Vimentin Is Involved in Peptidylarginine Deiminase 2-Induced Apoptosis of Activated Jurkat Cells

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca²⁺-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.     

Kisspeptin Effect on Endothelial Monocyte Activating Polypeptide II (EMAP-II)-Associated Lymphocyte Cell Death and Metastases in Colorectal Cancer Patients

Kisspeptin is an antimetastatic agent in some cancers that has also been associated with lymphoid cell apoptosis, a phenomenon favoring metastases. Our aim was to determine the association of kisspeptin with lymphocyte apoptosis and the presence of metastases in colorectal cancer patients. Blood was drawn from 69 colon cancer patients and 20 healthy volunteers. Tissue specimens from healthy and pathological tissue were immunohistochemically analyzed for kisspeptin and endothelial monocyte activating polypeptide II (EMAP-II) expression. Blood EMAP-II and soluble Fas ligand (sFasL) levels were examined by an enzyme-linked immunosorbent assay method. The kisspeptin and EMAP-II expression and secretion levels in the DLD-1 and HT-29 colon cancer cell lines were examined by quantitative real-time polymerase chain reaction, Western analysis and enzyme-linked immunosorbent assay, whereas lymphocyte viability was assessed by flow cytometry. The effect of kisspeptin on the viability of colon cancer cells was examined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Exogenous, synthetic and naturally produced, kisspeptin induces through the G-protein-coupled receptor 54 (GPR54; also known as the kisspeptin receptor) the EMAP-II expression and secretion in colon cancer cell lines, inducing in vitro lymphocyte apoptosis, as verified by the use of an anti-EMAP-II antibody. These results were reversed with the use of kisspeptin inhibitors and by kisspeptin-silencing experiments. Tumor kisspeptin expression was associated with the tumor EMAP-II expression (p < 0.001). Elevated kisspeptin and EMAP-II expression in colon cancer tissues was associated with lack of metastases (p < 0.001) in colon cancer patients. These data indicate the antimetastatic effect of tumor-elevated kisspeptin in colon cancer patients that may be mediated by the effect of kisspeptin on EMAP-II expression in colon cancer tumors in patients with normal serum EMAP-II levels. These findings provide new insight into the role of kisspeptin in the context of metastases in colon cancer patients.     

Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy.     

Crosstalk between medulloblastoma cells and endothelium triggers a strong chemotactic signal recruiting T lymphocytes to the tumor microenvironment

Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)" is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant "Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES)." This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications.     

Actions of TGF-β as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-β (TGF-β) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-β inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-β receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-β receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-β whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-β induces epithelial–mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-β acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-β regulates angiogenesis. Finally, TGF-β suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-β pathway. In conclusion, TGF-β signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.     

Bcl－2 over－expression and activation of protein kinase C suppresss the Trail－induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.     

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

INTRODUCTIONThaillll (TNF-related apoptosis inducing ligand)is a recently described member of the TNF family.Like other members of the TNF ligand family) availcould induce apoptosis of neoplastically transformedcells by priVating cell surface death receptors ThailRI and ThaiLR212].Trail has been demonstrated to play an important role in homeostasis of immune system includ.lug eradication of the old lymphocytel3], actiVationinduced T cell deathI41, regulation Of T cen eXPansion by…     

Cutting edge: the tumor counterattack hypothesis revisited: colon cancer cells do not induce T cell apoptosis via the Fas (CD95, APO-1) pathway

The counterattack hypothesis, suggesting that cancer cells express Fas ligand (FasL) and are able to kill Fas-expressing tumor-infiltrating activated T cells, was supported by reports of the killing of Jurkat cells by FasL-expressing human colon cancer cell lines. Through the use of an improved cytotoxic assay in which soluble FasL and FasL-transfected KFL9 cells were used as positive controls, we show that none of seven human colon cancer cell lines induce apoptosis of two Fas-expressing target cell lines, Jurkat and L1210-Fas cells. Moreover, in coculture experiments, cancer cell monolayers do not inhibit the growth of Fas-expressing lymphoid cells. Although FasL mRNA and protein were detected in the extracts of the colon cancer cell lines, flow cytometry and confocal microscopy failed to detect the protein on the surface of tumor cells. These results suggest that the counterattack of tumor-infiltrating T lymphocytes by cancer cells may not account for immune tolerance toward tumor cells.     

Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells

Although tumor progression involves processes such as tissue invasion that can activate inflammatory responses, the immune system largely ignores or tolerates disseminated cancers. The mechanisms that block initiation of immune responses during cancer development are poorly understood. We report here that constitutive activation of Stat-3, a common oncogenic signaling pathway, suppresses tumor expression of proinflammatory mediators. Blocking Stat-3 in tumor cells increases expression of proinflammatory cytokines and chemokines that activate innate immunity and dendritic cells, leading to tumor-specific T-cell responses. In addition, constitutive Stat-3 activity induces production of pleiotropic factors that inhibit dendritic cell functional maturation. Tumor-derived factors inhibit dendritic cell maturation through Stat-3 activation in progenitor cells. Thus, inhibition of antitumor immunity involves a cascade of Stat-3 activation propagating from tumor to dendritic cells. We propose that tumor Stat-3 activity can mediate immune evasion by blocking both the production and sensing of inflammatory signals by multiple components of the immune system.     

Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.     

TRAIL gene reorganizes the cytoskeleton and decreases the motility of human leukemic Jurkat cells

TRAIL can selectively induce rapid apoptosis of various types of tumor cells. We induced the expression of TRAIL in Jurkat cells, and measured the adhesion of cells to human umbilical vein endothelial cells (HUVECs) and laminin (LN) in a parallel plate flow chamber system and by using a colorimetric method. The apoptosis percentage, cycle distribution, intracellular Ca(2+) concentration, and adhesion molecule expression of the cells were detected by flow cytometry. Cytoskeleton was observed with a laser confocal microscopy. The roles of adhesion molecules in the cell interaction was defined by their function blocking. The results showed that TRAIL attenuated the adhesion of Jurkat cells to HUVECs and LN, as well as their transendothelial migration. The increased apoptosis and G1-phase cell percentages, decreased intracellular Ca(2+) concentration, depolymerized actin and impaired cell deformability could contribute to the decreased adhesion of Jurkat cells caused by TRAIL. Furthermore, CD11a was found to play a more important role than CD62L in the adhesion of Jurkat cells to HUVECs. These findings contribute to the knowledge on the role of TRAIL in tumor metastasis and provide mechanistic basis for the clinical application of TRAIL and tumor therapy.     

EMAP-II facilitates TNF-R1 apoptotic signalling in endothelial cells and induces TRADD mobilization

Endothelial monocyte-activating polypeptide-II (EMAP-II), a proinflammatory cytokine with antiangiogenic properties, renders tumours sensitive to tumour necrosis factor-alpha (TNF) treatment. The exact mechanisms for this effect remain unclear. Here we show that human endothelial cells (EC) are insensitive to TNF-induced apoptosis but after a short pre-treatment with EMAP-II, EC quickly undergo TNF-induced apoptosis. We further analysed this EMAP-II pre-treatment effect and found no increase of TNF-R1 protein expression but rather an induction of TNF-R1 redistribution from Golgi storage pools to cell membranes. In addition, we observed EMAP-II induced mobilization and membrane expression of the TNF-R1-Associated Death Domain (TRADD) protein. Immunofluorescence co-staining experiments revealed that these two effects occurred at the same time in the same cell but TNF-R1 and TRADD were localized in different vesicles. These findings suggest that EMAP-II sensitises EC to apoptosis by facilitating TNF-R1 apoptotic signalling via TRADD mobilization and introduce a molecular and antiangiogenic explanation for the TNF sensitising properties of EMAP-II in tumours.     

Dinitrosyl-iron triggers apoptosis in Jurkat cells despite overexpression of Bcl-2

Cells expressing the cytokine-inducible NO synthase are known to trigger apoptosis in neighboring cells. Paramagnetic dinitrosyl nonheme iron complexes (DNIC) were found in tumor tissue about 40 years ago; however, the role of these NO(+)-bearing species is not completely understood. In the human Jurkat leukemia cell line, the application of the model complex DNIC-thiosulfate (50-200 microM) induced apoptosis (defined by phosphatidylserine externalization) in a concentration- and time-dependent manner. In Jurkat cells, the pan-caspase inhibitor, zVADfmk (50 microM), and/or stable transfection of antiapoptotic protein, Bcl-2, was unable to afford protection against DNIC-induced apoptosis. The membrane-impermeable metal chelator, N-methyl-D-glucamine dithiocarbamate (MGD; 200 microM), in the presence of DNIC significantly increased apoptosis, but had no effect on its own. Electron paramagnetic resonance studies showed that MGD led to rapid transformation of the extracellular DNIC into the stable impermeable NO-Fe-MGD complex and to a burst-type release of nitrosonium (NO(+)) equivalents in the extracellular space. These results suggest that in Jurkat cells, DNIC-thiosulfate induces Bcl-2- and caspase-independent apoptosis, which is probably secondary to local nitrosative stress at the cell surface. We hypothesize that the local release of nonheme Fe-NO species by activated macrophages may play a role in the killing of malignant cells that have high Bcl-2 levels.     

Doxycycline induces Fas/Fas ligand-mediated apoptosis in Jurkat T lymphocytes.

Tetracyclines have been used in the treatment of chronic inflammatory diseases associated with local infiltration of inflammatory cells and matrix destruction as observed in rheumatoid arthritis and periodontal disease. Fas/Fas ligand (FasL)-mediated apoptosis plays an important role in maintaining T lymphocyte homeostasis and modulating immune response. The present study demonstrates that doxycycline inhibits Jurkat T lymphocyte proliferation and induces apoptosis. The phytohemagglutinin (PHA)-activated Jurkat cells are more susceptible to doxycycline-induced apoptosis. Furthermore, doxycycline-induced apoptosis is associated with increased Fas/FasL expression in Jurkat cells. The increase of apoptosis in Jurkat cells treated with doxycycline is consistent with the increase of FasL expression. These results suggest that doxycycline may downregulate the inflammatory process in certain diseases by eliminating activated T lymphocytes through Fas/FasL-mediated apoptosis.     

Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.     

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

Background Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3ζ expression. However, the hypothesis of tumor counterattack is still controversial. Methods We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG₁ peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3ζ, CD3ε, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3ζ and CD3ε in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied. Results Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3ε but not CD3ζ chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3ε, but not CD3ζ, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. Conclusions Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3ε in T-cells induced by NSCLC cells might lead to T-cell dysfunction.     

CD47 signals T cell death.

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3epsilon-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3epsilon and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1beta-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.