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.

Forkhead family transcription factors are critical regulators of cell cycle progression and apoptosis in hematopoietic cells. Here, we show that FOXO3a (also known as FKHRL1) is a new substrate of caspase-3-like proteases during apoptosis in T lymphocytes. FOXO3a was cleaved in vivo by caspases in leukemic Jurkat cells following engagement of Fas (CD95) receptor, staurosporine, and etoposide treatment, but not following engagement of CD99, a caspase-independent cell death inducer. Caspasemediated cleavage of FOXO3a was also observed in CD4 þ peripheral T cells subjected to activation-induced cell death. The expression of the death adapter FADD and caspase-8 was required for Fas-induced FOXO3a cleavage, but activation of survival pathways by overexpression of FLICE-inhibitory protein or phorbol myristate acetate treatment prevented it. FOXO3a was cleaved in vitro by caspase-3-like proteases at the consensus sequence DELD 304 A, releasing the N-terminal DNAbinding domain of FOXO3a from its C-terminal transactivating domain. Whereas full-length FOXO3a enhanced Forkhead response element-dependent transcription and apoptosis in Jurkat cells, both fragments were inactive to promote gene activation and cell death. In contrast, a caspase-resistant FOXO3a mutant exhibited enhanced transcriptional and proapoptotic activities. Together, these results indicate that the proteolytic cleavage of FOXO3a by caspases may represent a novel regulatory mechanism of FOXO3a activity during death receptors signaling.

CD99/E2 is an integral transmembrane protein which forms, together with Xga, a distinct family whose genes are located in the pseudoautosomal region. The number of T cells that firmly bound to vascular endothelial cells under physiological shear stress increased 2–14‐fold upon CD99 stimulation, and bound cells became much more resistant to detachment forces and spread. T cell arrest occurred within 1 min and was dependent on the α4β1‐VCAM‐1 pathway. In contrast, the αLβ2‐ICAM‐1 pathway remained unactivated. This was observed with T cell lines and with activated peripheral blood lymphocytes, and was limited within the resting peripheral CD4+ T cells to the memory subset, while virgin cells were unaffected. This discloses a stepwise regulation of the T cell extravasation cascade.