Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-X L , an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-X L was mediated through both its CED-4-like domain and the Cterminal domain containing WD-40 repeats. Expression of Bcl-X L inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-X L purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-X L failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-X L regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-X L associates with caspase-9 and Apaf-1, and show that Bcl-X L inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized antibody, hD1V1G2/G4 (hD1), directed against the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to explore its capacity to serve as a target receptor for vaccination purposes. hD1 was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). We observed that the chimeric antibody-protein complex (hD1-KLH) bound specifically to DC-SIGN and was rapidly internalized and translocated to the lysosomal compartment. To determine the targeting efficiency of hD1-KLH, monocyte-derived DCs and peripheral blood lymphocytes (PBLs) were obtained from patients who had previously been vaccinated with KLHpulsed DCs. Autologous DCs pulsed with hD1-KLH induced proliferation of patient PBLs at a 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1- KLH-targeted IntroductionDendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a key role in regulating antigen-specific immunity. DCs capture antigens, process them into peptides, and present these to T cells. 1 The interaction between DC and T-cell controls the type and magnitude of the resulting immune response. Recently, preclinical and clinical studies have exploited DCs in an attempt to improve vaccine efficacy. 2 Most of these studies involve ex vivo antigen loading of autologous monocyte-derived DCs that are readministrated to the patient, a laborious and costly procedure. A more direct strategy involves targeting of antigens specifically to antigen uptake receptors on the DC in vivo. Potential candidate receptors highly expressed by DCs include Fc receptors [3][4][5] and members of the C-type lectin family. 6,7 Whereas Fc receptors are expressed by many different cell types, the expression of some members of the C-type lectin family are more DC restricted. 8 C-type lectins bind sugar residues in a calcium-dependent manner via a highly conserved carbohydrate recognition domain. C-type lectin receptors expressed by DCs are implicated in immunoregulatory processes, such as antigen capture, DC trafficking, and DC-T-cell interactions. 8 Based on the location of the amino (N) terminus, 2 types of membrane-bound C-type lectins can be distinguished on DCs. Type I C-type lectins have their N terminus located outside, while type II C-type lectins have their N terminus located inside the cell. Several studies have been conducted on antigen targeting to C-type lectin receptors for vaccination purposes, mainly focusing on the type I C-type lectins mannose receptor (MR) 9 and DEC-205. 6,10,11 Vaccines based on natural MR ligands have been shown to effectively induce humoral and cellular responses. 9 However, these ligands lack specificity for the MR, and may target multiple lectins with overlapping binding sp...

The Caenorhabditis elegans survival gene ced-9 regulates ced-4 activity and inhibits cell death, but the mechanism by which this occurs is unknown. Through a genetic screen for CED-4-binding proteins, CED-9 was identified as an interacting partner of CED-4. CED-9, but not loss-of-function mutants, associated specifically with CED-4 in yeast or mammalian cells. The CED-9 protein localized primarily to intracellular membranes and the perinuclear region, whereas CED-4 was distributed in the cytosol. Expression of CED-9, but not a mutant lacking the carboxy-terminal hydrophobic domain, targeted CED-4 from the cytosol to intracellular membranes in mammalian cells. Thus, the actions of CED-4 and CED-9 are directly linked, which could provide the basis for the regulation of programmed cell death in C. elegans.

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.