CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (⌬⌿m). Using CD47 as bait in a yeast twohybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRP␣1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis.

Extracellular signal-regulated kinase (ERK) activation is important for both thymocyte development and T cell function. Classically, signal transduction from the T cell antigen receptor (TCR) to ERK is thought to be regulated by signaling from Ras guanine nucleotide exchange factors (GEFs), through the small G protein Ras, to the three-tiered Raf–MAPK/ERK kinase (MEK)–ERK kinase cascade. Developing and mature T cells express four members of two RasGEF families, RasGRP1, RasGRP4, son of sevenless 1 (Sos1), and Sos2, and several models describing combined signaling from these RasGEFs have been proposed. However, recent studies suggest that existing models need revision to include both distinct and overlapping roles of multiple RasGEFs during thymocyte development and novel, Ras-independent signals to ERK that have been identified in peripheral T cells.

CD99, a unique integral membrane protein present on the surface of all human T cells, has previously been shown to regulate cell function and fate. In peripheral T cells, it triggers immediate activation of alpha4b1 integrin and cell arrest on inflamed vascular endothelium, whereas it mediates an apoptotic signal in double-positive thymocytes undergoing the selection process. Two isoforms of CD99 exist, a long form corresponding to the full-length protein and a short form harboring a deletion in the intracytoplasmic segment. Here, we show that while peripheral T cells display exclusive expression of the long form, double-positive thymocytes express both isoforms. Moreover, differential expression of these two CD99 molecules can lead to distinct functional outcomes. Expression of the long form in a CD99-deficient Jurkat T cell line is sufficient to promote CD99-induced cell adhesion, whereas coexpression of the two isoforms is required to trigger T-cell death. When coexpressed, the two proteins form covalent heterodimers, which locate within glycosphingolipidic rafts and induce sphingomyelin degradation. Cholesterol depletion experiments show that this localization is required for the induction of apoptosis. Thus, the surface expression pattern of CD99 isoforms determines T-cell functional outcomes.

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