Antigen-specific immunotherapy with apitopes suppresses generation of FVIII inhibitor antibodies in HLA-transgenic mice.

Hemophilia A (HA) is a blood clotting disorder that is caused by various genetic deficiencies in the factor VIII (FVIII)-encoding F8 gene. Patients receiving FVIII-replacement therapy are at risk for developing neutralizing antibodies (FVIII inhibitors), rendering the FVIII-replacement therapy ineffective. Immunological tolerance toward FVIII can be achieved through immune tolerance induction protocols in some patients, but this is a lengthy and costly desensitization program. Long-term eradication of inhibitors in patients with HA could be achieved by antigen-specific immunotherapy targeting CD4+ T-cells, because formation of FVIII inhibitors is T-cell dependent. Here, we report a peptide-based antigen-specific immunotherapy that is designed to specifically reestablish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified 2 FVIII immunodominant peptides in immunized HLA-DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimized for tolerogenicity. These modified peptide analogs were initially screened for recognition using FVIII-specific T-cell hybridoma clones from FVIII-immunized HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1* allelic variants. The combination of these 2 FVIII apitopes (ATX-F8-117), administered according to a dose-escalation protocol, promoted T-cell tolerance toward FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates anti-FVIII T-cell and B-cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to suppress and treat inhibitor formation in susceptible patients with HA.

Regulatory CD4+ T cells and the control of autoimmune disease.

The immune system is a delicately balanced network of interacting cells. In recent years, the concept of immune regulation/suppression has been firmly established, and both natural and induced regulatory cells play vital roles in protection from autoimmune disease. Recent work has revealed the diverse nature of regulatory CD4+ T (Treg) cells and the molecules involved in their function. Innate and adaptive responses to infection are able to override the suppressive properties of such regulatory cells, whereas several reports point to deficiencies in regulatory cell function in autoimmune disease. Protocols have been developed that allow the expansion of Treg cells in vitro and their antigen-specific induction in vivo. A full understanding of Treg differentiation and function will facilitate the development of improved strategies for prevention and treatment of autoimmune diseases.

Natural and induced regulatory T cells.

Mucosal antigen delivery can induce tolerance, as shown by suppression of subsequent responses to antigen. Our previous work showed that both intranasal and oral routes of antigen delivery were effective but indicated that the intranasal route might be more reliable. Intranasal peptide administration induced cells that could mediate bystander suppression of responses to associated antigenic epitopes. Here, we discuss further investigation into the nature of intranasal, peptide-induced tolerance. Cells from mice treated with intranasal peptide became anergic and shut down secretion of cytokines such as IL-2, but still secreted IL-10. This latter cytokine was required for suppression of immune responses in vivo even though suppression of responses in vitro was IL-10 independent. Intranasal peptide induced a subset of CD25(-), CTLA-4(+) regulatory cells that suppressed naive cell function in vitro and in vivo. We provide evidence that these cells arise from CD25(-) precursors and differentiate independently from natural CD25(+) regulatory cells. IL-10-secreting regulatory cells are also found in the peripheral blood of humans and can be induced by soluble peptide administration. This route of tolerance induction offers promise as a means of antigen-specific immunotherapy of allergic and autoimmune conditions in humans.

Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10-secreting Th1 cells.

Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)-specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10-secreting CD4(+) T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4(+) T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-gamma to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN-gamma and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

Natural and induced regulatory T cells: targets for immunotherapy of autoimmune disease and allergy.

Recent advances in immunology have greatly increased our understanding of immunological tolerance. In particular, there has been a resurgence of interest in mechanisms of immune regulation. Immune regulation refers to the phenomenon, previously known as immune suppression, by which excessive responses to infectious agents and hypersensitivities to otherwise innocuous antigens such as self antigens and allergens are avoided. We now appreciate that various distinct cell types mediate immune suppression and that some of these may be induced by appropriate administration of antigens, synthetic peptides and drugs of various types. The induction of antigen specific immunotherapy for treatment of autoimmune and allergic diseases remains the 'holy grail' for treatment of these diseases. This goal comes ever closer as understanding of the mechanisms of immune suppression and in particular antigen specific immunotherapy increases. Here we review evidence that immune suppression is mediated by various different subsets of CD4 T cells.

Antigen-induced IL-10+ regulatory T cells are independent of CD25+ regulatory cells for their growth, differentiation, and function.

Recent studies have emphasized the importance of T cells with regulatory/suppressor properties in controlling autoimmune diseases. A number of different types of regulatory T cells have been described with the best characterized being the CD25(+) population. In addition, it has been shown that regulatory T cells can be induced by specific Ag administration. In this study, we investigate the relationship between peptide-induced, CD4(+) regulatory T cells and naturally occurring CD4(+)CD25(+) cells derived from the Tg4 TCR-transgenic mouse. Peptide-induced cells were FoxP3(-) and responded to Ag by secreting IL-10, whereas CD25(+) cells failed to secrete this cytokine. Both cell types were able to suppress the proliferation of naive lymphocytes in vitro although with distinct activation sensitivities. Depletion of CD25(+) cells did not affect the suppressive properties of peptide-induced regulators. Furthermore, peptide-induced regulatory/suppressor T cells could be generated in RAG(-/-), TCR-transgenic mice that do not spontaneously generate CD25(+) regulatory cells. These results demonstrate that these natural and induced regulatory cells fall into distinct subsets.

Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance.

Repetitive antigen stimulation induces peripheral T cell tolerance in vivo. It is not known, however, whether multiple stimulations merely suppress T cell activation or, alternatively, change the transcriptional program to a distinct, tolerant state. In this study, we have discovered that STAT3 and STAT5 were activated in response to antigen stimulation in vivo, in marked contrast to the suppression of AP-1, NF-kappaB and NFAT. In addition, a number of transcription factors were induced in tolerant T cells following antigen challenge in vivo, including T-bet, Irf-1 and Egr-2. The altered transcription program in tolerant cells associates closely with the suppression of cell cycle progression and IL-2 production, as well as with the induction of IL-10. Studies of T-bet and Egr-2 show that the function of T-bet in peptide treatment-induced regulatory T cells is not associated with Th1 differentiation, but correlates with the suppression of IL-2, whereas expression of Egr-2 led to an up-regulation of the cell cycle inhibitors p21(cip1) and p27(kip). Our results demonstrate a balanced transcription program regulated by different transcription factors for T cell activation and/or tolerance during antigen-induced T cell responses. Persistent antigen stimulation can induce T cell tolerance by changing the balance of transcription factors.

IL-2 overcomes the unresponsiveness but fails to reverse the regulatory function of antigen-induced T regulatory cells.

Intranasal administration of peptide Ac1-9[4Y], based on the N-terminal epitope of myelin basic protein, can induce CD4(+) T cell tolerance, and suppress experimental autoimmune encephalomyelitis induction. The peptide-induced regulatory T (PI-T(Reg)) cells failed to produce IL-2, but expressed IL-10 in response to Ag and could suppress naive T cell responses in vitro. Analysis of Jak-STAT signaling pathways revealed that the activation of Jak1, STAT3, and STAT5 were induced in tolerant T cells after Ag stimulation in vivo. In addition, the expression of suppressor of cytokine signaling 3 was induced in tolerant T cells, suggesting that cytokines regulate the tolerant state of the PI-T(Reg) cells. Stimulation of PI-T(Reg) cells in vitro with IL-10 induced Jak1 and STAT3 activation, but not STAT5, suggesting that IL-10 is important, but not the only cytokine involved in the development of T cell tolerance. Although IL-2 expression was deficient, stimulation with IL-2 in vitro induced Jak1 and STAT5 activation in PI-T(Reg) cells, restored their proliferative response to antigenic stimulation, and abrogated PI-T(Reg)-mediated suppression in vitro. However, the addition of IL-2 could not suppress IL-10 expression, and the IL-2 gene remained inactive. After withdrawal of IL-2, the PI-T(Reg) cells regained their nonproliferative state and suppressive ability. These results underline the ability of the immune system to maintain tolerance to autoantigens, but at the same time having the ability to overcome the suppressive phenotype of tolerant T cells by cytokines, such as IL-2, during the protective immune response to infection.

Experimental autoimmune encephalomyelitis induction in naive mice by dendritic cells presenting a self-peptide.

Self-reactive T cells escape deletion in the thymus and are found in the peripheral repertoire. Because bone-marrow-derived dendritic cells (BM-DC) are potent activators of antigen-specific T cells, these cells could theoretically activate self-reactive T cells leading to autoimmunity. We investigated whether BM-DC could induce the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our results show that transfer of BM-DC presenting a self-peptide from the myelin oligodendrocyte glycoprotein (MOG35-55) into naive mice induced EAE 7-14 days later. MOG35-55-specific T cells of the Th1 phenotype were present in the lymph nodes and spleens of mice that received live peptide-pulsed BM-DC. Heat-killed or formaldehyde-fixed BM-DC presenting MOG35-55 could induce neither clinical signs of EAE nor a measurable T-cell response in vitro. These data show that live BM-DC presenting a self-antigen can induce the organ-specific autoimmune disorder EAE in a non-transgenic system. Therefore, this new EAE model could be used as a more clinically relevant model for the human disease multiple sclerosis. These findings could also have implications for the use of DC immunotherapy in a clinical setting.

Role for IL-10 in suppression mediated by peptide-induced regulatory T cells in vivo.

Regulatory CD4(+) T cells were induced in the Tg4 TCR transgenic mouse specific for the N-terminal peptide (Ac1-9) of myelin basic protein by intranasal administration of a high-affinity MHC-binding analog (Ac1-9[4Y]). Peptide-induced tolerant cells (PItol) were anergic, failed to produce IL-2, but responded to Ag by secretion of IL-10. PItol cells were predominantly CD25(-) and CTLA-4(+) and their anergic state was reversed by addition of IL-2 in vitro. PItol cells suppressed the response of naive Tg4 cells both in vitro and in vivo. The in vitro suppression mediated by these cells was not reversed by cytokine neutralization and was cell-cell contact-dependent. However, suppression of proliferation and IL-2 production by PItol cells in vivo was abrogated by neutralization of IL-10. These results emphasize an important role for IL-10 in the function of peptide-induced regulatory T cells in vivo and highlight the caution required in extrapolating mechanisms of T regulatory cell function from in vitro studies.

Induction of experimental autoimmune encephalomyelitis in the absence of c-Jun N-terminal kinase 2.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-dependent, organ-specific autoimmune model commonly used to investigate mechanisms involved in the activation of autoreactive T(h)1 cells. Mitogen-activated protein kinases such as c-Jun N-terminal kinase (Jnk) 1 and 2 play an important role in the differentiation of naive precursors into T(h)1 or T(h)2 effector cells. To investigate the role of Jnk2 on autoimmunity, Jnk2(-/-) and wild-type mice were immunized with the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide and the onset of EAE studied. Surprisingly, Jnk2(-/-) mice were as susceptible to EAE as wild-type mice, regardless of whether low or high antigen doses were used to induce disease. In vitro stimulation of lymph node cells from Jnk2(-/-) and wild-type mice resulted in comparable proliferation in response to MOG35-55, Mycobacterium tuberculosis and concanavalin A. MOG35-55-specific T cells lacking Jnk2 showed a T(h)1 cytokine profile with IFN-gamma, but no IL-4 or IL-5 production. No differences in the types of infiltrating cells or myelin destruction in the central nervous system were found between Jnk2(-/-) and wild-type mice, indicating that lack of Jnk2 does not alter the effector phase of EAE. Our results suggest that, despite involvement in T(h)1/T(h)2 differentiation in vitro, Jnk2 is necessary neither for the induction nor effector phase of MOG35-55-induced EAE and nor is it required for antigen-specific IFN-gamma production.