Youngmee Bae

CD95/Fas Signaling in T Lymphocytes Induces the Cell Cycle Control Protein p21cip-1/WAF-1, Which Promotes Apoptosis

Ligation of CD95 on T lymphocytes resulted in the up-regulation of a cell cycle control protein, p21cip-1/WAF-1, an inhibitor of cyclin-dependent kinases. This up-regulation was completely blocked by the cysteine protease inhibitor Z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), whereas DEVD-CHO (succinyl-Asp-Glu-Val-Asp-aldehyde), a caspase 3 inhibitor, had no effect. In Faslpr-cg mice, a point mutation in the death domain of CD95 results in failure to recruit FADD (Fas-associated death domain), and in the present study this mutation prevented both CD95-mediated apoptosis and p21cip-1/WAF-1 induction. During apoptotic cell death due to irradiation, p21cip-1/WAF-1 is up-regulated by a p53-dependent pathway that responds to DNA damage. However, CD95-induced up-regulation of p21cip-1/WAF-1 in T cells was p53-independent. T cells deficient in p21cip-1/WAF-1 were less susceptible to CD95-induced apoptosis. We conclude that in T cells, ligation of CD95 and activation of caspases cause the induction of p21cip-1/WAF-1, which acts to promote cell death.

CD99 is essential for leukocyte diapedesis in vivo

Recruitment of leukocytes into inflamed tissue requires migration of leukocytes from the blood stream across the endothelial lining and the basement membrane of the local blood vessels. CD99 in humans is a 32-kDa highly O-glycosylated cell surface protein expressed on most leukocytes. The authors recently found CD99 to be expressed in leukocytes and at human endothelial cell contacts. Human CD99 is involved in homophilic interaction between the two cell types and participates in the transendothelial migration of monocytes and polymorphonuclear neutrophils (PMNs) in vitro. To test the role of CD99 in vivo, the authors cloned murine CD99 (muCD99), expressed it in vitro, and generated a blocking monoclonal antibody against it. We first showed that muCD99 is expressed on mouse leukocytes as well as enriched at the endothelial cell borders. Transfection of cells with muCD99 imparts on them the ability to aggregate in a CD99-dependent homophilic manner. Cells expressing muCD99 did not bind to cells expressing murine or human platelet endothelial call adhesion molecule (PECAM) or human CD99. In the thioglycollate peritonitis model of inflammation, anti-CD99 monoclonal antibody blocked the recruitment of neutrophils and monocytes by over 40% and 80%, respectively, at 18 h. Microscopy showed that this blocking occurred at the luminal surface of venules. The authors conclude that CD99 plays a major role in the emigration of leukocytes in vivo.

In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

Administration of an ICAM-1–specific antibody arrests dendritic cells in a semi-immature state and facilitates antigen-specific T cell tolerance to islet allografts in humanized mice and Rhesus monkeys.

PKB/Akt inhibits ceramide-induced apoptosis in neuroblastoma cells by blocking apoptosis-inducing factor (AIF) translocation

Ceramide is a sphingolipid that is abundant in the plasma membrane of neuronal cells and is thought to have regulatory roles in cell differentiation and cell death. Ceramide is known to induce apoptosis in a variety of different cell types, whereas the physiological significance of gangliosides, another class of sphingolipids, in these processes is still unclear. We examined the mechanisms of ceramide‐induced cell death using a human neuroblastoma cell line. Treatment of the human neuroblastoma cell line SH‐SY5Y with ceramide induced dephosphorylation of the PKB/Akt kinase and subsequent mitochondrial dysfunction. In addition, ceramide‐induced neuronal cell death was not completely blocked by inhibition of caspase activity. This incomplete inhibition appeared to be attributable to the translocation of apoptosis‐inducing factor to the nucleus. Furthermore, overexpression of active PKB/Akt or Bcl‐2 successfully blocked ceramide‐induced neuronal cell death through inhibition of the translocation of apoptosis‐inducing factor. J. Cell. Biochem. 102: 1160–1170, 2007.

The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas‐mediated apoptosis in Jurkat T cells. Augmentation of Fas‐mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z‐VAD‐fmk or z‐IETD‐fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.

TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-Linking

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4−CD8− double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.

Identification of antigenic peptide recognized by the anti-JL1 leukemia-specific monoclonal antibody from combinatorial peptide phage display libraries

AbstractnPurpose. In the present study an antigen-mimetic peptide of the anti-JL1 leukemia-specific monoclonal antibody (mAb) was identified and characterized.nMethods. From combinatorial peptide phage display libraries displaying the random linear heptapeptides and dodecapeptides, we selected clones with affinity to anti-JL1 mAb through repeated rounds of panning on a mAb-coated ELISA plate. The antigenicity and immunogenicity of the peptide epitopes were then studied using chemically synthesized peptides.nResults. The selected clones had the LXPSIP consensus sequence. Two synthetic peptides LPPSIPFGLTVGGGGS and LLPSIPNQAYLGGGGS specifically reacted with anti-JL1 mAb in ELISA. These two peptides were found to inhibit the interaction between anti-JL1 mAb and JL1 antigen-positive Molt-4 cells. Although the immune sera raised against the keyhole limpet hemocyanin-conjugated peptides failed to react with Molt-4 cells, it showed strong reactivity to the peptide epitope. However, one mAb raised by peptide immunization successfully bound to Molt-4 cells.nConclusion. An epitope-mimetic peptide of anti-JL1 mAb was found using combinatorial peptide phage display libraries. It induced strong humoral response against itself, but only a limited fraction of this humoral response was cross-reactive with the original JL1 antigen.

A cell surface molecule, JL1; a specific target for diagnosis and treatment of leukemias

We previously reported a novel differentiation antigen, which is specifically expressed in stage II double positive (CD4+CD8+) human cortical thymocytes (Park et al, J Exp Med 1993; 178: 1447–1451). This study was designed to investigate the expression pattern of JL1 in various types of leukemic cells from patients and normal hematopoietic cells to evaluate the possibility as a tool for diagnosis and treatment of leukemia. The expression of JL1 antigen was observed in 75.6% of leukemic cases (117 out of 154 leukemic patients tested) on flow cytometric analysis. The percentage of JL1-positive cases of T lineage acute lymphoblastic leukemia (T-ALL) (92.6%) was higher than that of other types of leukemias (75%). The presence of JL1 antigen was also confirmed by immunoblotting and immunoprecipitation. Since the JL1 antigen is selectively expressed on the surface of human leukemic cells but not on the mature human peripheral blood cells, normal bone marrow cells and various types of normal tissues, JL1 could be an excellent candidate for an immunodiagnostic and immunotherapeutic tool for hematopoietic malignancies such as leukemia.

CD43 cross-linking increases the Fas-induced apoptosis through induction of Fas aggregation in Jurkat T-cells

CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells.

Anti-adhesive functions of CD43 expressed on colon carcinoma cells through the modulation of integrins.

CD43 has conflicting roles in both pro- and anti-adhesive function in cell-to-cell adhesion in hematopoietic cells. We examined the role of CD43 glycoprotein in a colorectal carcinoma cell line. We expressed human CD43 antigen on HT-29 cells, a colon adenocarcinoma cell line, and compared the adhesion to the extracellular matrix with that of mock-transduced cells in vitro. CD43 expression inhibited the adhesion to extracellular matrix, such as collagen type IV and laminin. As the expression of β1 integrin was downregulated in CD43-expressing HT-29 cells, the anti-adhesive effect of CD43 might be implicated in its expression. Our findings suggest that the anti-adhesive function of CD43 in colon carcinoma cells plays a role in the tumorigenesis and metastasis of colorectal carcinoma cells.