The nonpolymorphic soluble HLA-G1 (sHLA-G1) isoform has been reported to be secreted by trophoblast cells at the materno-fetal interface, suggesting that it may act as immunomodulator during pregnancy. In this paper, we report that affinity-purified β₂-microglobulin-associated sHLA-G1 triggered apoptosis in activated, but not resting CD8⁺ peripheral blood cells. We demonstrate by Western blotting that sHLA-G1 enhanced CD95 ligand expression in activated CD8⁺ cells. Cytotoxicity was inhibited by preincubation of the cells with a CD95 antagonist mAb (ZB4) or a soluble recombinant CD95-Fc, indicating that apoptosis is mediated through the CD95/CD95 ligand pathway. Finally, we show that such sHLA-G1-induced apoptosis depends on the interaction with CD8 molecules, with cell death being blocked by various CD8 mAbs.

The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.

Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90° light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP‐16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis.

HLA-G is a major histocompatibility complex class Ib molecule whose constitutive tissue distribution is restricted mainly to trophoblast cells at the maternal-fetal interface during pregnancy. In this study, we demonstrated the ability of the soluble HLA-G1 (sHLA-G1) isoform to inhibit fibroblast growth factor-2 (FGF2)-induced capillary-like tubule formation. Using a rabbit corneal neovascularization model, we further showed that sHLA-G1 inhibits FGF2-induced angiogenesis in vivo. We also demonstrated that sHLA-G1 induces endothelial cell apoptosis through binding to BY55/CD160, a glycosylphosphatidylinositolanchored receptor expressed by endothelial cells. Furthermore, we showed that the specific CL1-R2 anti-CD160 monoclonal antibody mimics sHLA-G1-mediated inhibition of endothelial cell tube formation and induction of apoptosis. Thus, the engagement of CD160 in endothelial cells may be essential for the inhibition of angiogenesis. sHLA-G1/CD160-mediated antiangiogenic property may participate in the vascular remodeling of maternal spiral arteries during pregnancy, and, given that we found that CD160 is strongly expressed in the vasculature of a murine tumor, it offers an attractive therapeutic target for preventing pathologic neovascularization.