技术资料

Killer immunoglobulin-like receptors (KIR) bind self-major histocompatibility complex class I molecules,allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes,but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression,3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5' gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor,5-aza-2'-deoxycytidine,induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus,NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles. View Publication

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses with different coreceptor usage profiles were isolated from 29 South African patients with advanced AIDS. All 24 R5 isolates were inhibited by the CCR5-specific agents,PRO 140 and RANTES,while the two X4 viruses and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor,AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells that did not express CCR5. When tested using coreceptor-transfected cell lines,one R5 virus was also able to use CXCR6,and another R5X4 virus could use CCR3,BOB/GPR15,and CXCR6. The R5X4 and X4 viruses contained more-diverse V3 loop sequences,with a higher overall positive charge,than the R5 viruses. Hence,some HIV-1 subtype C viruses are able to use CCR5,CXCR4,or both CXCR4 and CCR5 for entry,and they are sensitive to specific inhibitors of entry via these coreceptors. These observations are relevant to understanding the rapid spread of HIV-1 subtype C in the developing world and to the design of intervention and treatment strategies. View Publication

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study,we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs),evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti-vascular endothelial growth factor (anti-VEGF) and/or anti-interleukin 6 (anti-IL-6) antibodies neutralized this inhibitory effect,confirming that VEGF and IL-6,at least in part,mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo,immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor alpha (TNF-alpha) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs,and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6),DCs only (SI = 9.3),or MM lysate-pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM. View Publication

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated,and was due,at least in part,to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1,diminished TGF-beta production,and decreased lymphocyte apoptosis. Moreover,the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition,CD69 engagement induced NK and T cell production of TGF-beta,directly linking CD69 signaling to TGF-beta regulation. Furthermore,anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors. View Publication

Isolation and analysis of intact fetal cells in maternal blood is an attractive method of non-invasive prenatal diagnosis; however,detection levels are not optimal. The poor sensitivity and inconsistent recovery of fetal cells is compounded by small numbers of circulating fetal cells and loss of fetal cells during enrichment procedures. Optimizing selection criteria by utilizing less complicated methods for target cell enrichment is essential. We report here salutary results using a simple density-based depletion method that requires neither MACS (magnetic-activated cell sorting) nor flow cytometric separation for enrichment of progenitor cells. Maternal blood samples (n = 81) were obtained from women prior to invasive prenatal genetic diagnostic procedures and processed randomly within 24 h using one of two density-based enrichment methods. For progenitor cell enrichment,samples (n = 49) were labeled with a RosetteSep progenitor antibody cocktail to remove unwanted mature T-cells,B-cells,granulocytes,natural killer cells,neutrophils and myelomonocytic cells. For CD45-negative cell enrichment,samples (n = 14) were labeled with RosetteSep CD45 antibody to remove unwanted maternal white cells. The desired cellular fraction was collected and analyzed by either fluorescent in situ hybridization (FISH) or real-time PCR for the presence of intact fetal cells and to quantify Y-chromosome-specific DYS1 sequences,respectively. Overall,FISH and real-time PCR correct detection rates for the progenitor cell enrichment approach were 53% and 89% with 3% (1 out of 30 cases) and 0% false-positive detection,respectively. Fetal sequences were detected in the range from 0.067 to 1.167 genome equivalents per milliliter of blood. No fetal cells were detected using the CD45-negative enrichment method. Flow cytometric analysis of cord blood showed that a unique myeloid population of cells was recovered using RosetteSep trade mark progenitor enrichment compared with the CD45-negative enrichment method. Sensitivity of the RosetteSep progenitor enrichment approach for detection of fetal cells in this pilot study shows great promise with recovery of cells that are suitable for FISH and automated microscope scanning. This simple and rapid method may also allow expansion in culture and characterization of the fetal cell type(s) that circulate in maternal blood,hence,greatly improving reliability of non-invasive prenatal diagnosis. View Publication

Inter-alpha inhibitor protein (IalphaIp) is an endogenous serine protease inhibitor in human plasma. Circulating IalphaIp levels were lower in 51 patients with severe sepsis than in healthy volunteers. Mean levels were 688+/-295 mg/L in patients with severe sepsis who survived (n=32),486+/-193 mg/L in patients with sepsis who died (n=19),and 872+/-234 mg/L in control subjects (n=25). IalphaIp levels were lower in patients with shock versus those without (540+/-246 [n=33] vs. 746+/-290 [n=18] mg/L; P=.0102). IalphaIp levels were inversely correlated with 28-day mortality rates and Acute Physiology and Chronic Health Evaluation II scores and directly correlated with antithrombin III,protein C,and protein S levels. The administration of IalphaIp (30 mg/kg body weight intravenously) increased the 50% lethal dose in mice by 100-fold after an intravenous challenge of Escherichia coli. Thus,human IalphaIp may be a useful predictive marker and potential therapeutic agent in sepsis. View Publication

The deregulation of the immune response is a critical component in inflammatory disease. Recent in vitro data show that T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of cytokine signaling. Furthermore,tc-ptp(-/-) mice display immune defects and die within 5 weeks of birth. We report here that tc-ptp(-/-) mice develop progressive systemic inflammatory disease as shown by chronic myocarditis,gastritis,nephritis,and sialadenitis as well as elevated serum interferon-gamma. The widespread mononuclear cellular infiltrates correlate with exaggerated interferon-gamma,tumor necrosis factor-alpha,interleukin-12,and nitric oxide production in vivo. Macrophages grown from tc-ptp(-/-) mice are inherently hypersensitive to lipopolysaccharide,which can also be detected in vivo as an increased susceptibility to endotoxic shock. These results identify T-cell protein tyrosine phosphatase as a key modulator of inflammatory signals and macrophage function. View Publication

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model,we investigated whether expression of CD1a and langerin,an LC-specific C-type lectin,imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin,placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. View Publication

In the current study,we evaluated whether the capacity of HIV to modulate major histocompatibility complex (MHC) class I molecules has an impact on the ability of autologous natural killer (NK) cells to kill the HIV-infected cells. Analysis of HIV-infected T-cell blasts revealed that the decrease in MHC class I molecules on the infected cell surface was selective. HLA-A and -B were decreased on cells infected with HIV strains that could decrease MHC class I molecules,whereas HLA-C and -E remained on the surface. Blocking the interaction between HLA-C and -E and their corresponding inhibitory receptors increased NK cell killing of T-cell blasts infected with HIV strains that reduced MHC class I molecules. Moreover,we demonstrate that NK cells lacking HLA-C and -E inhibitory receptors kill T-cell blasts infected with HIV strains that decrease MHC class I molecules. In contrast,NK cells are incapable of destroying T-cell blasts infected with HIV strains that were unable to reduce MHC class I molecules. These findings suggest that NK cells lacking inhibitory receptors to HLA-C and -E kill HIV-infected CD4+ T cells,and they indicate that the capacity of NK cells to destroy HIV-infected cells depends on the ability of the virus to modulate MHC class I molecules. View Publication

Although structurally unrelated,the human killer cell Ig-like (KIR) genes and the rodent lectin-like Ly49 genes serve similar functional roles in NK cells. Moreover,both gene families display variegated,monoallelic expression patterns established at the transcriptional level. DNA methylation has been shown to play an important role in maintenance of expression patterns of KIR genes,which have CpG island promoters. The potential role of DNA methylation in expression of Ly49 genes,which have CpG-poor promoters,is unknown. In this study,we show that hypomethylation of the region encompassing the Pro-2 promoter of Ly49a and Ly49c in primary C57BL/6 NK cells correlates with expression of the gene. Using C57BL/6 x BALB/c F1 hybrid mice,we demonstrate that the expressed allele of Ly49a is hypomethylated while the nonexpressed allele is heavily methylated,indicating a role for epigenetics in maintaining monoallelic Ly49 gene expression. Furthermore,the Ly49a Pro-2 region is heavily methylated in fetal NK cells but variably methylated in nonlymphoid tissues. Finally,in apparent contrast to the KIR genes,we show that DNA methylation and the histone acetylation state of the Pro-2 region are strictly linked with Ly49a expression status. View Publication

To search for new indicators of self-renewing hematopoietic stem cells (HSCs),highly purified populations were isolated from adult mouse marrow,micromanipulated into a specially designed microscopic array,and cultured for 4 days in 300 ng/ml Steel factor,20 ng/ml IL-11,and 1 ng/ml flt3-ligand. During this period,each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (textgreater1% contribution to lymphoid and myeloid lineages). In a first experiment,6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity,demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions. View Publication

Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here,we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen,blood,and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally,we compared infection of macrophages,CD4(+) T cells,and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node,blood,and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit. View Publication