CTLA-4 Fusion Protein Research Articles

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the β-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

Establishment of a glycoengineered CHO cell line for enhancing antennary structure and sialylation of CTLA4-Ig

Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (−10.9% and −18.6%, respectively), while bi- and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.

Improved Overall Survival of Patients Treated with Abatacept in Combination with a Calcineurin Inhibitor and Methotrexate Following 7/8 HLA-Matched Unrelated Allogeneic Hematopoietic Stem Cell Transplantation: Analysis of the Center for International Blood and Marrow Transplant Research Database

Improved Overall Survival of Patients Treated with Abatacept in Combination with a Calcineurin Inhibitor and Methotrexate Following 7/8 HLA-Matched Unrelated Allogeneic Hematopoietic Stem Cell Transplantation: Analysis of the Center for International Blood and Marrow Transplant Research Database

Therapeutic Concepts for Treatment of Patients with Non-infectious Uveitis Biologic Disease Modifying Antirheumatic Drugs

Biologic disease modifying antirheumatic drugs (bDMARDs) can be highly efficient in the treatment of various non-infectious uveitis entities. Currently, the TNF-α-inhibitor Adalimumab is the only in-label therapeutic option, whereas, all other bDMARDs need to be given as an off-label therapy. bDMARDs are indicated in diseases refractory to conventional synthetic DMARD therapy and/or systemic steroids, or in patients in whom treatment with those is not possible due to side effects. Therapeutic mechanisms currently employed are cytokine-specific (interferons, inhibition of TNF-α or of interleukin [IL]-1-, IL-6- or IL-17-signalling), inhibit T cell costimulation (CTLA-4 fusion protein), or act via depletion of B cells (anti-CD20). All bDMARDs need to be administered parenterally, and therapy is initiated by the treating internal specialist only after interdisciplinary coordination of all treating subspecialties and after exclusion of contraindications. Regular clinical and laboratory monitoring is mandatory for all patients while under bDMARD therapy.

Costimulatory Pathways in Kidney Transplantation: Pathogenetic Role, Clinical Significance and New Therapeutic Opportunities

Costimulatory pathways play a key role in immunity, providing the second signal required for a full activation of adaptive immune response. Different costimulatory families (CD28, TNF-related, adhesion and TIM molecules), characterized by structural and functional analogies, have been described. Costimulatory molecules modulate T cell activation, B cell function, Ig production, cytokine release and many other processes, including atherosclerosis. Patients suffering from renal diseases present significant alterations of the costimulatory pathways, which might make them particularly liable to infections. These alterations are further pronounced in patients undergoing kidney transplantation. In these patients, different costimulatory patterns have been related to distinct clinical features. The importance that costimulation has gained during the last years has led to development of several pharmacological approaches to modulate this critical step in the immune activation. Different drugs, mainly monoclonal antibodies targeting various costimulatory molecules (i.e. anti-CD80, CTLA-4 fusion proteins, anti-CD154, anti-CD40, etc.) were designed and tested in both experimental and clinical studies. The results of these studies highlighted some criticisms, but also some promising findings and now costimulatory blockade is considered a suitable strategy, with belatacept (a CTLA-4 fusion protein) being approved as the first costimulatory blocker for use in renal transplantation. In this review, we summarize the current knowledge on costimulatory pathways in the setting of kidney transplantation. We describe the principal costimulatory molecule families, their role and clinical significance in patients undergoing renal transplantation and the new therapeutic approaches that have been developed to modulate the costimulatory pathways.

Safely targeting autoimmunity in type 1 diabetes: the MonoPepT1De trial

urrent treatment of type 1 diabetes (T1D) centres on exogenous insulin to replace the deficit due to autoimmune destruction of β-cells. Despite considerable advances in insulin therapy and delivery, patients still have the daily concern of glucose excursions which lead to the serious complications of hyperglycaemia, risks of hypoglycaemia and the significant psychological impact of the disease. Immunotherapy for diabetes aims to target the disease at its core, halting autoimmune destruction of β-cells and removing dependence on exogenous insulin. Early studies in the 1980s provided proof of principle that immunosuppression with agents such as cyclo sporine, 1 azathioprine and prednisolone 2 could lead to remission of β-cell destruction and metabolic improvements, at least in the short term. More recent studies show varying degrees of success in prolonging β-cell function by targeting different aspects of the autoimmune response via drugs such as anti-CD3 3,4 and anti-CD20 5 monoclonal antibodies and CTLA-4 fusion protein. 6 Yet this type of immunosuppression/immune modulation remains generalised and continues to raise concerns over side effects of non-specific targeting of the immune system. Ideal timing for immunotherapy is at or preferably before clinical onset, the point at which there is greatest scope for β-cell salvage. Such early treatment targets a young age group leading to greater concerns over the potential impact of long-term risks such as toxicity, viral reactivation and malignancy, especially as current insulin treatment is relatively simple and safe. Antigen-specific immunotherapy (ASI) offers a safer targeted approach to modulating the autoimmune process towards a tolerogenic response instead of global suppression. The aim is to re-establish tolerance by administration of an autoantigen, which can be in the form of a whole protein or as peptide immunotherapy (PIT), using short peptides derived from key β-cell autoantigens. The use of ASI has been successfully pioneered in clinical allergy 7‐10 with interest fuelling research across autoimmune and inflammatory diseases (Figure 1). Use of peptides has potential benefits over whole antigen administration such as ease of use, enabling a pure highly concentrated epitope to be delivered at lower cost than whole antigen without the risk of complications of biological activity. 11 In PIT, natural pro

Biologika zur Therapie der juvenilen idiopathischen Arthritis - Update 2007

Mit der Erstzulassung des TNF-α-Antagonisten Etanercept zur Behandlung der juvenilen polyartikulären Arthritis vor nunmehr 8 Jahren wurden die Behandlungsmöglichkeiten revolutionär erweitert. Die heute zur Verfügung stehenden drei TNF-Antagonisten Etanercept, Adalimumab und Infliximab erscheinen gleichwertig effektiv bei der Behandlung der Arthritis, wenngleich keine Studien zum Direktvergleich zur Verfügung stehen. Die profunde Entzündungshemmung bewirkt eine Verminderung subjektiver Beschwerden, Morgensteifigkeit, Gelenkschmerzen, Müdigkeit/Fatigue, Verhinderung von Knorpel- und Knochendestruktionen und Aufholwachstum bei der weit überwiegenden Mehrheit der Patienten bei überraschend guter Verträglichkeit. Extraartikuläre Manifestationen, wie z. B. die chronische Uveitis und die systemischen Manifestationen im Rahmen eines Still-Syndroms lassen sich nicht mit gleicher Zuverlässigkeit kontrollieren. Adalimumab scheint aber für den Einsatz bei der JIA-assoziierten Uveitis besonders geeignet. Aufgrund der Verschiedenheit der Zytokindysregulation beim Still-Syndrom ist die Blockade der proinflammatorischen Zytokine Interleukin-1β und Interleukin-6 mit dem Interleukin-1-Rezeptoranatonisten Anakinra oder dem Interleukin-1-Rezeptor Fusionsprotein Rilonacept bzw. mit Tocilizumab, einem Antikörper gegen den Interleukin-6-Rezeptor, bereits in klinischen Studien erfolgreich untersucht. Neben der Zytokinblockade ist die Ausschaltung der Aktivierung von T-Zellen durch Blockade kostimulatorischer Moleküle mit Abatacept, einem CTLA4-IgG-Fusionsprotein, erfolgreich. Die vorliegende Übersicht soll den aktuellen Stand dieser neuen Therapiestrategien der differenziellen Zytokinblockade und der Aktivierungsblockade bei der Therapie der juvenilen idiopathischen Arthritis darstellen.

A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation

By ligating CD80/CD86 (B7) molecules, the synthetic immunomodulatory reagent CTLA4-Ig (soluble synthetic CTLA4 fusion protein) induces expression of the enzyme indoleamine 2,3-dioxygenase (IDO) in some dendritic cells (DCs), which acquire potent T cell regulatory functions as a consequence. Here we show that this response occurred exclusively in a population of splenic DCs co-expressing the marker CD19. B7 ligation induced activation of the transcription factor signal transducer and activator of transcription (STAT1) in sorted CD19+, but not CD19(NEG), DCs. STAT1 activation occurred even when DCs lacked receptors for type II IFN (IFNgamma); however, STAT1 activation and IDO up-regulation were not observed when DCs lacked receptors for type I IFN (IFNalphabeta). Thus, IFNalpha, but not IFNgamma, signaling was essential for STAT1 activation and IDO up-regulation in CD19+ DCs following B7 ligation. Consistent with these findings, B7 ligation also induced sorted CD19+, but not CD19(NEG), DCs to express IFNalpha. Moreover, recombinant IFNalpha induced CD19+, but not CD19(NEG), DCs to mediate IDO-dependent T cell suppression, showing that IFNalpha signaling could substitute for upstream signals from B7. These data reveal that a minor population of splenic DCs expressing the CD19 marker is uniquely responsive to B7 ligation, and that IFNalpha-mediated STAT1 activation is an essential intermediary signaling pathway that promotes IDO induction in these DCs. Thus, CD19+ DCs may be a target for regulatory T cells expressing surface CTLA4, and may suppress T cell responses via induction of IDO.

Local or short‐term systemic costimulatory molecule blockade prolongs rat corneal allograft survival

Costimulatory molecule blockade with antibody-based immunosuppressive agents has been shown to prolong the survival of many types of allograft. The effects were evaluated of local costimulatory molecule blockade with different CTLA4-Ig constructs and of systemic, short-term treatment with an anti-CD28 monoclonal antibody on orthotopic corneal allograft survival in the rat. Adult Fischer-344 rats underwent Wistar-Furth orthotopic corneal grafts. The rats were treated with two different CTLA4-fusion proteins administered intraocularly in the perioperative period, or systemically with anti-CD28 monoclonal antibody JJ319. Corneal graft survival was determined by daily slit-lamp examination. The day of rejection was defined as the first postoperative day on which the iris margin was no longer clearly visible through the corneal graft. Local administration of CTLA4-fusion protein with mutated immunoglobulin constant region domains via a single perioperative intraocular injection prolonged corneal graft survival modestly but significantly (P < 0.05), in contrast to a CTLA4-fusion protein with wild-type immunoglobulin domains, which had no effect on graft survival (P > 0.5). Systemic short-term administration of 400 microg total of an anti-CD28 monoclonal antibody also prolonged corneal graft survival significantly (P < 0.05) and was more effective than systemic administration of 2 mg total of CTLA4-fusion protein (P < 0.05). Local administration of CTLA4-fusion protein with mutated (non-functional) immunoglobulin domains or systemic administration of anti-CD28 monoclonal antibody can prolong corneal allograft survival in the rat.

Ovine dendritic cells transduced with an adenoviral CTLA4eEGFP fusion protein construct induce hyporesponsiveness to allostimulation

CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) 'in frame' with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA), respectively, using anti-GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross-reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF-56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4-EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.

Antitumor immunopreventive effect in mice induced by DNA vaccine encoding a fusion protein of alpha-fetoprotein and CTLA4.

To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4, and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Murine alpha-fetoprotein (mAFP) gene was cloned from total RNA of Hepa1-6 cells by RT-PCR. A DNA vaccine was constructed by fusion murine alpha-fetoprotein gene and extramembrane domain of murine CTLA4 gene. The DNA vaccine was identified by restriction enzyme analysis, sequencing and expression. EL-4 (mAFP) was developed by stable transfection of EL-4 cells with pmAFP. The frequency of cells producing IFN-gamma in splenocytes harvested from the immunized mice was measured by ELISPOT. Mice immunized with DNA vaccine were inoculated with EL-4 (mAFP) cells in back to observe the protective effect of immunization on tumor. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. 1.8 kb mAFP cDNA was cloned from total RNA of Hepa1-6 cells by RT-PCR. The DNA vaccine encoding a fusion protein of mAFP-CTLA4 was constructed and confirmed by restriction enzyme analysis, sequencing and expression. The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR. The ELISPOT results showed that the number of IFN-gamma-producing cells in pmAFP-CTLA4 group was significantly higher than that in pmAFP, pcDNA3.1 and PBS group. The tumor volume in pmAFP-CTLA4 group was significantly smaller than that in pmAFP, pcDNA3.1 and PBS group, respectively. The hepatic and kidney functions in each group were not altered. AFP-CTLA4 DNA vaccine can stimulate potent specific CTL responses and has distinctive antitumor effect on AFP-producing tumor. The vaccine has no impact on the function of mouse liver and kidney.

Targeting and blocking B7 costimulatory molecules on antigen-presenting cells using CTLA4Ig-conjugated liposomes: in vitro characterization and in vivo factors affecting biodistribution.

CTLA4Ig, a fusion protein of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain, inhibits the essential costimulatory signal for full T cell activation via blocking the interaction between CD28 and B7 molecules and renders T cell nonresponsiveness. CTLA4Ig has been used to control deleterious T cell activation in many experimental systems. We hypothesized that by conjugating CTLA4Ig to liposomes the efficacy of CTLA4Ig could be enhanced through multivalent ligand effect, superior targetability, and modification of the fate of ligated costimulatory molecules. Consistent with this hypothesis, liposome-conjugated CTLA4Ig bound to B7 and blocked their binding sites more efficiently than free CTLA4Ig, lowering the half maximal dose for B7 blocking by an order of the magnitude. These results were similar both in B7-1 expressing p815 cells and in activated macrophages. Moreover, CTLA4Ig-liposomes underwent rapid internalization upon cell surface binding through B7 molecules. In allogenic mixed lymphocyte reaction assays, the CTLA4Ig-liposomes were tested to show effective inhibition of T cell proliferation. In vivo, however, when CTLA4Ig-liposomes were injected into mice, a significant fraction was localized to the reticuloendothelial system (RES), presumably because of its binding to Fc receptors expressed on tissue macrophages. The Fc receptor-mediated uptake could be alleviated by coinjection of anti-FcR monoclonal antibody. In the mouse engrafted with pancreatic islets of Langerhans underneath the capsule of one kidney, despite the increased localization in RES, enhanced accumulation of CTLA4Ig-conjugated liposome was observed in the engrafted kidney compared to the contralateral kidney. We show that the conjugation of CTLA4Ig to liposome could increase the efficiency of the targeting by increasing the binding avidity at cellular level and by increasing the concentration at the target site in in vivo system. The biodistribution and circulation time data suggested that the CTLA4Ig-liposomes could be improved upon minimizing the FcR-mediated uptake by Fc receptor-bearing cells. Thus, the strategy of conjugating CTLA4Ig to liposomes could be exploited for immune intervention in transplantation and autoimmune diseases for the efficient blocking of costimulation.

Immunomodulation with CTLA4-Ig in islet transplantation.

The need for permanent, nonspecific, and potentially harmful immunosuppression remains a major obstacle for islet transplantation. The response of a type 1 diabetic recipient to an islet graft includes a specific allogenic immune response and the recurrence of autoimmunity. Free or encapsulated in an immunoisolation device, islet cells are exposed to immune aggression, initiated by donor antigen-presenting cells or by indirect, host antigen-presenting cell-mediated antigen presentation. CTLA4-Ig is a genetically engineered fusion protein of human CTLA4 and the IgG 1 Fc region. It prevents T-cell activation by binding to human B7, which costimulates T cells through CD28. Interesting data were reported in experimental islet transplantation, suggesting that CTLA4-Ig may be slightly but significantly beneficial to islet allograft survival, although studies in autoimmune diabetes are scarce. The main limitations include transient and low levels of expression when CTLA4-Ig is delivered locally, a predominant effect on the direct recognition pathway, and the lack of effect on memory cells. Clinical trials in islet transplantation could be discussed in nonuremic patients, with steroid-free and anticalcineurin-free regimens, in combination with another costimulation blocker, rapamycin, and an anti-interleukin 2 receptor antibody, and with a strategy directed against the recurrence of autoimmunity.

Different Costimulation Signals Used by CD4 + and CD8 + Cells That Independently Initiate Rejection of Allogenic Hepatocytes in Mice

The current study evaluated the role of CD40/CD40 ligand (CD40L) and CD28/B7 costimulation signals during alloimmune responses independently mediated by CD4 + or CD8 + T cells. Allogeneic hepatocytes were transplanted into CD8 or CD4 knock out (KO) mice under cover of costimulatory blockade. Rejection of FVB/N (H-2 q) hepatocytes occurred by day 10 posttransplant in untreated CD8 or CD4 KO (H-2 b) mice. Treatment of CD8 or CD4 KO mice with anti-CD40L monoclonal antibody (mAb; MR1) resulted in significant prolongation of hepatocyte survival indicating that CD40/CD40L interactions were critical in both CD4 + and CD8 + T-cell initiated hepatocyte rejection. Anti-CD40L mAb also prolonged hepatocyte survival in B-cell KO (H-2 b) mice, indicating that the efficacy of CD40/CD40L blockade in preventing hepatocyte rejection was B-cell (and antibody) independent. In contrast, treatment with CTLA4 fusion protein (CTLA4Ig), prolonged hepatocyte survival in CD8 KO but not CD4 KO mice, showing that CD28/B7 interactions were important in CD4 + but not CD8 + T-cell initiated hepatocyte rejection. Under selected circumstances, such as in CD40 KO mice, both CD4 + and CD8 + T cells mediate hepatocyte rejection in the absence of CD40/CD40L costimulation and without a significant contribution from CD28/B7 costimulation signals. These results highlight the disparate roles of CD40/CD40L and CD28/B7 costimulation signals in CD4 + versus CD8 + T-cell mediated immune responses to allogeneic hepatocytes. The CD4 + T-cell independent, CD40L-sensitive, CD28/B7-independent pathway of CD8 + T-cell activation in response to transplantation antigens is novel. (Hepatology 2000;32:1018-1028.)

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

Gammadelta T cells present antigen to CD4+ alphabeta T cells.

Gammadelta and alphabeta TCR+ T cells share many properties and their interactions are likely to be coordinated and regulated. We provide evidence that cattle gammadelta T cells are able to present antigen to CD4+ T cells. To help elucidate their function gammadelta T cell lines were propagated for extensive characterization. Cells expressed high levels of MHC class II and production of co-stimulatory molecules as evidenced by the binding of a CTLA-4 fusion protein and synthesis of CD80 transcripts. These properties and the presence of a well-developed endosomal compartment indicated the cells might function as antigen-presenting cells. Resting CD4+ T cells from calves immunized with ovalbumin or respiratory syncytial virus-antigen proliferated in response to gammadelta T cells pulsed with antigen that appeared to be endocytosed via clathrin-coated pits or specific receptors.

In situ expression of B7 and CD28 receptor families in skin lesions of patients with lupus erythematosus.

To examine the expression of costimulatory molecules of the B7 and CD28 receptor families in active skin lesions of patients with systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus (SCLE), and chronic discoid lupus erythematosus (CDLE). The in situ expression of B7-1, B7-2, BB-1, and CD28 was studied by immunohistochemistry, and B7-1 and B7-2 RNA expression was examined by reverse transcription-polymerase chain reaction. Only in lesional skin from SLE, SCLE, and CDLE patients did dermal and epidermal antigen-presenting cells (APC) express B7-1 and B7-2, particularly when in apposition to CD28+ T cells. These B7-1+ and B7-2+ APC bound CTLA-4 fusion protein. In lesional (but not in nonlesional) skin, keratinocytes expressed BB-1. The majority of infiltrating T cells were CD28+. B7-1 and B7-2 RNA were expressed in lesional skin from SLE, SCLE, and CDLE patients; when dermis was separated from epidermis, only faint B7-1 and B7-2 RNA signals were detectable in the epidermis, indicating that dermal but not epidermal cells were the major source of B7-1 and B7-2 RNA. During treatment, both B7-1 and B7-2 protein and RNA expression were reduced. These in situ findings suggest that costimulation via the B7-CD28 pathway may be important for the generation and/or propagation of T cell activity in skin lesions of humans with lupus erythematosus. Thus, the manipulation of this pathway (e.g., by CTLA-4 fusion protein) could be an important target for the development of future therapies for LE.

Long-term acceptance of major histocompatibility complex-mismatched cardiac allograft induced by a low dose of CTLA4IgM plus FK506.

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2b) neonatal hearts were transplanted to CBA/J (H-2b) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.

Immunomodulation of transplant rejection using monoclonal antibodies and soluble receptors.

The main objective of our studies has been to optimize the effects of monoclonal antibodies (MAbs) and other immunosuppressive reagents to enhance organ graft survival. One such agent is OKT3, a MAb that is directed against the CD3 component of the human T-cell receptor (TCR) complex. Treatment of a rejection episode with OKT3 results in a rapid and efficient clearing of circulating T cells and reversal of most rejection episodes. Its wider use in transplantation and in the treatment of immune-mediated disease is limited by adverse reactions that follow the initial dose, the production of neutralizing Abs, and the transient nature of the immunosuppression. We have engineered CDR-grafted "humanized" anti-CD3 MAbs that lack Fc-receptor binding activity through mutagenesis of amino acids in the Fc portion of the MAb. This results in an immunosuppressive anti-CD3 MAb that is less antigenic and one that does not induce the first-dose side effects. In addition, we have pursued a goal of developing a therapy that will induce donor-specific tolerance while maintaining overall recipient immune competency. Because antigen-specific T-cell activation depends not only on TCR-ligand interaction, but also on additional costimulatory signals mediated by accessory molecules such as CD28, blocking the binding of CD28 on T cells to its ligand B7, during TCR engagement, might modulate transplantation responses. Using a soluble fusion protein of human CTLA4, CTLA4-Ig, that binds B7 with high affinity, inhibition of human pancreatic islet rejection that occurs, at least in part, by affecting T-cell recognition of human B7+ antigen-presenting cells has been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an additional ligand, distinct from B7, for the CTLA-4 receptor.

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complex complexes and costimulatory signals provided by antigen-presenting cells (APCs). The best-characterized costimulatory molecule to date is the B7 antigen, a member of the immunoglobulin family that binds two receptors, CD28 and CTLA-4, expressed on the T-cell surface. Using the anti-mouse B7 (mB7) monoclonal antibody (mAb) 16-10A1, which we recently developed, we found that mB7 is indeed an important costimulatory ligand for the antigen-specific activation of murine T cells by B lymphocytes. Three lines of evidence suggest, however, the existence of at least one additional ligand for the CTLA-4 receptor. First, a soluble fusion protein of human CTLA-4 and the IgG1 Fc region, termed CTLA4Ig, blocks better than the anti-mB7 mAb the allogeneic stimulation of T cells by unfractionated splenic APCs. Second, saturating amounts of anti-mB7 mAb do not significantly block binding of fluorescein isothiocyanate-conjugated CTLA4Ig to activated splenic APCs. Furthermore, CTLA4Ig but not the anti-mB7 mAb reacts with the M12 and M12.C3 cell lines. The identification of an additional ligand for CTLA-4 may have applications to the treatment of autoimmune disease and transplant-associated disorders.