CD4 mAb induced apoptosis of peripheral T cells: multiparameter subpopulation analysis by flow cytometry using Attractors.

Studies describing the induction of apoptosis for CD4 mAbs do not delineate between epitope-dependent and Fc-driven epitope cross-linking induced cell death. Keliximab and clenoliximab are two CD4 mAbs that differ only in their heavy chain isotypes, being an IgG1 and a modified IgG4, respectively. These antibodies suppress CD4 T cell responses in vitro and in vivo and have been in human clinical trials for the treatment of RA and asthma. Here we compared the apoptotic activity of these mAbs to differentiate between the contributions of epitope-dependent vs. Fc-driven epitope cross-linking induced cell death in vitro as a link to differential CD4 cell depletion in vivo. We developed a simple flow cytometry procedure that measures apoptosis within intact and compromised subpopulations of PBMCs within a few hours of culture. Attractors software was used to quantitate the percentage of apoptotic CD4 T cells, which generate reactive oxygen species (ROS), express external phosphatidyl serine (PS) and cleaved fluorescein diacetate (FDA), within the intact and compromised lymphocyte populations. Treatment of freshly isolated PBMCs with keliximab resulted in the appearance of characteristic apoptotic condensed CD4 T cells that contained reactive oxygen species, were annexin V positive and had intact esterase activity. Apoptosis was evident within 3 h and continued throughout the 72-h culture period. In contrast, clenoliximab alone did not induce apoptosis. The use of multiparameter flow cytometry and Attractors to analyze subpopulations based on scatter properties and biochemical processes during apoptosis provides a sensitive assay in which to quantitate and characterize the induction of cell death. Depletion of CD4 T cells in vivo by keliximab may reflect, in part, antibody-mediated apoptosis of these cells that is dependent on Fcgamma receptors.

Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth.

Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.

Antibodies to TR2 (herpesvirus entry mediator), a new member of the TNF receptor superfamily, block T cell proliferation, expression of activation markers, and production of cytokines.

TR2 (TNFR-related 2) is a recently identified member of the TNFR family with homology to TNFRII. We have demonstrated previously that TR2 mRNA is expressed in resting and activated human T cells and that TR2-Ig partially inhibits an allogeneic mixed leukocyte proliferation response. We now characterize TR2 further by the use of specific mAbs. Flow-cytometry analysis using TR2 mAbs confirmed that resting PBL express high levels of cell surface TR2, and that TR2 is widely expressed on all freshly isolated lymphocyte subpopulations. However, stimulation of purified T cells with either PHA or PHA plus PMA resulted in decreased surface expression within 48 h of activation before returning to resting levels at 72 h. TR2 mAbs inhibited CD4+ T cell proliferation in response to stimulation by immobilized CD3 or CD3 plus CD28 mAbs. Assay of culture supernatants by ELISA showed inhibition of TNF-alpha, IFN-gamma, IL-2, and IL-4 production, which, for IL-2 and TNF-alpha was also confirmed by intracellular cytokine staining. Furthermore, expression of activation markers on CD4+ T cells, including CD25, CD30, CD69, CD71, and OX40 (CD134), was inhibited. TR2 mAbs inhibited proliferation in a three-way MLR, and a response to soluble recall Ag, tetanus toxoid. In conclusion, these results suggest that TR2 is involved in the activation cascade of T cell responses and TR2 mAbs prevent optimal T cell proliferation, cytokine production, and expression of activation markers.

Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL.

TRAIL is a tumor necrosis factor-related ligand that induces apoptosis upon binding to its death domain-containing receptors, DR4 and DR5. Two additional TRAIL receptors, TRID/DcR1 and DcR2, lack functional death domains and function as decoy receptors for TRAIL. We have identified a fifth TRAIL receptor, namely osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homologue that inhibits osteoclastogenesis and increases bone density in vivo. OPG-Fc binds TRAIL with an affinity of 3.0 nM, which is slightly weaker than the interaction of TRID-Fc or DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apoptosis of Jurkat cells. Conversely, TRAIL blocks the anti-osteoclastogenic activity of OPG. These data suggest potential cross-regulatory mechanisms by OPG and TRAIL.