c-FLIP efficiently rescues TRAF-2-/- cells from TNF-induced apoptosis.

For the past 20 years, it has been known that preparations of Tumor Necrosis Factor alpha (TNF) fail to induce apoptosis due to cytoprotective responses that render cells resistant to its cytotoxic activity. Here we show that TRAF-2-/- embryonic fibroblasts express reduced levels of the anti-apoptotic molecule c-FLIP due to extensive degradation of the protein. Reconstitution of TRAF-2-/- EF with c-FLIP is sufficient for resistance to TNF toxicity. Our results strengthen the role of c-FLIP in protecting cells from the cytotoxic effect of TNF and have implication for the treatment of inflammatory and proliferative disorders.

Caspase recruitment domain (CARD)-dependent cytoplasmic filaments mediate bcl10-induced NF-kappaB activation.

Mucosa-associated lymphoid tissue (MALT) lymphomas are associated with overexpression and constitutive activity of bcl10, a caspase recruitment domain (CARD)-containing protein that activates NF-kappaB. Here, we show that arrangement of overexpressed bcl10 protein in cytoplasmic filaments is essential for recruitment of signal transducer molecules-involved NF-kappaB activation. We also show that cytoskeleton elements regulate bcl10 signaling.Thus, organized assemblage of proteins in ordered structures linked to the cytoskeleton network may represent a general mechanism for intracellular signaling.

c-E10 is a caspase-recruiting domain-containing protein that interacts with components of death receptors signaling pathway and activates nuclear factor-kappaB.

Members of the tumor necrosis factor receptor superfamily induce apoptosis via interaction with FADD and regulate cell growth and differentiation through TRADD and TRAFs molecules. While screening for molecules involved in the regulation of death receptor signaling, we identified a novel protein, c-E10. c-E10 contains an amino-terminal caspase-recruiting domain (CARD) and shares a sequence homologous with E10, a viral CARD-containing protein that binds to c-E10. In transfection experiments c-E10 oligomerizes, binds to the cytoplasmic portion of TRAIL receptor 1 (DR4) and coprecipitates with TRADD. Expression of c-E10 under the control of a doxycycline-dependent transcriptional transactivator results in NF-kappaB activation, which is inhibited by dominant negative forms of TRAF2 and NIK kinase. Thus, our results suggest that c-E10 is an adapter protein that activates NF-kappaB through a molecular pathway involved in death receptor signaling.

Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction.

ALG-2 is a 22-kDa calcium-binding protein necessary for cell death induced by different stimuli in 3DO T-cell hybridoma. 3DO cell clones depleted of ALG-2 protein exhibit normal caspases activation, suggesting that ALG-2 function is required downstream or is independent of caspase proteases activity for apoptosis to occur. Using the yeast two-hybrid screening system, we have isolated and characterized the mouse cDNA encoding for ALG-2 interacting protein 1 (AIP1), a novel protein that interacts with ALG-2. ALG-2 and AIP1 colocalize in the cytosol and the presence of calcium is an indispensable requisite for their association. Sequence alignment shows that AIP1 is highly similar to BRO1, a yeast protein related to components of the Pkc1p-MAP kinase cascade. Overexpression of a truncated form of AIP1 protects two different cell types from death induced by trophic factors withdrawal; thus, our data indicate that AIP1 cooperates with ALG-2 in executing the calcium-dependent requirements along the cell death pathway.

Cross-linking CTX, a novel thymocyte-specific molecule, inhibits the growth of lymphoid tumor cells in Xenopus.

CTX, a new Xenopus Ig superfamily molecule present on some cortical thymocytes and lymphoid tumor cells, is expressed at the cell surface under six differently glycosylated isoforms as shown by two-dimensional gel analysis and by endo F glycosidase treatment. Following chemical cross-linking before immunoprecipitation, a large fraction of surface CTX forms non-covalently linked dimers at the cell surface. This finding, which is consistent with the presence of a J segment with diglycine beta bulge in the V region of the molecule, suggests that this dimer has the same conformation as a T-cell receptor (TCR) or an Ig molecule. The V8 digest patterns of the monomers and dimers are identical. While this suggests that the dimer is a homodimer of two CTX chains, it does not distinguish whether each CTX chain is encoded by the same or different gene loci. When tumor cells were added to culture wells that had been coated with the anti-CTX monoclonal antibody X71, 30-50% underwent rapid (within 30 min) morphological changes followed by growth inhibition as determined by a decrease in thymidine incorporation and by direct cell counting. No apoptosis, calcium flux or external calcium requirement was noted after cross-linking of CTX. These results suggest that CTX can function as a receptor, and that its interaction with a ligand influences the control of cell proliferation through a signalling pathway that is distinct from the TCR machinery.

Effects of thymectomy and tolerance induction on tumor immunity in adult Xenopus laevis.

Major-histocompatibility-complex homozygous partially inbred adults of the ff strain of Xenopus reject transplants of tumor cells of ff strain origin; ff tadpoles do not. Thymectomy, performed 5 days after fertilization, abrogated the adult tumor-rejection response suggesting that in this system tumor rejection is immunologically mediated by T cells. Thymectomy later in larval life did not alter tumor rejection, but it did reduce T-cell numbers. Tolerance to minor-histocompatibility(H) antigens segregating within the ff family, which was induced by grafting adult skin to metamorphosing larvae, did not affect the tumor-rejection capacity of the tolerant adult hosts. This suggests that the ff-2 tumor expresses (a) tumor-specific antigen(s). Immunization of larvae with tumor cells did not induce tolerance to skin grafts transplanted during adult life. Indeed, such grafts were rejected in accelerated fashion, suggesting that memory cells generated in the larvae persist through metamorphosis.

Ontogeny of the alloimmune response against a transplanted tumor in Xenopus laevis.

Xenopus laevis lymphoid tumor cells of the ff genotype grow after transplantation in inbred ff tadpoles or young post-metamorphic animals, but do not grow in fully grown ff adults. The ability to grow is lost progressively after metamorphosis and is apparently due to an immune response of the adult host against minor histocompatibility antigens (non-MHC encoded) expressed by the tumor cells. The difference in alloimmune responses between the larval and the adult immune system of the amphibian Xenopus has been subsequently investigated with this new in vivo model. The resistance of the host against transplanted tumor cells rises during the post-metamorphic development in parallel with the second histogenesis observed in the thymus, the expression of MHC class II by peripheral T cells and the recovery of T cell effector functions such as MLR, and can be abrogated by sub-lethal irradiation. Pre-immunization of ff adults with irradiated ff-2 cells specifically accelerates subsequent ff skin graft rejection, which implies the generation of memory against antigenic determinants common between the ff skin and the tumor cells. Similarly, both anti-ff alloserum and anti-ff-2 serum contain antibodies specifically precipitating two surface proteins (180-200 kDa) from ff-2 cells. One of these proteins is also detected on normal ff thymocytes and splenic T cells. On the other hands, ff-2 tumor cells (MHC I+II-) are not rejected by class I-negative tadpoles (class I expression on the tumor cell surface is even increased), and no anti-tumor antibody response can be detected. However, tumor growth has been reduced in tadpoles following priming with irradiated ff-2 cells, although immunization is not sufficient to prevent ultimate tumor development and tadpole death. Moreover, priming with irradiated ff-2 cells at larval stages does interfere with tumor growth in transplanted young post-metamorphic adults, suggesting that long-lived memory has been generated and has been maintained through metamorphosis. These results suggest that the lack of tumor rejection by larvae results from an incomplete effector function rather than an absence of recognition. Full responsiveness against minor H antigens cannot be elicited before adulthood.

Lymphoid tumors of Xenopus laevis with different capacities for growth in larvae and adults.

Three new lymphoid tumors offering an assortment of variants in terms of MHC class I expressions, MHC class II expression, and Ig gene transcription have been discovered in the amphibian Xenopus. One was developed in an individual of the isogenic LG15 clone (LG15/0), one in a frog of the LG15/40 clone (derived from a small egg recombinant of LG15), and one (ff-2) in a male ff sib of the individual in which MAR1, the first lymphoid tumor in Xenopus was found 2 years ago. These tumors developed primarily as thymus outgrowths and were transplantable in histocompatible tadpoles but not in nonhistocompatible hosts. Whereas LG15/0 and LG15/40 tumor cells also grow in adult LG15 frogs, the ff-2 tumor, like the MAR1 cell line, is rejected by adult ff animals. Using flow cytometry with fluorescence-labeled antibodies and immunoprecipitation analysis, we could demonstrate that, like MAR1, these three new tumors express on their cell surface lymphopoietic markers recognized by mAbs F1F6 and RC47, as well as T-cell lineage markers recognized by mAbs AM22 (CD8-like) and X21.2, but not by immunologobulin (Ig) nor MHC class II molecules. Another lymphocyte-specific marker AM15 is expressed by 15/0 and 15/40 but not ff-2 tumor cells. The ff-2 tumor cell expresses MHC class I molecule in association with beta 2-microglobulin on the surface, 15/40 cells contain cytoplasmic class I alpha chain that is barely detected at the cell surface by fluocytometry, and 15/0 cells do not synthesize class I alpha chain at all. The three new tumors all produce large amounts of IgM mRNA of two different sizes but no Ig protein on the membrane nor in the cytoplasm. All tumor cell types synthesize large amount of Myc mRNA and MHC class I-like transcripts considered to be non classical.

B lymphocytes from the autoimmune-prone mouse strain MLR/lpr manifest an intrinsic defect in tetraparental MRL/lpr in equilibrium DBA/2 chimeras.

Data are presented showing that MRL/lpr in equilibrium DBA/2 tetraparental (allophenic) chimeras, unlike conventional lpr/lpr----+/lpr bone marrow chimeras, fail to develop graft-vs-host disease; instead they develop full-blown lymphoproliferation and autoantibody formation typical of unmanipulated MRL/lpr mice. The increase in the splenic and especially the lymph node mass is comprised predominantly of MRL/lpr-derived cells and all of the serum IgG2a is MRL/lpr derived. This dominance of MRL/lpr lymphoid activity occurred even in chimeras where greater than 90% of the skin and/or bone marrow cells were of the DBA/2 type. These results demonstrate the failure of the lpr environment to recruit normal B and T cells into the autoimmune process, the inability of normal cells to suppress MRL/lpr disease, and indicate further that the lpr mutation has an intrinsic effect on lymphocytes of both the B and T lineages.