyzed NK cells isolated from decidual tissue for their surface phenotype and functional capability. We show that decidual NK (dNK) cells express normal surface levels of certain activating receptors,including NKp46,NKG2D,and 2B4,as well as of killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A inhibitory receptor. In addition,they are characterized by high levels of cytoplasmic granules despite their CD56(bright) CD16- surface phenotype. Moreover,we provide evidence that in dNK cells,activating NK receptors display normal triggering capability whereas 2B4 functions as an inhibitory receptor. Thus,cross-linking of 2B4 resulted in inhibition of both cytolytic activity and interferon-gamma (IFN-gamma) production. Clonal analysis revealed that,in the majority of dNK cell clones,the 2B4 inhibitory function is related to the deficient expression of signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) mRNA. Moreover,biochemical analysis revealed low levels of SAP in the dNK polyclonal population. This might suggest that dNK cells,although potentially capable of killing,are inhibited in their function when interacting with cells expressing CD48. View Publication

Monitoring genome-wide,cell-specific responses to human disease,although challenging,holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements,including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations,combined with cell-specific pathway analyses,offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes),monocyte (2,801 genes),and total leukocyte (3,437 genes) transcriptomes,with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1,CD152,NRP-1,and Lag3) and concomitant decreases in stimulatory receptors (CD28,CD4,and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus,genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly,application of cell separation,genome-wide expression,and cell-specific pathway analyses can be used to discover pathway alterations in human disease. View Publication

The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member,IL-23,shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17-producing T cells. We introduce a novel compound,STA-5326,that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level,and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4+CD45Rbhigh T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon-gamma (IFN-gamma) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326,indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis. View Publication

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition,vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy. View Publication

Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors,their interaction with the host immune system,and their host range. We have investigated the capacity of a panel of GPs of both retroviral (amphotropic murine leukemia virus [MLV-A]; gibbon ape leukemia virus [GALV]; RD114,feline endogenous virus) and nonretroviral (fowl plague virus [FPV]; Ebola virus [EboV]; vesicular stomatitis virus [VSV]; lymphocytic choriomeningitis virus [LCMV]) origins to pseudotype lentiviral vectors derived from simian immunodeficiency virus (SIVmac251). SIV vectors were efficiently pseudotyped with the FPV hemagglutinin,VSV-G,LCMV,and MLV-A GPs. In contrast,the GALV and RD114 GPs conferred much lower infectivity to the vectors. Capitalizing on the conservation of some structural features in the transmembrane domains and cytoplasmic tails of the incorporation-competent MLV-A GP and in RD114 and GALV GPs,we generated chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP. Importantly,SIV-derived vectors pseudotyped with these GALV/TR and RD114/TR GP chimeras had significantly higher titers than vectors coated with the parental GPs. Additionally,RD114/TR-pseudotyped vectors were efficiently concentrated and were resistant to inactivation induced by the complement of both human and macaque sera,indicating that modified RD114 GP-pseudotyped lentiviral vectors may be of particular interest for in vivo gene transfer applications. Furthermore,as compared to vectors pseudotyped with other retroviral GPs or with VSV-G,RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells. View Publication

We have recently shown that proteasome inhibitor PS-341 induces apoptosis in drug-resistant multiple myeloma (MM) cells,inhibits binding of MM cells in the bone marrow microenvironment,and inhibits cytokines mediating MM cell growth,survival,drug resistance,and migration in vitro. PS-341 also inhibits human MM cell growth and prolongs survival in a SCID mouse model. Importantly,PS-341 has achieved remarkable clinical responses in patients with refractory relapsed MM. We here demonstrate molecular mechanisms whereby PS-341 mediates anti-MM activity by inducing p53 and MDM2 protein expression; inducing the phosphorylation (Ser15) of p53 protein; activating c-Jun NH(2)-terminal kinase (JNK),caspase-8,and caspase-3; and cleaving the DNA protein kinase catalytic subunit,ATM,and MDM2. Inhibition of JNK activity abrogates PS-341-induced MM cell death. These studies identify molecular targets of PS-341 and provide the rationale for the development of second-generation,more targeted therapies. View Publication

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis,an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA),the most potent known inhibitor of Kv1.3,for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited textgreater80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments,ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (textgreater600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation,peaking at 15-20 h and then declining to baseline over the next 7 days,in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues,and to visualize the distribution of functional channels in intact cells. View Publication