TRAF4 functions as an intermediate of GITR-induced NF-kappaB activation.

Members of the tumor necrosis factor receptor (TNFR) family regulate the activation, differentiation, and function of many cell types, including cells of the immune system. TNFR-associated factors (TRAFs) function as adapter molecules controlling signaling pathways triggered by TNFR family members, such as activation of nuclear factor kappaB (NF-kappaB). Despite intensive research, the function of TRAF4 in signaling pathways triggered by TNFR-related proteins remains enigmatic. Intriguingly, our functional studies indicated that TRAF4 augments NF-kappaB activation triggered by glucocorticoid-induced TNFR (GITR), a receptor expressed on T cells, B cells, and macrophages. Further analyses revealed that TRAF4-mediated NF-kappaB activation downstream of GITR depends on a previously mapped TRAF-binding site in the cytoplasmic domain of the receptor and is inhibited by the cytoplasmic protein A20. GITR is thought to inhibit the suppressive function of regulatory T cells (Treg cells) and to promote activation of T cells. Taken together, our studies provide the first indications that TRAF4 elaborates GITR signaling and suggest that TRAF4 can modulate the suppressive functions of Treg cells.

Reactive oxygen species are downstream products of TRAF-mediated signal transduction.

Members of the TNFR (tumor necrosis factor receptor) superfamily are involved in regulating activation and differentiation of cells as well as cell survival and programmed cell death/apoptosis. Multimerization of TNFRs can lead to recruitment of TRAFs (TNFR-associated factors) by the receptors resulting in activation of kinases and transcription factors, such as c-Jun N-terminal kinase and nuclear factor kappaB (NF-kappaB). Signal transduction triggered by TNF-alpha also induces an increase in intracellular reactive oxygen species (ROS). ROS have been suggested to play a role in NF-kappaB activation, which is thought to promote cell survival. However, oxidation of proteins and lipids by ROS can also result in apoptosis. The processes generating intracellular ROS and the mechanism(s) regulating the cellular redox status have not been fully elucidated. We investigated whether TRAFs play a role in controlling intracellular ROS levels. Our results indicate that recruitment of TRAFs to the plasma membrane of human embryonic kidney (HEK) 293 cells is crucial for activation of signaling pathways, which regulate ROS production in mitochondria. TRAF-mediated changes in ROS levels enhanced NF-kappaB activation but were not dependent on NF-kappaB-inducing kinase. Consistent with its anti-apoptotic function, Bcl-x(L) interfered with TRAF-mediated ROS generation but not NF-kappaB activation. Taken together, our results suggest a novel role of TRAFs in signal transduction pathways triggered by TNFR-related proteins, which balance cell survival and apoptosis by regulating the electron transport in mitochondria.

Cutting edge: distinct motifs within CD28 regulate T cell proliferation and induction of Bcl-XL.

CD28 provides an important costimulatory signal in T cell activation that regulates multiple cellular processes including proliferation and survival. Several signal transduction pathways are activated by CD28; however, the precise biochemical mechanism by which CD28 regulates T cell function remains controversial. Retroviral gene transfer into primary T cells from TCR-transgenic, CD28-deficient mice was used to determine the specific sequences within CD28 that determine function. Discrete regions of the cytoplasmic domain of CD28 were identified that differentially regulate T cell proliferation and induction of the anti-apoptotic protein Bcl-X(L). Mutation of C-terminal proline residues abrogated the proliferative and cytokine regulatory features of CD28 costimulation while preserving Bcl-X(L) induction. Conversely, mutation of residues important in phosphatidylinositol 3-kinase activation partially inhibited proliferation but prevented induction of Bcl-X(L.) Thus the ability of CD28 to regulate proliferation and induction of Bcl-X(L) map to distinct motifs, suggesting independent signaling cascades modulate these biologic effects.

Translocation of TRAF proteins regulates apoptotic threshold of cells.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are involved in signaling pathways triggered by members of the TNF receptor (TNFR) family and other cell surface proteins. After recruitment to a receptor, TRAFs initiate formation of multiprotein complexes that induce downstream events, such as translocation of transcription factor nuclear factor kappaB (NF-kappaB) and activation of c-Jun N-terminal kinase (JNK). Several proteins in these complexes play important roles in regulation of apoptosis. However, the fate of TRAF-containing complexes once assembled in response to receptor multimerization is not understood. In this report, we demonstrate that crosslinking of TNFR family members or interaction of TRAF2 with the cytoplasmic protein A20 leads to intracellular translocation of TRAF2. This redistribution leads to depletion of the cytoplasmic pool of TRAF2. The ratio between soluble and insoluble TRAF2 determines the sensitivity of cells to TNF-alpha-induced apoptosis and may play an important role in limiting further TRAF-dependent signal transduction.

Regulation of T cell apoptosis.

Proliferative expansion of lymphoid cells is required for effective immune responses against invading microorganisms, but after the infection is controlled, the expanded effector cells must be eliminated to prevent non-adaptive accumulation of cells. Higher vertebrates have developed extensive networks of signal transduction pathways to ensure controlled activation and expansion of cells during immune responses and apoptotic deletion of lymphoid cells that are no longer needed at the end of immune responses. Extracellular signals received by cell surface receptors that trigger intracellular signaling cascades are essential elements that control both processes. These signal transduction pathways converge to regulate cell fate at both transcriptional and post-transcriptional levels. Here we review the role of pathways, especially those triggered by TNF receptor-related molecules, that determine the fate of T cells during development and activation. In addition, we introduce the possibility that these same pathways may be abnormally programmed and so lead to immune cell accumulation during inflammatory diseases such as asthma.

Lymphocyte survival--the struggle against death.

Cell proliferation and cell death must be closely regulated to maintain the integrity of the immune system during the lifetime of multicellular organisms. Proliferative expansion of lymphoid cells is required for effective immune responses against invading microorganisms. However, following infection eradication, expanded effector cells must be eliminated to prevent non-adaptive accumulation of cells. Therefore, higher vertebrates have developed an extensive network of signal transduction pathways that allow integration of cell survival and cell death stimuli. This network functions to ensure the controlled activation and expansion of cells during an immune response and the deletion of lymphoid cells that are no longer needed at the end of an immune response. Extracellular signals appear to control both mechanisms. Ultimate responses are integrated through cell surface receptors that are linked to intracellular signaling cascades. These signal transduction pathways converge to regulate cell fate at both transcriptional and post-transcriptional levels. In this review, the role of pathways triggered by TNFR-related molecules that determine the fate of lymphoid cells during development and activation is summarized.

4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kappaB.

Members of the tumor necrosis factor (TNF)-nerve growth factor (NGF) receptor family have been shown to be important costimulatory molecules for cellular activation. 4-1BB and Ox40 are two recently described members of this protein family which are expressed primarily on activated T cells. To gain insight into the signaling pathways employed by these factors, yeast two-hybrid library screens were performed with the cytoplasmic domains of 4-1BB and Ox40 as baits. TNF receptor-associated factor 2 (TRAF2) was identified as an interacting protein in both screens. The ability of both 4-1BB and Ox40 to interact with TRAF2 was confirmed in mammalian cells by coimmunoprecipitation studies. When the binding of the receptors to other TRAF proteins was investigated, 4-1BB and Ox40 displayed distinct binding patterns. While 4-1BB bound TRAF2 and TRAF1, Ox40 interacted with TRAF3 and TRAF2. Using deletion and alanine scanning analysis, we defined the elements in the cytoplasmic domains of both receptors that mediate these interactions. The 4-1BB receptor was found to have two independent stretches of acidic residues that can mediate association of the TRAF molecules. In contrast, a single TRAF binding domain was identified in the cytoplasmic tail of Ox40. The cytoplasmic domains of both receptors were shown to activate nuclear factor kappaB in a TRAF-dependent manner. Taken together, our results indicate that 4-1BB and Ox40 bind TRAF proteins to initiate a signaling cascade leading to activation of nuclear factor kappaB.

Development of a bispecific F(ab')2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity.

A bispecific F(ab')2 antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab' fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5'-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of "clump-like" cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively.

Transient expression of CD44 variant isoforms in the ontogeny of the rat: ectoderm-, endoderm- and mesoderm-derived cells express different exon combinations.

Expression on rat tumor cells of CD44 variant isoforms containing exons v4-v7 or v6-v7 has been described as sufficient for initiation of the metastatic cascade. The question arose as to whether physiological programs may be reactivated by particular CD44 isoforms. With this in mind, expression of mRNAs for the CD44 isoforms was surveyed during ontogeny of the rat. Using available monoclonal antibodies, expression of the CD44 standard isoform (CD44s) and of an epitope of CD44 exon v6 (CD44v6) were evaluated by immunohistology also. While CD44s was expressed in cells of all three germ layers, CD44v6 expression was restricted to distinct epithelial layers and cells of the hematopoietic system. During ontogeny, expression of CD44v6 was first noted in the neural tube and the leading epithelial layer of the limb buds. Later, anti-CD44v6 (1.1ASML) stained basal layers of the epidermis, the epithelium of the gut, and the acini of the submandibular gland. Strong, but transient expression of CD44v6 was seen during lung development, in hematopoietic stem cells of the liver, in thymic epithelia and early thymic immigrants. Expression in these organs was downregulated shortly before or after birth. As revealed by Southern blotting after use of the reverse transcriptase polymerase chain reaction (RT-PCR), most CD44v6-positive organs contained more than one CD44 variant isoform, and the expression patterns differed between individual organs. Hematopoietic cells preferentially expressed exons v4-v7, endodermal tissue exons v4-v10 and only in the epidermis were exons v1-v10 detected. The temporally regulated expression during ontogeny and the different exon compositions suggest a pivotal role of CD44 isoforms particularly in hematopoesis and in pattern formation by instructive epithelia.

Expression of CD44 variant isoforms in malignant melanoma.

In a variety of human tumors, expression of splice variants of the adhesion molecule CD44 (CD44v) has been described as correlating with tumor progression. Here, we report on the expression of CD44v in melanocytes, nevi, primary melanomas, and cutaneous and lymph node metastases. Thirteen nevi, 65 primary melanomas of varying thickness, 39 cutaneous and 15 lymph node metastases, and melanocytes and a panel of melanoma lines were tested for surface expression of the standard form of CD44 and the variant exons v5, v6, v7, v7-v8, and v10 by immunohistology or fluorescence-activated cell sorting. Melanocytes did not express any variant isoform of CD44. However, nevi, as well as primary melanoma and melanoma metastases, stained to a varying degree with anti-CD44v5, anti-CD44v7-v8, and anti-CD44v10. Exons v6 and v7 were not detected on any of these tissue specimens. Compared with nevi, expression of exon v10 was up-regulated in thick primary tumors and skin metastases. Lymph node metastases displayed elevated levels of exon v5. Expression of CD44v in melanoma lines (n = 20) differed, inasmuch as many lines did not express variant isoforms; in particular, exon v10. Interestingly, however, the few CD44v5-positive melanoma lines metastasized in the nu/nu mouse. Because benign as well as malignant growth of melanocytes was accompanied by expression of CD44 variant isoforms, a linkage between expression of CD44 variant isoforms and malignant transformation or tumor progression was excluded. Considering the function of distinct isoforms, one might speculate that expression of exon CD44v5, which was up-regulated in lymph node metastases compared with nevi and primary melanoma, provided a growth stimulus. Exon v10 is present at high density in epidermal cells. The de novo expression of this exon in nevi and the increased expression in thick melanoma and skin metastases would be in line with the assumption of an anchoring advantage in the surrounding epidermal tissue.

Prevention of tumor metastasis formation by anti-variant CD44.

A splice variant of CD44 (CD44v) originally discovered on metastases of a rat pancreatic adenocarcinoma (BSp73ASML) has been shown by transfection to confer metastatic behavior to nonmetastatic tumor cells (Günthert U., M. Hofmann, W. Rudy, S. Reber, M. Zöller, I. Haussmann, S. Matzku, A. Wenzel, H. Ponta, and P. Herrlich. 1991. Cell. 65:13). A monoclonal antibody (mAb), 1.1ASML, to the metastasis-specific domain of the CD44v molecule retards growth of lymph node and lung metastases of the metastatic tumor line BSp73ASML, and can efficiently prevent formation of metastases by the transfected line. The antibody is only effective when given before lymph node colonization. Anti-CD44v does not downregulate the expression of CD44v, and prevention of metastatic growth by anti-CD44v is not due to activation of any kind of immune defense. We suggest that the mAb interferes with proliferation of metastasizing tumor cells in the draining lymph node, most probably by blocking a ligand interaction. The interference with metastatic spread will greatly facilitate the exploration of the function of CD44v and, in particular, may also open new strategies for the therapy of human metastases.

Expression of CD44 isoforms carrying metastasis-associated sequences in newborn and adult rats.

Expression of a splice variant of CD44, recognised by the monoclonal antibody (Mab) 1.1ASML, confers metastatic potential to non-metastasising tumour cells (Cell 1991, 65, 13-24). To explore whether the metastasis-associated variant of CD44 (CD44v) is expressed under physiological conditions, tissues of newborn and adult rats were stained with the Mab 1.1ASML. The 1.1ASML epitope is, indeed, expressed on the basal layer of the epidermis and the hair follicles as well as on cryptic epithelia in the gut. In addition, ductal epithelia of the pancreatic gland of newborn rats express CD44v. This pattern of expression differs from that of standard lymphocyte CD44 (CD44s). The anti-CD44s mAB Ox50 predominantly stains connective tissue. Although different variants of CD44 may express the epitope recognised by 1.1ASML, cells expressing CD44v share properties with metastasising tumour cells: the stage of proliferation and a restricted degree of mobility. Thus, during metastatic progression tumour cells may reactivate the expression of gene segments which serve highly specialised functions in embryonic and adult tissues.

Participation in normal immune responses of a metastasis-inducing splice variant of CD44.

A variant of the glycoprotein CD44 (CD44v) that shares sequences with variants causally involved in metastasis formation is transiently expressed on B and T lymphocytes and macrophages after antigenic stimulation and in the postnatal period. Antibodies to the variant hinder in vivo activation of both B and T cells. The observation that a protein domain that is expressed on CD44 and required for the lymphatic spread of tumor cells can catalyze an essential step in the process of lymphocyte activation supports the idea that metastasizing tumor cells mimic lymphocyte behavior.