The 4-1BB (a TNFR superfamily member) is an inducible costimulatory molecule that can exert regulatory effects on T cells independently of CD28 stimulation. The in vitro expression of 4-1BB (CD137) is induced following activation of T cells with various stimuli, including anti-TCR mAbs, lectins, and a combination of PMA and ionomycin. To delineate further the physiological role of 4-1BB in immunity, mice deficient in this receptor were generated. These mutant mice developed normally, and were viable and fertile. Humoral responses to vesicular stomatitis virus were comparable with those seen in wild-type mice, whereas the IgG2a and IgG3 isotype responses to keyhole limpet hemocyanin were somewhat reduced in the mutant mice. The 4-1BB-deficient mice demonstrated enhanced T cell proliferation in response to mitogens or anti-CD3 even in the environment of reduced ability to secrete growth-supporting cytokines (IL-2 and IL-4). Although T cells from 4-1BB-deficient mice showed enhanced proliferation, the T cell immune responses of these animals, such as cytokine production and CTL activity, were diminished. In addition, 4-1BB deletion appears to play a role in the regulation of myeloid progenitor cell growth, leading to an increase in these precursor cells in peripheral blood, bone marrow, and spleen.

A newly identified member of the tumor necrosis factor receptor (TNFR) superfamily shows activities associated with osteoclastogenesis inhibition and fibroblast proliferation. This new member, called TR1, was identified from a search of an expressed sequence tag database, and encodes 401 amino acids with a 21-residue signal sequence. Unlike other members of TNFR, TR1 does not contain a transmembrane domain and is secreted as a 62 kDa glycoprotein. TR1 gene maps to chromosome 8q23-24.1 and its mRNA is abundantly expressed on primary osteoblasts, osteogenic sarcoma cell lines, and primary fibroblasts. The receptors for TR1 were detected on a monocytic cell line (THP-1) and in human fibroblasts. Scatchard analyses indicated two classes of high and medium-high affinity receptors with a kD of approximately 45 and 320 pM, respectively. Recombinant TR1 induced proliferation of human foreskin fibroblasts and potentiated TNF-induced proliferation in these cells. In a coculture system of osteoblasts and bone marrow cells, recombinant TR1 completely inhibited the differentiation of osteoclast-like multinucleated cell formation in the presence of several bone-resorbing factors. TR1 also strongly inhibited bone-resorbing function on dentine slices by mature osteoclasts and decreased 45Ca release in fetal long-bone organ cultures. Anti-TR1 monoclonal antibody promoted the formation of osteoclasts in mouse marrow culture assays. These results indicate that TR1 has broad biological activities in fibroblast growth and in osteoclast differentiation and its functions.

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Pmel 17 is preferentially expressed in pigment cells in a manner suggestive of involvement in melanin biosynthesis. The gene is identical to the silver (si) pigmentation locus in mice. We now produced a recombinant glutathione-S-transferase-human Pmel 17 infusion protein and raised polyclonal antibodies against it to confirm the ultrastructural location and presumed site of action predicted by the deduced primary structure of Pmel 17/silver, and to authenticate the specificity of the DHICA converting function as inherent to the silver-locus protein. Full-length Pmel 17 cDNA also produced in insect cells in a baculovirus expression vector to ensure that activity did not originate from a co-precipitated protein. Natural hPmel 17 from human melanoma cells has an approximate molecular size of 100 kDa. By immunoperoxidase electron microscopic cytochemistry, the antigen was localized to the limiting membranes of premelanosomes and presumed premelanogenic cytosolic vesicles and, to a minor extent, in the premelanosomal matrix. In an in vitro assay, both the natural and the recombinant Pmel 17 accelerated the conversion of DHICA to melanin. This activity was inhibited by the anti-Pmel 17 polyclonal antibodies, indicating that the acceleration of DHICA conversion by natural protein is genuine and cannot be due to contaminating complexed proteins. We suggest that in situ Pmel 17/silver is a component of a postulated premelanosomal/melanosomal complex of membrane-bound melanogenic oxidoreductive enzymes and cofactors, in analogy to the electron transfer chain in mitochondria.

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