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Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti‐CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39‐expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39‐expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co‐stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39‐CD40 to drive proliferation and that soluble gp39 alone in a non‐membrane bound form is able to provide co‐stimulatory signals to B cells.

gp39, a cytokine expressed on the surface of activated T cells, is essential for T cell-dependent antibody responses in vivo. We cloned and sequenced 1.2 kilobases of the 5' flank region of the human gp39 gene promoter and determined its transcription start site. When used in reporter gene assays, this DNA segment conferred promoter activity in response to T cell activation. gp39 promoter function in transfectants was inhibited by cyclosporin A, as is expression of the endogenous gp39 gene in T-lineage cells. At least 0.5 kilobase of the 5' flank region was required for promoter activity. Two putative binding sites for the NF-AT family of transcriptional activator proteins were identified at -259 to -265 and -62 to -69 with respect to the transcription start site. Both sites contributed significantly and independently to promoter activity in response to T cell activation. Additionally, when incubated in vitro with nuclear protein purified from activated human CD4 T cells, both of these sites preferentially bound the NF-AT family member, NF-ATp. These results suggest that NF-ATp, via binding to at least two cis-elements, is essential for the induction of gp39 gene expression in response to T cell activation.

Common variable immunodeficiency (CVI) is characterized by hypogammaglobulinemia and recurrent bacterial infections due to failure of CVI B cells to differentiate in vivo into immunoglobulin-secreting plasma cells. We hypothesized that T-cell dysfunction resulting in abnormal contact-mediated B-cell activation may play a prominent role in the failure of CVI B cells to produce specific antibody. We have previously shown that B-cell proliferation and IgE production after stimulation with anti-CD40 and interleukin (IL) 4 were normal in 22 CVI patients evaluated, indicating that CVI B cells respond to signals delivered via CD40. Here we report that CD40 ligand (gp39) mRNA expression by activated lymphocytes from CVI patients (n = 31) as a group was significantly depressed (P < 0.0001) compared with normal controls (n = 32). gp39 mRNA expression by activated lymphocytes from 13 CVI patients fell below the normal control range. T-cell surface expression of functional gp39 protein was correspondingly low in those patients with gp39 mRNA levels below normal control range and normal in patients with gp39 mRNA levels within normal control range. In CVI patients as a group, gp39 mRNA levels correlated with IL-2 mRNA levels (P < 0.002, r = 0.6) and production (P < 0.001, r = 0.7) but not with gene expression or production of other lymphokines evaluated, suggesting an as-yet-undetermined association between gp39 and IL-2 gene regulation. Of the 13 patients whose activated T cells exhibited gp39 mRNA expression below the normal control range, 2 had normal T-cell-derived lymphokine production, whereas the remaining 11 exhibited broader T-cell dysfunction, resulting in IL-2 deficiency, and in some patients deficient production of other lymphokines as well, reflecting a heterogeneity in the underlying mechanisms leading to depressed gp39 expression in these patients. The observation that both gene and surface expression of gp39 by activated T cells is depressed in a subgroup of CVI patients suggests that inefficient signaling via CD40 may be responsible, in part, for failure of B-cell differentiation in these patients.

Native sulfatides, as well as many sulfated glycolipids, have been shown to avidly bind to the selectin receptors. In vivo, native sulfatides significantly block activity in selectin-dependent inflammatory responses. The fact that nonsulfated galactocerebrosides did not inhibit selectin-mediated adhesion identified a critical role for the anionic sulfate residue. We therefore initiated a program to evaluate the activity of position isomers. This study showed a binding selectivity for the positions 2 and 3 of the sulfate group on the carbohydrate ring as well as enhanced activity for the disulfated analogs. Furthermore, it was discovered that the attachment of lipophilic substituents on the carbohydrate ring was tolerated, consistent with the presence of a lipophilic pocket in the binding cavity. This resulted in compounds with a 6-fold increased potency.

The interaction between activated vascular endothelium and T cells has been shown to play an important role in the recruitment and activation of T cells at sites of inflammation. Here we report the expression of CD40 by vascular endothelial cells and its regulation by inflammatory agents. Using the soluble recombinant CD40 ligand, sgp39, we show that the interaction of CD40 with its ligand can lead to endothelial cell activation, which in turn leads to leukocyte adhesion. This adhesion is partly mediated by the expression of E-selectin. In addition to E-selectin expression, sgp39 induces the expression of intercellular adhesion molecule 1 and augments the tumor necrosis factor alpha-induced expression of vascular cell adhesion molecule 1. The effects of sgp39 on endothelial cells can be blocked with anti-gp39 monoclonal antibody (mAb), anti-CD40 mAb, or soluble CD40. Staining of tissues from healthy human skin using anti-CD40 mAb showed very weak expression of CD40 by the endothelium, while skin involved in inflammatory disease showed marked upregulation of CD40 expression. These studies suggest that interactions between cell surface proteins expressed by activated T cells with their receptors on vascular endothelium can stimulate the vasculature at sites of inflammation and may be involved in normal inflammatory responses and in inflammatory disease.

The recently isolated 4-1BB cDNA clone encodes a cell surface protein expressed by activated T cells. Its extracellular domain is homologous to members of the nerve growth factor receptor super family and its cytoplasmic domain contains a sequence homologous to the binding site for the T-cell-specific tyrosine kinase p56lck found in the cytoplasmic domains of CD4 and CD8 alpha. At present the function of 4-1BB is not known. We prepared a 4-1BB-immunoglobulin fusion protein (4-1BB Rg). This protein was used in immunohistochemical studies to identify tissues that express the 4-1BB ligand. 4-1BB Rg bound to virtually all tissues examined, suggesting that extracellular components might function as its ligands. To explore this possibility, 4-1BB was expressed in COS cells and found to mediate the binding of fibronectin, vitronectin, laminin, and collagen VI but not of collagen I. The binding of extracellular matrix proteins to 4-1BB was not mediated by Arg-Gly-Asp (RGD) or CS-1 amino acid sequences. Experiments with overlapping proteolytic fragments of fibronectin showed that 4-1BB interacts with multiple regions of fibronectin. The interaction between extracellular matrix proteins and 4-1BB was completely blocked by the anionic carbohydrate polymer fucoidan and was partially blocked by the anionic carbohydrate polymer dextran sulfate and the glycosaminoglycan heparin sulfate but was unaffected by desulfated heparin. These results suggest that carbohydrates may play a role in mediating the 4-1BB-extracellular matrix protein adhesion.

Expression of gp39 on activated T cells provides a co-stimulatory signal in peripheral lymphoid tissue that regulates humoral and cell-mediated immunity. The function of gp39 and its receptor CD40 in thymus remains uncertain. Here we report that overexpression of gp39 in transgenic mouse thymus caused a dose-dependent decline in thymocyte numbers (> 500 fold), loss of cortical epithelium and expansion of CD40+ medullary cells. Transplantation of transgenic bone marrow into normal mice indicated that gp39 significantly diminished thymocyte viability in the context of a 'normal' thymic environment. The peripheral tissues of transgenic mice also accumulated abnormalities in a transgene dose-dependent manner that involved inflammation and lymphoid tissue hypertrophy. Animals with the highest transgene copy numbers acquired a lethal inflammatory bowel disease marked by the infiltration of gp39+ T cells and CD40+ cells into diseased tissues. Examination of cells overexpressing gp39 suggested that these defects were caused, in part, by the saturation of a mechanism that sequesters gp39 inside non-activated cells and thus protects the immune system from inappropriate gp39-CD40 interaction. These results establish a regulatory role for gp39 in thymus function and a causal relationship in mediating chronic inflammatory disease.

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10-stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti‐CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39‐expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39‐expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co‐stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39‐CD40 to drive proliferation and that soluble gp39 alone in a non‐membrane bound form is able to provide co‐stimulatory signals to B cells.