Mechanisms of tolerance induction: blockade of co-stimulation

Induction of tolerance to transplantation antigens is believed to be a promising way to achieve long-term allograft survival without a deleterious immunosuppressive regimen. T-cell activation, which is an essential feature of graft rejection, requires a first signal provided by T-cell receptor (TCR) ligation and a second signal provided by engagement of co-stimulatory molecules with their respective ligands on antigen-presenting cells. The coordinated triggering of these two independent signalling systems ensures the full T-cell activation, including proliferation and acquisition of effector function. TCR occupancy in the absence of co-stimulatory signals leads to a sustained loss of antigen responsiveness called clonal anergy, which could be of major importance in transplantation. In vivo, co-stimulation blockade was indeed shown to allow for long-term allograft survival in several transplantation models. However, the current continuous identification of new co-stimulatory molecules suggests that a functional redundancy of the system exists and that tolerance to transplantation antigens might be achieved more easily through the combined blockade of two or several co-stimulatory signals. In this review, we analyse the biological effects of the disruption of some co-stimulation pathways in vitro and in vivo and discuss their potential interest for tolerance induction.     

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.     

New cytokine therapeutics for inflammatory bowel disease

Conventional therapy for inflammatory bowel diseases rely on corticosteroids and 5-aminosalicylates combined with immunosuppressive agents for maintenance. These drugs are not always effective and may inflict serious side effects. Other therapies are therefore awaited. Infliximab, a monoclonal antibody against the pro-inflammatory cytokine TNF-alpha has been successfully applied as a treatment for Crohn's disease. The mechanism of action of this drug extends beyond the level of TNF-alpha scavenging and includes induction of apoptosis of effector cells. Numerous anti-TNF antibodies have been developed and are currently evaluated in clinical trials. Other targets for monoclonal antibodies include integrins and cytokines involved in T-cell differentiation and activation. Likewise recombinant proteins that moderate TNF bioactivity and lymphocyte function have been developed. The therapeutic effect of recombinant interleukin-10 seems to be dependent on local delivery of the protein. Antisense therapy targeting lymphocyte migration has also been tested in IBD. Finally, the conventional drug thalidomide and possibly MAP-kinase inhibitors may become novel treatment entities for IBD.     

Co-stimulation agonists as a new immunotherapy for autoimmune diseases

Lymphocytes are important in the pathogenesis of many autoimmune diseases. Blocking co-stimulatory signals for T-cell activation has been widely used as an approach to treating autoimmunity, but it has encountered limited clinical success. Some agonistic monoclonal antibodies to co-stimulatory molecules greatly enhance immune responses mediated by T cells, such as antiviral, anti-tumor and alloresponses. Surprisingly, recent studies have demonstrated that these agonists have profound therapeutic effects on autoimmune diseases by potentially depleting autoreactive lymphocytes or by inhibiting their function. These findings imply that signaling through co-stimulatory molecules can have diametric outcomes in modulating immune responses, thereby providing a novel approach to the treatment of autoimmune diseases.     

T-cell therapies for T-cell lymphoma

T-cell lymphomas represent a subpopulation of non-Hodgkin lymphomas with poor outcomes when treated with conventional chemotherapy. A variety of novel agents have been introduced as new treatment strategies either as first-line treatment or in conjunction with chemotherapy. Immunotherapy has been demonstrated to be a promising area for new therapeutics, including monoclonal antibodies and adoptive cellular therapeutics. T-cell therapeutics have been shown to have significant success in the treatment of B-cell malignancies and are rapidly expanding as potential treatment options for other cancers including T-cell lymphomas. Although treating T-cell lymphomas with T-cell therapeutics has unique challenges, multiple targets are currently being studied both preclinically and in clinical trials.     

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells

Background aimsThe targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues.MethodsTo explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs.ResultsA lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity.ConclusionsThese data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target.     

How the immune system protects the host from infection.

The immune system is made up of sets of interacting cells. The first to respond in all cases are the antigen-presenting cells (APCs), which are equipped with receptors for microbial patterns. Engagement of these receptors induces co-stimulatory molecules on the surface of the APCs, and allows it to stimulate potent CD4 T-cell responses, and also CD8 T-cell responses. This in turn leads to B-cell-derived antibody responses. The entire response is controlled by suppressor T cells, as predicted many years ago by Richard Gershon.     

Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway

LIGHT was recently described as a member of the tumor necrosis factor (TNF) 'superfamily'. We have isolated a mouse homolog of human LIGHT and investigated its immunoregulatory functions in vitro and in vivo. LIGHT has potent, CD28-independent co-stimulatory activity leading to T-cell growth and secretion of gamma interferon and granulocyte-macrophage colony-stimulating factor. Gene transfer of LIGHT induced an antigen-specific cytolytic T-cell response and therapeutic immunity against established mouse P815 tumor. In contrast, blockade of LIGHT by administration of soluble receptor or antibody led to decreased cell-mediated immunity and ameliorated graft-versus-host disease. Our studies identify a previously unknown T-cell co-stimulatory pathway as a potential therapeutic target.     

Regulation of immunity by anti-T-cell antibodies.

Current pharmacologic approaches to immune suppression leave much to be desired. The prevention of allograft rejection and the suppression of autoimmunity generally require treatment with corticosteroids or cytotoxic drugs, or both, which may not be sufficiently effective and which frequently cause serious immediate and long-term complications. With the advent of monoclonal antibody technology, it has become possible to identify and selectively inhibit distinct elements in the immune system that contribute to pathologic immune responses. This achievement has led to new therapeutic strategies that may be safer and more effective than the immunosuppressive therapies currently available. Many of these strategies focus on subsets of T cells because of the critical importance of T cells in immune responses. Monoclonal antibodies directed against CD4 + T cells, T-cell activation antigens, and T-cell receptor families have all shown promise in animal models and, in some cases, in preliminary human trials. The challenge now is to translate this promise into practical new forms of immunosuppressive therapy.     

Increased frequency of activated T-cells in the Helicobacter pylori-infected antrum and duodenum

Helicobacter pylori colonize the human stomach and duodenum. The infection has been shown to induce a strong T-cell response in the stomach, whereas the response within the duodenum has been poorly characterized. Furthermore, it remains to be elucidated whether the T-cell response may contribute to ulcer formation in the host. In this study, the frequency of different T-cell subsets, their degree of activation and expression of co-stimulatory receptors in biopsies from the duodenum as well as the antrum were studied by immunohistochemistry and flow cytometry. It was also evaluated whether there are differences in the T-cell responses between duodenal ulcer patients and asymptomatic carriers that might explain why only 10-15% of the infected subjects develop duodenal ulcers. The frequencies of CD4+, CD8+ and CD45RO+, i.e. memory T-cells, were significantly increased in the antrum, and the number of CD25+ cells was considerably higher in both the antrum and duodenum of duodenal ulcer patients and asymptomatic carriers as compared to uninfected individuals. Interestingly, the levels of immunosuppressive CTLA-4+ cells were significantly higher in the duodenum of duodenal ulcer patients, as compared to the asymptomatic carriers. H. pylori cause activation of T-cells in the duodenum as well as in the stomach. Our observation of higher levels of CTLA-4+ cells in the duodenum of duodenal ulcer patients than in the asymptomatic carriers suggests that a suppressive T-cell response may be related to the development of duodenal ulcers.     

Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population

T-cell co-stimulation delivered by the molecules B7-1 or B7-2 through CD28 has a positive effect on T-cell activation, whereas engagement of cytotoxic T-lymphocyte antigen 4 (CTLA-4) by these molecules inhibits activation. In vivo administration to mice of blocking monoclonal antibodies or Fab fragments against CTLA-4 can augment antigen-specific T-cell responses and, thus, therapy with monoclonal antibody against CTLA-4 has potential applications for tumor therapy and enhancement of vaccine immunization. The effects of B7-1 and B7-2 co-stimulation through CD28 depend on the strength of the signal delivered through the T-cell receptor (TCR) and the activation state of T cells during activation. Thus, we sought to determine whether these factors similarly influence the effect of B7-mediated signals delivered through CTLA-4 during T-cell activation. Using freshly isolated human T cells and Fab fragments of a monoclonal antibody against CTLA-4, we demonstrate here that CTLA-4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation. Thus, for whole T-cell populations, blocking a negative signal may paradoxically inhibit immune responses. These results provide a theoretical framework for clinical trials in which co-stimulatory signals are manipulated in an attempt to modulate the immune response in human disease.     

Co-stimulatory members of the TNFR family: keys to effective T-cell immunity?

Interactions between co-stimulatory ligands and their receptors are crucial for the activation of T cells, the prevention of tolerance and the development of T-cell immunity. It is now evident that members of the immunoglobulin-like CD28-B7 co-stimulatory family cannot fully account for an effective long-lasting T-cell response or the generation of memory T cells. Several members of the tumour-necrosis factor receptor (TNFR) superfamily--OX40, 4-1BB, CD27, CD30 and HVEM (herpes-virus entry mediator)--are poised to deliver co-stimulatory signals both early and late after encounter with antigen. The roles of these molecules in initiating and sustaining the T-cell response and in promoting long-lived immunity are discussed.     

Hybrid-cell vaccines for cancer immune therapy

Hybrid cells generated by fusing allogenic-presenting cells, such as dendritic cells, with tumor cells are a new tool in cancer immunotherapy which are designed to enhance the immunogenicity of antigenic tumors by presenting the whole spectrum of tumor-associated antigens, by providing the co-stimulatory molecules required for T-cell activation, and by the expression of allogenic MHC molecules for recruitment and activation of T-cell help. This approach has been successfully tested in animal models as well as in clinical phaseI/II trials with various tumors. Besides clinical repsonses, induction of tumor-specific cytolytic T-cells were observed. The electrofusion protocol described here has the advantage of high fusion efficiency, high hybrid-cell viability, as well as high reproducibility, and can be used for various tumor cell types after minor adjustments are made to the instrument settings in order to process large numbers of dendritic cells with consistent efficiencies.     

The new immunosuppression

New knowledge on how lymphocytes become tolerant to antigens is now enabling novel tolerance-harnessing strategies to enter the clinical arena. In the field of transplantation, monoclonal antibodies used either to deplete lymphocytes or to block T-cell function can induce tolerance in mice and non-human primates. Understanding the mechanisms underlying tolerance should enable application to the clinic. Harnessing of tolerance mechanisms may enable more judicious use of conventional immunosuppressive agents even to the point of maintenance monotherapy, so limiting drug side effects and ensuring compliance.     

Osthole enhances the immunosuppressive effects of bone marrow-derived mesenchymal stem cells by promoting the Fas/FasL system

Thanks to the advantages of easy harvesting and escape from immune rejection, autologous bone marrow-derived mesenchymal stem cells (BMSCs) are promising candidates for immunosuppressive therapy against inflammation and autoimmune diseases. However, the therapy is still challenging because the immunomodulatory properties of BMSCs are always impaired by immunopathogenesis in patients. Because of its reliable and extensive biological activities, osthole has received increased clinical attention. In this study, we found that BMSCs derived from osteoporosis donors were ineffective in cell therapy for experimental inflammatory colitis and osteoporosis. In vivo and in vitro tests showed that because of the down-regulation of Fas and FasL expression, the ability of osteoporotic BMSCs to induce T-cell apoptosis decreased. Through the application of osthole, we successfully restored the immunosuppressive ability of osteoporotic BMSCs and improved their treatment efficacy in experimental inflammatory colitis and osteoporosis. In addition, we found the immunomodulatory properties of BMSCs were enhanced after osthole pre-treatment. In this study, our data highlight a new approach of pharmacological modification (ie osthole) to improve the immune regulatory performance of BMSCs from a healthy or inflammatory microenvironment. The development of targeted strategies to enhance immunosuppressive therapy using BMSCs may be significantly improved by these findings.     

The thymus and central tolerance

T-cell differentiation in the thymus generates a peripheral repertoire of mature T cells that mounts strong responses to foreign antigens but is largely unresponsive to self-antigens. This state of specific immunological tolerance to self-components involves both central and peripheral mechanisms. Here we review the process whereby many T cells with potential reactivity for self-antigens are eliminated in the thymus during early T-cell differentiation. This process of central tolerance (negative selection) reflects apoptosis and is a consequence of immature T cells receiving strong intracellular signalling through T-cell receptor (TCR) recognition of peptides bound to major histocompatibility complex (MHC) molecules. Central tolerance occurs mainly in the medullary region of the thymus and depends upon contact with peptide-MHC complexes expressed on bone-marrow-derived antigen-presenting cells (APCs); whether tolerance also occurs in the cortex is still controversial. Tolerance induction requires a combination of TCR ligation and co-stimulatory signals. Co-stimulation reflects interaction between complementary molecules on T cells and APCs and probably involves multiple molecules acting in consort, which may account for why deletion of individual molecules with known or potential co-stimulatory function has little or no effect on central tolerance. The range of self-antigens that induce central tolerance is considerable and, via low-level expression in the thymus, may also include tissue-specific antigens; central tolerance to these latter antigens, however, is likely to be limited to high-affinity T cells, leaving low-affinity cells to escape. Tolerance to alloantigens and the possibility of using central tolerance to promote acceptance of allografts are discussed.     

Molecular targets for existing and novel immunosuppressive drugs

Anti-neoplastic cytostatic antiproliferative agents, such as methotrexate, 6-mercaptopurine and cyclophosphamide, were originally used as immunosuppressive drugs. Although these agents induced only modest anti-rejection activity, they caused serious non-specific bone marrow suppression, impairing host resistance and increasing the incidence of infections. Unlike these non-selective agents, cyclosporine A, tacrolimus and sirolimus act more selectively on different stages of the T-lymphocyte (T-cell) and B-lymphocyte (B-cell) activation cycles; however, cyclosporine and tacrolimus are nephrotoxic, whereas sirolimus causes hypertriglyceridaemia. Thus, despite this progress, continued efforts must be made to develop and test new, potentially very selective agents. The agent 15-deoxyspergualin moderately inhibits both mitogen-stimulated T-cell proliferation and the generation of cytotoxic T lymphocytes (CTLs) but does not affect the production of interleukin 2 (IL-2). Another drug, FTY720, has a unique action to prevent rejection, by altering the homing of lymphocytes to the lymphoid compartments. The newest members of the family of antiproliferative agents, namely mycophenolate mofetil, leflunomide and brequinar, are potentially more selective than their predecessors. However, the most promising agents are produced using antisense technology. This approach involves the design of antisense oligodeoxynucleotides; these novel drugs are designed to block allograft rejection by blocking selected messenger RNA (mRNA). This review outlines the mechanisms of action, the limitations of application and the molecular or cellular targets of traditional agents, newly developed drugs and also antisense technology, which is an example of a new application of molecular medicine.