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Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2b mice. CD40L-mediated interactions were also necessary for development of protective H-2b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.

CD40 ligand (CD40L) plays a crucial role in T cell-dependent B cell responses, but whether its abundance is a limiting factor in their development is unclear. This question was addressed in transgenic mice expressing the murine CD40L gene under the control of the IL-2-promoter (CD40Ltg+). The fraction of activated T cells from the CD40Ltg+ mice with detectable levels of surface CD40L was modestly greater (1.1- to 2-fold) than littermate controls and paralleled an approximately 1.8-fold increase in CD40L mRNA abundance. In response to trinitrophenol (TNP)-keyhole limpet hemocyanin and tetanus/diphtheria vaccine, CD40Ltg+ mice developed higher titers of high-affinity IgG and IgG1 Ab than wild-type mice. In contrast, the Ab response of CD40Ltg+ and control mice was similar in response to the T-independent Ag TNP-Ficoll. These results suggest that a modest increment in expression of CD40L accelerates the development of T-dependent responses, and that CD40L plays a limiting role in the induction of high-affinity Ab and Ab-class switching.

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.

Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced beta-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.

Murine lupus in NZB/NZW F1 (B/W) mice can be retarded by sustained administration of CTLA4Ig and by brief treatment early in life with mAb that block CD40/gp39 interactions. We sought to determine whether brief therapy with CTLA4Ig could provide sustained benefit in B/W mice and whether a synergistic effect could be derived by blockade of both the B7/CD28 and the CD40/gp39 pathways. We found that a short course of CTLA4Ig at the onset of disease produced only short-term benefit. However, when CTLA4Ig was combined with anti-gp39, there was long-lasting inhibition of autoantibody production and renal disease. Ten months after the 2-wk course of therapy, 70% of these mice were alive, compared with only 18% and 0% of those that received only anti-gp39 or CTLA4Ig, respectively. These findings demonstrate that brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways can produce benefit that lasts long after treatment has been discontinued.

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.

Interleukin-6 (IL-6) is the major cytokine to date mediating antigen (Ag)- or mitogen-driven B cell differentiation. Recently, CD40 ligand (CD40L), with the co-stimulatory cytokines IL-4 and IL-10, has been shown to trigger immunoglobulin (Ig) secretion and class switching. In the present report, we have examined the role of IL-6 in mediating B cell differentiation and Ig secretion triggered with CD40L and/or these cytokines. Culture of splenic B cells with CD40L triggered (1) significant (5.4-fold) increases in IL-6 secretion; (2) differentiation, evidenced by sequential loss of B cell (CD20, CD21) and acquisition of plasma cell (CD38, PCA-1) surface antigens (Ags); and (3) Ig secretion. Interleukin-4 increased both IL-6 and IgG secretion stimulated by CD40L. Interleukin-10+ CD40L triggered 100-fold increments in IgG, IgA and IgM secretion, but IL-10 suppressed IL-6 secretion triggered with CD40L +/- IL-4. Exogenous IL-6 can further increase IgG secretion induced by CD40L + IL-10; moreover, the anti-IL-6 monoclonal antibody partially blocked IgG secretion triggered by CD40L +/- IL-4 or IL-10. Finally, IL-10 suppressed differentiation of B cells induced by CD40L. These studies suggest that CD40L augments Ig secretion in at least two mechanisms: by triggering IL-6 secretion and related differentiation, and by priming B cells for responsiveness to IL-10.

The binding of CD40 ligand on activated T cells to CD40 on resting B cells induces the expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86). The induction of B7 molecules by CD40 ligand-CD40 interaction represents a critical step in rendering B cells competent for antigen presentation. The CBA/N mouse has a defect in CD40 signalling which has been attributed to a mutation in Bruton's tyrosine kinase. We have compared the ability of murine CD40 ligand to induce B7-1 and B7-2 expression on B cells isolated from CBA/N and wild-type CBA/J mice. We find that the CBA/N defect partially impairs both B7-1 and B7-2 induction via CD40. Subsequent experiments investigated the roles of different second messenger systems in B7-1 and B7-2 induction in normal B cells. In M12 B lymphomas either CD40 cross-linking or cAMP treatment can induce B7 molecules. Here we report that treatment with dibutyryl-cAMP also induces B7 molecules in normal B cells provided that they have been preactivated by CD40 cross-linking. We also find that PMA and ionomycin treatment of B cells induces B7-2 but not B7-1 expression. Our data therefore show roles for BTK, cAMP and PMA/ionomycin in B7 induction, as well as providing further evidence for differential regulation of B7-1 and B7-2 induction in B cells.

Studies in vivo have documented the importance of CD40-gp39 interactions in the development of T-dependent antibody responses to foreign and auto-antigens. In this report, we demonstrate that allograft rejection is also associated with strong induction of CD40 and gp39 transcripts. When treatment was initiated at the time of transplant, MR1, a mAb specific for gp39, induced markedly prolonged survival of fully disparate murine cardiac allografts in both naive and sensitized hosts. However, when therapy was delayed until postoperative day 5, anti-gp39 failed to prolong graft survival. Allografts from recipients treated with MR1 from the time of transplantation showed decreased expression of transcripts for the macrophage effector molecule, inducible nitric oxide synthase, but essentially unaltered expression of B7 molecules and T cell cytokine transcripts (interleukin [IL]-2, interferon-gamma, IL-10, and IL-4) relative to control allografts. In addition, alloantibody responses in the MR1-treated mice were profoundly inhibited. However, our studies using B cell-deficient mice indicated that the ability of MR1 to prolong allograft survival was not dependent on B cells. These data suggest that blockade of CD40-gp39 interactions may inhibit allograft rejection primarily by interfering with T cell help for effector functions, rather than by interference with T cell activation.

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.

Recombinant immunoglobulin (Ig) fusion proteins of cell surface and intracellular proteins have wide applications. For example, fusion proteins have been used in the isolation, identification and study of ligands and the effects of binding or blocking a receptor-ligand pair, either in vivo or in vitro. For some applications, removal of the immunoglobulin Fc region is advantageous. We have developed two vectors for the expression of Ig fusion proteins that contain recognition sequences for protease cleavage using thrombin. In one vector, the sequence encoding the thrombin cleavage site is located at the junction of the DNA fragment encoding the protein or protein fragment to be studied and the hinge and constant regions of the immunoglobulin, allowing the generation of a monomeric form of the protein of interest. In the second vector, the sequence encoding the thrombin cleavage site is located between the sequences encoding the hinge and constant regions of the immunoglobulin, allowing for the generation of covalent dimers of the recombinant protein without the constant Fc domains. We have used these vectors to produce the constructs encoding two forms of the extracellular domain of CD40, CD40ThrIg and CD40HinThrIg, allowing production of a monomeric and dimeric form of recombinant CD40. Cleavage is efficient and complete. Following cleavage, there was no detectable binding of the monomeric form of CD40 to a soluble form of gp39, the ligand of CD40, while the dimeric form was able to bind. These vectors have been constructed to allow facile substitution with other sequences to generate cleavable forms of other proteins of interest.

CD40 is a glycoprotein of about 50 kDa that plays a crucial role in B cell growth and differentiation. It is found on the surface of B cells, follicular dendritic cells, monocytes, and some endothelial, epithelial, and carcinoma cells. Engagement of CD40 with anti-CD40 mAbs, gp39 expressed on the cell surface or soluble forms of gp39, primes B cells to efficiently respond to subsequent stimulatory signals leading to B cell proliferation, differentiation, and isotype switching. Peripheral monocytes also express CD40 on the cell surface and expression in increased following treatment with IFN-gamma. Using a soluble murine CD8/human gp39 fusion protein (sgp39) we have found that CD40 plays a crucial role in the regulation of monocyte function. Stimulation of human peripheral monocytes with sgp39 induced homotypic aggregation and significantly increased the expression of several cell-surface proteins including CD54, MHC class II, CD86, and CD40. Soluble gp39 also dramatically enhanced monocyte survival, preventing the onset of apoptosis that normally occurs upon withdrawal of serum. Finally, in the absence of any costimulatory molecules, sgp39 stimulated monocytes to produce TNF-alpha, IL-1 beta, IL-6, and IL-8. These results suggest that ligation of CD40 on human monocytes induces phenotypic changes that would be expected to influence T cell activation by the monocyte and also to enhance or prolong inflammatory responses.

The interaction between gp39 (CD40L, TRAP, T-BAM) on activated T cells and mast cells and CD40 on antigen-presenting cells modulates immune responses. Gp39 and CD40 are homologous to tumor necrosis factor (TNF) and its receptor (TNFR), respectively. The TNF-beta/TNFR interaction has been analyzed on the basis of mutagenesis experiments and crystal structures. Using the interaction of TNF-beta/TNFR as a guide, we previously reported a site-directed mutagenesis study in which we identified residues in gp39 (K143, Y145) and CD40 (Y82, D84, N86) involved in gp39/CD40 interactions. Here we describe the use of the TNF-beta/TNFR complex crystal structure as a template to prepare molecular models of gp39, CD40, and their approximate interaction. The application of these models has allowed us to extend our mutagenesis analysis of gp39/CD40 interactions. These experiments have led to the identification of additional gp39 (Y146, R203, Q220) and CD40 (E74, E117) residues that contribute to the gp39/CD40 interaction. We also further explored the importance of gp39 residue Y145 and CD40 residue Y82 for the gp39/CD40 interaction by conservatively replacing these residues with Phe. The results of these studies have enabled us to approximately outline the binding sites in gp39 and CD40. It appears that the gp39/CD40 interaction is centered on at least two clusters of residues and involves residues of two adjacent gp39 monomers. The molecular regions involved in the gp39/CD40 interaction essentially correspond to those in the homologous TNF-beta/TNFR system.

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.

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.

The interaction between the CD40 ligand (gp39), expressed by activated T cells, and CD40, constitutively expressed by B cells, is critical for an effective antibody response to T cell dependent antigens. Patients with X-linked hyper IgM (HIM) syndrome fail to express a functional CD40 ligand due to a mutation within the gene for gp39. As a direct consequence, HIM patients, when immunized with T dependent antigens, produce only small amounts of IgM antibody without the development of immunologic memory, amplification and switch from IgM to IgG. Mutations affecting the gene for the HIM syndrome are localized throughout the coding region of gp39 and consist predominantly of point mutations. The resulting amino acid substitutions interfere directly with the receptor binding site or lead to stop codons or deletions secondary to splice site mutations. Expression of gp39 by activated T cells from patients with common variable immunodeficiency (CVI) is low in approximately half of the patients and is associated with depressed expression of IL-2. These findings suggest that inefficient signaling via CD40 may be responsible in part for failure of B cell differentiation in CVI.

CD40 functional responses can be triggered by binding of mAb G28-5. Here we show that G28-5 induces partial CD40 responses and functions as a partial antagonist of natural CD40 ligand, gp39, by preventing gp39 binding. Fab fragments of G28-5 retain inhibitory activity but lose crosslinking-dependent stimulatory activity. The synergistic interaction of CD40 signals with PMA or CD20 show differential requirements for CD40 crosslinking and different sensitivity to cyclosporine A, suggesting that CD40 receptor may use different effector mechanisms for synergy with calcium-dependent CD20 signals or with calcium-independent signals from PMA. Activation of NF-kappa B occurred in RAJI cells by G28-5 or by gp39 treatment, and was CD40 crosslinking-dependent. These results suggest that activation of NF-kappa B is involved in some CD40 receptor signals and may be related to CD40 effects on stimulation or inhibition of apotosis.

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.

The prominent role of the CD40 receptor in B cell responses led us to investigate the role of the gp39-CD40 interaction in a group of primary immunodeficient patients with defective antibody production. Here we report that patients with hyper-IgM syndrome (HIM) have a defective gp39-CD40 interaction. B cells from HIM patients express functional CD40, but their T cells do not bind CD40-Ig. These patients expressed normal levels of gp39 mRNA, but these mRNAs encode defective gp39 proteins owing to mutations in the extracellular domain of gp39. Soluble recombinant forms of gp39 containing these mutations were unable to bind CD40 and drive normal B cell proliferation. The gene encoding gp39 was mapped to Xq26, the X chromosome region where the gene responsible for HIM had previously been mapped. These data suggest that a defect in gp39 is the basis of X-linked HIM.