Therapeutic intervention with inhibitors of co-stimulatory pathways in autoimmune disease.

Many humanized antibodies and fusion proteins targeting T-cell co-stimulatory molecules are now in late-stage clinical development (phase II, phase III) or have recently completed phase III clinical trials. Both Amevive, an LFA-3-Ig fusion protein targeting CD2, and Xanelim, a humanized anti-CD11a antibody, have shown efficacy in pivotal phase III trials in patients with plaque psoriasis. These new medicines are poised to enter clinical use in 2002.

Use of monoclonal antibodies for expression cloning.

This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell-surface and intracellular proteins. The first protocol describes the cloning of cDNAs encoding cell-surface antigens. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double-stranded cDNA.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis.

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.

Cutting edge: CD40 ligand is a limiting factor in the humoral response to T cell-dependent antigens.

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.

The role of the CD40 pathway in alloantigen-induced hyporesponsiveness in vivo.

Resting B (rB) cells are known to be incompetent APCs in vitro, which alone can induce specific unresponsiveness to single minor histocompatibility (miH) Ags and, when combined with CD40 pathway blockade, can induce hyporesponsiveness to MHC molecules in vivo. Here we show that anti-CD40 ligand (CD40L) mAb does not prevent the expression of B7-2 on allogeneic rB cells in vivo but did prolong donor-specific cardiac allograft survival. Moreover, pretreatment with professional APCs combined with anti-CD40L mAb induced hyporesponsiveness to alloantigens in vivo. rB cells from CD40 knockout mice were unable to induce unresponsiveness, while graft prolongation was achieved in CD40L knockout recipients pretreated with wild-type rB cells. These data suggest that CD40-CD40L interactions in the recipient play a critical role in the induction of hyporesponsiveness to alloantigens in vivo and that the effect of the CD40 pathway may be independent of its effect on the B7 costimulatory pathway.

Mutations of the CD40 ligand gene and its effect on CD40 ligand expression in patients with X-linked hyper IgM syndrome.

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.

Transient inhibition of CD28 and CD40 ligand interactions prolongs adenovirus-mediated transgene expression in the lung and facilitates expression after secondary vector administration.

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.

Central nervous system toxoplasmosis with an increased proportion of circulating gamma delta T cells in a patient with hyper-IgM syndrome.

Hyper-IgM syndrome represents a diverse group of immunodeficiencies characterized by normal or high serum IgM concentrations with decreased or absent IgG, IgA, and IgE. The X-linked form of hyper-IgM syndrome is caused by mutations in the CD40 ligand gene, preventing its expression on activated T cells. The CD40 ligand--CD40 interaction is critical for effective isotype switching and for initiating antigen-specific Tf cell responses. In addition to recurrent pyogenic infections, patients with the CD40L defect also have opportunistic infections. An increased proportion of circulating gamma-delta T cells, shown to be important early during primary infections, has been demonstrated in numerous infectious diseases including toxoplasmosis. Here, we report a patient with hyper-IgM syndrome and CNS toxoplasmosis, who showed a marked increase in gamma-delta T cells in his peripheral blood and who has responded well to treatment of his toxoplasmosis and to high-dose immunoglobulin replacement therapy.

Development of an immunoassay for BMS-191352, a single-chain immunotoxin, and its application to toxicokinetic studies.

BMS-191352 is a single-chain fusion protein composed of the variable regions of chimeric BR96 monoclonal antibody and the binding defective form of Pseudomonas Exotoxin A (PE40). The immunotoxin exhibits potent cytotoxicity against tumor cells expressing the Lewis antigen. A sensitive and specific double antibody sandwich ELISA has been developed and validated for the determination of BMS-191352 in rat and dog EDTA plasma. A monoclonal anti-PE40 antibody (EXA2-1H8) was used to capture BMS-191352 in plasma samples. The captured BMS-191352 was then detected using a biotinylated monoclonal BR96 antiidiotypic antibody (757-4-1) followed by the addition of streptavidin-horseradish peroxidase conjugate and chromogen 3,3',5,5'-tetramethylbenzidine. The optical density was measured at 450 nm. The standard curve range in rat and dog plasma was 2-32 ng/mL. The RSD for the inter- and intra-assay precision was within 9.2% and the accuracy was greater than 89.0%. The ELISA method was applied to the analysis of BMS-191352 in plasma samples from toxicokinetic studies conducted in rats and dogs. These studies revealed that the systemic exposure of BMS-191352 was dose proportional and the kinetics of BMS-191352 were linear between the dose range of 1.8-7.2 mg/m2 in the dog.

Prolonged acceptance of concordant and discordant xenografts with combined CD40 and CD28 pathway blockade.

BACKGROUND: The prompt and vigorous immune response to xenogenic tissue remains a significant barrier to clinical xenotransplantation. Simultaneous blockade of the CD28 and CD40 costimulatory pathways has been shown to dramatically inhibit the immune response to alloantigen. METHODS: . In this study, we investigated the ability of simultaneous blockade of the CD28 and CD40 pathways to inhibit the immune response to xenoantigen in the rat-to-mouse and pig-to-mouse models. RESULTS: Simultaneous blockade of the CD28 and CD40 pathways produced marked inhibition of the cellular response to xenoantigen in vivo and produced long-term acceptance of xenogeneic cardiac and skin grafts (rat-to-mouse), and markedly suppressed an evoked antibody response to xenoantigen. In addition, this strategy significantly prolonged the survival of pig skin on recipient mice. CONCLUSIONS: Long-term hyporesponsiveness to xenoantigen across both a concordant and discordant species barrier, measured by the stringent criterion of skin grafting, can be achieved using a noncytoablative treatment regimen.

Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype.

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow-derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-gamma, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3' portion of WASP interfere with crucial functions of the protein and cause classic WAS.

Sulfated galactocerebrosides as potential antiinflammatory agents.

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 activity. This resulted in compounds with a 6-fold increased potency.

BMS-190394, a selectin inhibitor, prevents rat cutaneous inflammatory reactions.

Selectin binding is the first step in extravasation of leukocytes through the endothelium. Infiltration of leukocytes is a hallmark of an inflammatory response. Blockade of selectin-dependent adhesion, therefore, represents a specific mechanism-based anti-inflammatory strategy. We have used the natural product sulfatide, one of the selectin ligands, as a template to design a novel selectin antagonist. BMS-190394, a structural analog of sulfatide, is an inhibitor of cell binding to P-, E- and L-selectin-Ig fusion proteins. BMS-190394 also inhibits binding mediated by native P-selectin expressed on the surface of activated platelets. Pharmacokinetic analysis of BMS-190394 showed that the compound remained in circulation with a T1/2 of 7 hr, long enough to inhibit the development of an acute inflammatory response. The in vitro activity and pharmacokinetic profile of this selectin-blocking compound led to the determination of its in vivo anti-inflammatory activity. BMS-190394 was a potent inhibitor of the dermal immune complex-induced reverse passive Arthus reaction in rats when delivered by the i.v. or i.p. route. The ED50 of the compound in the reverse passive Arthus reaction compares favorably to that for dexamethasone. BMS-190394 was also an effective inhibitor of the delayed-type hypersensitivity reaction in the rat. Compared with previous reports of the use of antibodies and complex oligosaccharides to inhibit the activity of the selectins, this low-molecular-weight inhibitor of the selectins presents a novel class of anti-inflammatory agents.

Long-term inhibition of murine lupus by brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways.

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.

Thymus dysfunction and chronic inflammatory disease in gp39 transgenic mice.

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.

Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver.

Although recombinant adenovirus vectors offer a very efficient means by which to transfer genetic information into cells in vivo, antigen-dependent immunity limits the duration of gene expression and prevents retreatment. Recombinant murine CTLA4Ig and anti-CD40 ligand antibody block costimulatory interactions between T cells and antigen presenting cells. We previously reported that murine CTLA4Ig prolongs adenoviral-mediated gene transfer, but does not allow for secondary expression after readministration of the vector. In studies described here, when anti-CD40 ligand and recombinant murine CTLA4Ig were coadministered around the time of primary vector administration (i) prolonged adenovirus-mediated gene expression (length of experiment up to 1 year) from the livers of >90% of treated mice was observed, and (ii) secondary adenovirus-mediated gene transfer was achieved in >50% of the mice even after the immunosuppressive effects of these agents were no longer present. Nearly two-thirds of these mice had persistent secondary gene expression lasting for at least 200-300 days. Neither agent alone allowed transduction after secondary vector administration. Treated mice had decreased immune responses to the vector as shown by markedly decreased production of neutralizing antibodies, diminished spleen proliferation responses and IFN-gamma production in vitro, and reduced T cell infiltrates in the liver. These results suggest that it may be possible to obtain persistence as well as secondary adenoviral-mediated gene transfer with transient immunosuppressive therapies.

Agonistic activity of a CD40-specific single-chain Fv constructed from the variable regions of mAb G28-5.

A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.

CD40 is functionally expressed on human keratinocytes.

The CD40/gp39 pathway is known to be an important feature of B/T cell collaboration leading to T cell-dependent activation, proliferation or differentiation of B cells. Additionally, CD40 is involved in the regulation of B cell survival and apoptosis. Recently, CD40 has been shown to be expressed functionally on non-hematopoietic cells, i.e. endothelial cells. Here, we demonstrate that human keratinocytes (KC) cultured in vitro express CD40 constitutively. The surface expression of CD40 is markedly up-regulated following stimulation with interferon (IFN)-gamma, but not with tumor necrosis factor-alpha or interleukin (IL)-1 beta. This process is regulated at the CD40 mRNA level as demonstrated by Northern blot analysis. Furthermore, ligation of CD40 via soluble gp39, the CD40 ligand, enhances intercellular adhesion molecule (ICAM)-1 and Bcl-x up-regulation on IFN-gamma-stimulated KC, but not lymphocyte function-associated antigen (LFA)-3, B7-2, HLA-DR, or Fas expression. The release of IL-8 is also induced following CD40 ligation on KC. In psoriasis, a T cell-mediated inflammatory skin disease, KC have a markedly enhanced expression of CD40. This expression co-localizes with the expression of ICAM-1, Bcl-x, and an influx of CD3+ T cells. These findings suggest a functional role of CD40 on KC in inflammatory skin disorders such as psoriasis and could make a therapeutic intervention by disrupting the CD40/gp39 pathway an approach to consider in these inflammatory skin diseases.

CD40 ligand triggers interleukin-6 mediated B cell differentiation.

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.

Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways.

The receptor-ligand pairs CD28-B7 and CD40-gp39 are essential for the initiation and amplification of T-cell-dependent immune responses. CD28-B7 interactions provide 'second signals' necessary for optimal T-cell activation and IL-2 production, whereas CD40-gp39 signals co-stimulate B-cell, macrophage, endothelial cell and T-cell activation. Nonetheless, blockade of either of these pathways alone is not sufficient to permit engraftment of highly immunogenic allografts. Here we report that simultaneous but not independent blockade of the CD28 and CD40 pathways effectively aborts T-cell clonal expansion in vitro and in vivo, promotes long-term survival of fully allogeneic skin grafts, and inhibits the development of chronic vascular rejection of primarily vascularized cardiac allografts. The requirement for simultaneous blockade of these pathways for effective inhibition of alloimmunity indicates that, although they are interrelated, the CD28 and CD40 pathways are critical independent regulators of T-cell-dependent immune responses.