Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

TCRαβ thymocytes differentiate to either CD8αβ cytotoxic T lymphocytes or CD4+ T helper cells. This functional dichotomy is controlled by key transcription factors, including the T helper master regulator, ThPOK, which suppresses the cytolytic program in MHC class II-restricted CD4+ thymocytes. ThPOK continues to repress CD8-lineage genes in mature CD4+ T cells, even as they differentiate to T helper effector subsets. Here we show that the T helper-fate was not fixed and that mature antigen-stimulated CD4+ T cells could terminate Thpok expression and reactivate CD8-lineage genes. This unexpected plasticity resulted in the post-thymic termination of the T helper-program and the functional differentiation of distinct MHC class II-restricted CD4+ cytotoxic T lymphocytes.

The thymus leukemia antigen (TL) is a nonclassical class I molecule, expressed abundantly on intestinal epithelial cells. We show that, in contrast to other major histocompatibility complex (MHC) class I molecules that bind CD8alphabeta, TL preferentially binds the homotypic form of CD8alpha (CD8alphaalpha). Thus, TL tetramers react specifically to CD8alphaalpha-expressing cells, including most intestinal intraepithelial lymphocytes. Compared with CD8alphabeta, which recognizes the same MHC as the T cell receptor (TCR) and thus acts as a TCR coreceptor, high-affinity binding of CD8alphaalpha to TL modifies responses mediated by TCR recognition of antigen presented by distinct MHC molecules. These findings define a novel mechanism of lymphocyte regulation through CD8alphaalpha and MHC class I.

In humans suffering from dialysis-related amyloidosis, the protein ␤2-microglobulin (␤2M) is deposited as an amyloid; however, an amyloid of ␤2M is unknown in mice. ␤2M sequences from human and mouse are 70% identical, but there is a seven-residue peptide in which six residues differ. This peptide from human ␤2M forms amyloid in vitro, whereas the mouse peptide does not. Substitution of the human peptide for its counterpart in the mouse sequence results in the formation of amyloid in vitro. These results show that a seven-residue segment of human ␤2M is sufficient to convert ␤2M to the amyloid state, and that specific residue interactions are crucial to the conversion. These observations are consistent with a proposed Zipper-spine model for ␤2M amyloid, in which the spine of the fibril consists of an anhydrous ␤-sheet. More than 20 proteins have been found to aggregate into amyloids, elongated unbranched fibrils that bind the aromatic dyes Congo red and ThioflavinT (ThT) and have a common cross ␤ x-ray diffraction pattern (1, 2). The proteins that form amyloids differ in size, function, sequence, and native structure, but all form aggregates similar in structure and properties (3-5). It has long been recognized from the cross-␤ diffraction pattern that amyloids are formed from ␤-sheets Ϸ10-12 Å apart, each made up of extended strands stacked Ϸ4.7 Å apart (6, 7). There is evidence that in some amyloids, the ␤-strands run parallel to each other (8-10), and in others they may run antiparallel (11,12).Some models for amyloid structure depict the entire native protein as refolding into the amyloid (13-16); we term these Entire-refolding models. Other models depict the interactions of amyloid to be formed from only a small segment of the protein, with the rest retaining a native-like structure (17)(18)(19)(20). Entirerefolding models are based in part on the idea that amyloid formation is an inexorable tendency of all proteins, and that variations in rate of achieving the amyloid state are mainly a matter of amino acid composition (21). In contrast, models that depict amyloid formation as having its basis in a ''gain of interaction'' (18) focus on the formation of a new intermolecular bond contributed by a segment of the entire protein. The formation of these intermolecular bonds would in principle depend on the amino acid sequence, not just the composition. In this paper, we focus on a particular gain-of-interaction model, called the Zipper-spine model, in which the new interaction is a spine of ␤-sheet (17).One of the most intensively studied amyloid-forming proteins is ␤2-microglobulin (␤2M), a normally soluble protein that aggregates into pathogenic fibrils either at low pH (22) or under physiological conditions when divalent copper is present (23). The Entire-refolding view of amyloid depicts dialysis-related amyloidosis pathogenesis as destabilization of the native structure of ␤2M followed by formation of a nucleating ␤2M species that forms amyloid fibrils (24-26). However, there is accumulating evidence that s...

The immune system preserves and makes use of autoreactive lymphocytes with specialized functions. Here we showed that one of these populations, CD8alphaalpha(+)TCRalphabeta(+) intestinal intraepithelial lymphocytes (IELs), arose from a unique subset of double-positive thymocytes. This subset of cells was precommitted to preferentially give rise to CD8alphaalpha(+)TCRalphabeta(+) IELs, but they required exposure to self-agonist peptides. The agonist-selected TCRalphabeta(+) thymocytes are CD4 and CD8 double-negative, and their final maturation, including the induction of CD8alphaalpha expression, appeared to occur only after thymus export in the IL-15-rich environment of the gut. These developmental steps, including precommitment of immature thymocytes, TCR-mediated agonist selection, and postthymic differentiation promoted by cytokines, define a unique pathway for the generation of CD8alphaalpha(+)TCRalphabeta(+) IEL.

Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric alpha chains of the CD8 molecule (CD8alphaalpha) are transiently induced on a selected subset of CD8alphabeta+ T cells upon antigenic stimulation. These CD8alphaalpha molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8alphaalpha.

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Death receptors belonging to the tumor necrosis factor receptor family (e.g., Fas/CD95, TNFR1, TRAIL-R1, TRAIL-R2, TRAMP/DR3, and DR6) play an important role in the regulation of lymphocyte homeostasis (35,42,44). Upon encounter of specific antigenic peptides presented by antigenpresenting cells, T cells become activated and enter the cell cycle. Concomitantly with proliferation, T cells differentiate into effector cells that are either cytolytic or able to provide help to B lymphocytes. Once the effector cells have successfully performed their prescribed function, the expanded pool of antigen-specific T-cell clones needs to be reduced to its original size. Both for the effector function of cytolytic T cells and in the elimination of expanded T cells, cell death signals transmitted by Fas/CD95 are of key importance (30). This is underscored by the observation that mice and humans lacking a functional Fas receptor or Fas ligand develop a lymphoproliferative disease and systemic autoimmunity accompanied by the production of autoantibodies (12,40,52,59).As with all death receptors, the prototypic death receptor Fas/CD95 contains within its cytoplasmic tail a 60-amino-acid death domain (DD) motif (35,44). Upon activation of Fas by its ligand, the DD undergoes homotypic interaction with a DD in the adaptor protein FADD, which then recruits the initiator caspase 8 via their mutual N-terminal death effector domains (DED) (3). A high local concentration of caspase 8 zymogens is thought to facilitate self-processing and cleavage to the active enzyme (34). Activated caspase 8 then initiates apoptosis by cleavage of the downstream effector caspases 3, 6, and 7 (10).A number of gammaherpesviruses and molluscipoxviruses encode a molecule termed FLIP (FLICE inhibitory protein) that can inhibit FasL-induced cell death (5, 53). v-FLIP resembles caspase 8 in containing two DED but lacks the enzymatic C-terminal portion. As such, v-FLIP can be recruited into the death-inducing signaling complex (DISC) of Fas, thereby competing with recruitment of caspase 8 to FADD. In this manner, v-FLIP may function to promote viral persistence and dissemination by inhibiting death receptor-mediated elimination of infected cells (56). A mammalian cellular homologue (c-FLIP) has been described that exists in at least two splice variants, c-FLIP S and c-FLIP L (15, 16, 20-22, 38, 47, 49). Like v-FLIP, the 26-kDa c-FLIP S has two DED and functions in a similar manner to inhibit death receptor-induced apoptosis (22). The full-length 55-kDa form of c-FLIP (c-FLIP L ) shows overall structural homology to caspase 8. It contains two DED that interact with FADD but bears a mutation in the caspase-like domain that renders it enzymatically inactive. Following Fas ligation, both c-FLIP L and caspase 8 are recruited into the DISC and are subsequently partially cleaved. The affinity for FADD of the c-FLIP L /caspase 8 heterodimer appears to be considerably greater than that of the caspase 8 homodimer; therefore, the ratio of c-FLIP L to caspase 8 is critical ...