Pathway of apoptosis induced in Jurkat T lymphoblasts by anti-HLA class I antibodies.

We demonstrated recently that human leukocyte antigen (HLA) class I human monoclonal antibodies (mAbs) are able to induce apoptosis of resting human lymphocytes as well as Jurkat lymphoblastic T cells. We now analyzed the signaling pathway involved in apoptosis mediated by human HLA class I allele-specific mAb OK2F3 and mouse monomorphic mAb W6/32. An inhibitor of a broad spectrum of caspases had only a moderate inhibiting effect, and an inhibitor of caspase 3 failed to inhibit HLA class I-mediated apoptosis. Although caspase 3 activation was not observed, internucleosomal DNA fragmentation was found in half of the apoptotic cells. Importantly, the mitochondrio-nuclear redistribution of apoptosis inducing factor (AIF), a caspase-independent mitochondrial death effector, was detected after 1 hour of treatment with human anti-HLA mAb and was associated with large-scale DNA fragmentation, whereas the release of cytochrome c, which is responsible for caspase-dependent internucleosomal fragmentation, followed AIF translocation and occurred after 2 hours. Our results indicate that apoptosis mediated through HLA class I molecules represents a unique mechanism of cell death in Jurkat T lymphoblasts that involves two parallel pathways, one caspase-independent and the other caspase-dependent. This study clarifies the precise mechanism of anti-HLA antibody-induced apoptosis which might have clinical implications.

Apoptosis-mediated selective killing of malignant cells by cardiac steroids: maintenance of cytotoxicity and loss of cardiac activity of chemically modified derivatives.

Cardiac glycosides are commonly used drugs in clinical medicine. We analyzed the cytotoxic effect of six steroids belonging to the bufadienolide family on malignant T lymphoblasts and normal peripheral blood mononuclear cells (PBMC). One compound was a natural bufadienolide glycoside (hellebrin) with cardiac activity. The other five compounds were chemically modified derivatives that did not contain cardioactive groups. We found that these steroids were able to cause time-dependent apoptosis in Jurkat T lymphoblasts, whereas they only minimally affected PBMC. Preferential killing of malignant cells was induced by the natural cardioactive substance hellebrin and by three of the five chemically modified non-cardioactive derivatives. The substances caused mitochondrial transmembrane potential disruption and internucleosomal DNA fragmentation in tumor cells. The cytoplasmic and nuclear events of bufadienolide-induced apoptosis were strongly inhibited in the presence of caspase 8, caspase 9, or caspase 3 inhibitors, as well as in the presence of the broad-spectrum caspase inhibitor Z-VAD-FMK. Overexpression of Bcl-2 significantly protected bufadienolide-treated cells from phosphatidylserine translocation, transmembrane potential disruption, and internucleosomal DNA fragmentation. Our results show that the analyzed bufadienolide derivatives preferentially kill malignant human lymphoblasts by initiating apoptosis via the classical caspase-dependent pathway. Apoptosis-inducing agents specific for tumor cells might be ideal anti-tumor drugs. The therapeutic use of bufadienolides has been hampered by their concomitant cardiac activity. The description of compounds without cardiac activity but with tumor-specific cytotoxicity suggests the potential of using them in cancer therapy.

Induction of apoptosis in human lymphocytes by human anti-HLA class I antibodies.

BACKGROUND: Ligation of MHC class I molecules expressed on T cells leads to both growth arrest and apoptosis. Although mouse anti-HLA class I monoclonal antibodies (mAbs) have been previously shown to cause apoptosis of human cells, an apoptosis-inducing capacity of human anti-HLA class I antibodies on human lymphocytes has not been reported. Because this is of potential clinical relevance, we tested human anti-HLA class mAbs for their capacity to induce apoptosis. METHODS: Twenty-five human HLA class mAbs and mouse mAb W6/32 were tested on resting human peripheral blood mononuclear cells or Jurkat lymphoblastic T cells using the Annexin-V binding and immunobinding assays. RESULTS: We demonstrated that HLA class I human mAbs are able to induce apoptosis as early as 1 hr after treatment of resting human peripheral blood mononuclear cells or Jurkat lymphoblastic T cells. The apoptosis-inducing effect and the binding of anti-HLA mAbs to the cells were strongly increased when lymphoblasts were prestimulated with cytokines, such as interferon-gamma and interleukin-2. Induction of apoptosis increased with the dosage and binding of anti-HLA mAbs. Caspase inhibitors did not affect apoptosis induced by MHC class I ligation. CONCLUSIONS: Our study showed for the first time that human HLA class I mAbs induce rapid and strong apoptosis in resting human peripheral blood mononuclear cells and Jurkat T lymphoblasts.