技术资料

Many cellular responses,such as autoimmunity and cytotoxicity,are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here,we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk,concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore,Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity,providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex,observed here with two types of inhibitory receptors,expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism. View Publication

The NKG2D receptor activates natural killer (NK) cell cytotoxicity and cytokine production on recognition of self-molecules induced by cellular stress under different conditions such as viral infections. The importance of NKG2D in the immune response to human cytomegalovirus (HCMV) is supported by the identification of several viral molecules that prevent the expression of NKG2D ligands by infected cells. In this study we report that,paradoxically,a significant,selective,and transient reduction of NKG2D expression on NK cells is detected during HCMV infection of peripheral blood mononuclear cells if needed. Antagonizing type I interferon (IFN),interleukin-12 (IL-12),and IFNgamma prevented HCMV-induced down-regulation of surface NKG2D. Moreover,treatment of purified NK cells with recombinant IFNbeta1 and IL-12 mimicked the effect,supporting a direct role of these cytokines in regulating NKG2D surface expression in NK cells. The loss of NKG2D expression selectively impaired NK-cell cytotoxicity against cells expressing NKG2D ligands but preserved the response triggered through other activating receptors. These results support that down-regulation of NKG2D expression on NK cells by cytokines with a key role in antiviral immune response may constitute a physiologic mechanism to control NK-cell reactivity against normal cells expressing NKG2D ligands in the context of inflammatory responses to viral infections. View Publication

Killer cell lectin-like receptor F1 (KLRF1) is an activating C-type lectin-like receptor expressed on human NK cells and subsets of T cells. In this study,we show that activation-induced C-type lectin (AICL) is a unique KLRF1 ligand expressed on tumor cell lines of hematopoietic and non-hematopoietic origins. We screened a panel of human tumor cell lines using the KLRF1 reporter cells and found that several tumor lines expressed KLRF1 ligands. We characterized a putative KLRF1 ligand expressed on the U937 cell line. The molecular mass for the deglycosylated ligand was 28 kDa under non-reducing condition and 17 kDa under reducing condition,suggesting that the KLRF1 ligand is a homodimer. By expression cloning from a U937 cDNA library,we identified AICL as a KLRF1 ligand. We generated mAbs against AICL to identify the KLRF1 ligands on non-hematopoietic tumor lines. The anti-AICL mAbs stained the tumor lines that express the KLRF1 ligands and importantly the interaction of KLRF1 with the KLRF1 ligand on non-hematopoietic tumors was completely blocked by the two anti-AICL mAbs. Moreover,NK cell degranulation triggered by AICL-expressing targets was partially inhibited by the anti-AICL mAb. Finally,we demonstrate that AICL is expressed in human primary liver cancers. These results suggest that AICL is expressed on tumor cells of non-hematopoietic origins and raise the possibility that AICL may contribute to NK cell surveillance of tumor cells. View Publication

Human natural killer (NK) cells express an abundant level of 2B4 and CD2 on their surface. Their counter-receptors,CD48 and CD58,are also expressed on the NK cell surface,raising a question about the functional consequences of potential 2B4/CD48 and CD2/CD58 interactions. Using blocking antibodies specific to each receptor,we demonstrated that both 2B4/CD48 and CD2/CD58 interactions were essential for the development of NK effector functions: cytotoxicity and cytokine secretion. However,only 2B4/CD48,but not CD2/CD58,interactions were shown to be critical for the optimal NK cell proliferation in response to interleukin (IL)-2. IL-2-activated NK cells cultured in the absence of 2B4/CD48 or CD2/CD58 interactions were severely impaired for their ability to induce intracellular calcium mobilization and subsequent ERK activation upon tumor target exposure,suggesting that the early signaling pathway of NK receptors leading to impaired cytolysis and interferon (IFN)-γ secretion was inhibited. Nevertheless,these defects did not fully account for the reduced proliferation of NK cells in the absence of 2B4/CD48 interactions,because anti-CD2 or anti-CD58 monoclonal antibody (mAb)-treated NK cells,showing defective signaling and effector functions,displayed normal proliferation upon IL-2 stimulation. These results propose the signaling divergence between pathways leading to cell proliferation and cytotoxicity/cytokine release,which can be differentially regulated by 2B4 and CD2 during IL-2-driven NK cell activation. Collectively,these results reveal the importance of homotypic NK-to-NK cell cross-talk through 2B4/CD48 and CD2/CD58 pairs and further present their differential and overlapping roles in human NK cells. 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

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. 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

Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps,similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently,CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99,albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid,natural killer (NK),or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors. View Publication

Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue,making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1$\$)-hESC lines using chEF1$\$ system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology,karyotype,and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers,OCT-3/4 and SSEA-4,in undifferentiated mHLA-G(EF1$\$)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors,expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1$\$)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives. View Publication