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CARMA1 (originally called CARD11) is a membrane-associated guanylate kinase family member that is required for T cell receptor (TCR)-induced NF-κB activation in T cell leukemia lines [1–5]. It uses its N-terminal caspase activation and recruitment domain (CARD) to interact with the CARD in the downstream adaptor Bcl-10. We show that primary B and T lymphocytes from knock-in mice expressing only a CARDless form of CARMA1 (ΔCARD) are defective at mitogen-induced NF-κB activation and fail to proliferate. CARMA1 mutant mice exhibited normal T but impaired B cell development; CD5 + peritoneal B cells were absent, and serum immunoglobulin levels were markedly reduced. A lacZ reporter gene knocked into the CARMA1 locus confirmed lymphocyte-specific expression of CARMA1. Thus, CARMA1 has an essential role in mediating B and T lymphocyte proliferation and requires its CARD to engage downstream signaling components.

BLyS (also called BAFF, TALL-1, THANK, and zTNF4), a TNF superfamily member, binds two receptors, TACI and BCMA, and regulates humoral immune responses [1–7]. These two receptors also bind APRIL [7–10], another TNF superfamily member. The results from TACI −/− and BCMA −/− mice suggest the existence of additional receptor(s) for BLyS. The TACI knockout gives the paradoxical result of B cells being hyperresponsive, suggesting an inhibitory role for this receptor [11, 12], while BCMA null mice have no discernable phenotype [13]. Here we report the identification of a third BLyS receptor (BR3; BLyS receptor 3). This receptor is unique in that, in contrast to TACI and BCMA, BR3 only binds BLyS. Treatment of antigen-challenged mice with BR3-Fc inhibited antibody production, indicating an essential role for BLyS, but not APRIL, in this response. A critical role for BR3 in B cell ontogeny is underscored by our data showing that the BR3 gene had been inactivated by a discrete, approximately 4.7 kb gene insertion event that disrupted the 3′ end of the BR3 gene in A/WySnJ mice, which lack peripheral B cells.

BLyS (also called TALL-1, THANK, or BAFF) [1] [2] [3] [4] is a member of the tumor necrosis factor (TNF) gene family that stimulates proliferation and immunoglobulin production by B cells. BLyS interacts with the TNF receptor (TNFR) homologue TACI (transmembrane activator and CAML-interactor) [5], and treatment of mice with a TACI-Fc fusion protein abolishes germinal center formation after antigenic challenge [6]. Here we report a novel interaction between BLyS and another TNFR homologue, BCMA (B cell maturation antigen) [7] [8]. Further, the TNF homologue APRIL [9], a close relative of BLyS, also bound to BCMA and TACI. BLyS or APRIL activated nuclear factor-kappaB (NF-kappaB) through TACI and BCMA, and each ligand stimulated immunoglobulin M (IgM) production by peripheral blood B cells. These results define a dual ligand-receptor system that may play an important role in humoral immunity.

Apoptosis, or programmed cell death, is the physiological process whereby individual cells are deliberately eliminated to achieve homeostasis and proper metazoan development. Numerous genes have recently been identified that are involved in apoptosis: some are believed to encode death effectors, whereas others encode positive or negative regulators of the cell-death machine. Precisely how these various proteins interact in the molecular mechanism of apoptosis remains to be discovered.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells provide immune surveillance against viruses and neoplasms, and play a central role in the pathogenesis of autoimmune disease, AIDS and graft rejection [1,2]. Thus, it is important to understand the precise molecular mechanism(s) whereby cytotoxic lymphocytes destroy susceptible target cells. Granule-mediated cytotoxicity requires a combination of both perforin and granzyme B [3]. Perforin polymerizes to form transmembrane channels and presumably allows granzyme B access to target cell substrates, which until recently, were unknown. One clue to the identity of the physiological substrate(s) activated by granzyme B comes from its unusual specificity for cleaving synthetic substrates after aspartate residues [4]. Members of the ICE/CED-3 family of cysteine proteases are prime candidates as they are important apoptotic effectors [5,6] and are expressed as zymogens, which can be processed to form active heterodimeric enzymes after cleavage at specific aspartate residues. Previous studies have shown that granzyme B proteolytically activates the cell death effector Yama/CPP32/apopain [7,8] (referred to here as Yama). Here we report that granzyme B also activates ICE-LAP3/Mch3/CMH-1 (referred to here as ICE-LAP3), which, along with Yama and Mch2, forms a subset of the ICE/CED-3 family of cysteine proteases most closely related to the Caenorhabditis elegans cell death gene, CED-3 [6,9]. Importantly, Jurkat T cells incubated with granzyme B and a sublytic concentration of perforin undergo apoptosis, which is preceded by the activation of endogenous ICE-LAP3. Thus, we propose that granzyme B mediates apoptosis by directly engaging the target cell's death effector machinery, which is probably composed of an arsenal of intracellular, CED-3-like cysteine proteases.

The Src-like adaptor protein (Slap) is a recently identified adaptor protein containing Src homology 3 (SH3) and SH2 domains. Slap is found in a wide range of cell types and was shown to interact with the Eck receptor tyrosine kinase in a yeast two-hybrid interaction screen [1]. Here, we found that Slap is expressed in NIH3T3 cells and could associate with the activated platelet-derived growth factor (PDGF) receptor. Using mutated versions of the PDGF receptor and phosphopeptide competition experiments, we determined that Slap has the highest affinity for the Src-binding site of the PDGF receptor. Our inability to produce cell lines that stably expressed Slap suggested that Slap inhibited cell growth. We further investigated this issue by transiently expressing Slap by microinjection. Overexpression of Slap by this method inhibited DNA synthesis induced by PDGF and serum, whereas overexpression of the adaptor proteins Grb2 and Shc did not. Finally, microinjection of a Slap antibody into NIH3T3 cells that had been stimulated with suboptimal doses of growth factors potentiated the effects of the growth factors. These data suggest that, unlike other adaptor proteins, Slap is a negative regulator of signalling initiated by growth factors.

Hypohydrotic Ectodermal Dysplasia (HED) is a genetic disease seen in humans and mice. It is characterized by loss of hair, sweat glands, and teeth. The predominant X-linked form results from mutations in ectodysplasin-A (EDA), a TNF-like ligand [1–4]. A phenotypically indistinguishable autosomal form of the disease results from mutations in the receptor for EDA (EDAR) [4, 5]. EDAR is a NF-κB-activating, death domain-containing member of the TNF receptor family [6–8]. crinkled, a distinct autosomal form of HED, was discovered in a mouse strain in which both the ligand (EDA) and receptor (EDAR) were wild-type, suggestive of a disruption further downstream in the signaling pathway [9, 10]. Employing a forward genetic approach, we have cloned crinkled (CR) and find it to encode a novel death domain-containing adaptor. crinkled binds EDAR through a homotypic death domain interaction and mediates engagement of the NF-κB pathway, possibly by recruiting TRAF2 to the receptor-signaling complex. This is an unprecedented example of naturally occurring mutations in ligand, receptor, or adaptor giving rise to the same phenotypic disease characterized by a defect in the proper development of epidermal appendages.