技术资料

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine,a potent inhibitor of PNP,was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK),we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization,phosphorylation of p53 at Ser15,and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation,changes in mitochondrial membrane potential,and PARP cleavage. Based on these data,a phase 2 clinical trial of forodesine has been initiated for CLL patients. View Publication

Depletion of CD4+CD25+ regulatory T cells (Treg) by treatment with alphaCD25 antibody synergizes with vaccination protocols to engender protective immunity in mice. The effectiveness of targeting CD25 to eliminate Treg is limited by the fact that CD25,the low-affinity interleukin-2 receptor,is up-regulated on conventional T cells. At present,foxp3 is the only product known to be exclusively expressed in Treg of mice. However,foxp3 is not expressed on the cell surface and hence cannot be targeted with antibodies. In this study,we tested the hypothesis that vaccination of mice against foxp3,a self-antigen expressed also in the thymus,is capable of stimulating foxp3-specific CTL that will cause the depletion of Treg and enhanced antitumor immunity. Vaccination of mice with foxp3 mRNA-transfected dendritic cells elicited a robust foxp3-specific CTL response and potentiated vaccine-induced protective immunity comparably with that of alphaCD25 antibody administration. In contrast to alphaCD25 antibody treatment,repeated foxp3 vaccination did not interfere with vaccine-induced protective immunity. Importantly,foxp3 vaccination led to the preferential depletion of foxp3-expressing Treg in the tumor but not in the periphery,whereas alphaCD25 antibody treatment led to depletion of Treg in both the tumor and the periphery. Targeting foxp3 by vaccination offers a specific and simpler protocol for the prolonged control of Treg that may be associated with reduced risk of autoimmunity,introducing an approach whereby specific depletion of cells is not limited to targeting products expressed on the cell surface. View Publication

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here,we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1),we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together,our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore,the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures. 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

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