Leslie P. Cousens

Interferon alpha/beta-mediated inhibition and promotion of interferon gamma: STAT1 resolves a paradox.

Induction of high systemic levels of type 1 interferons (IFNs) IFN-α and IFN-β is a hallmark of many viral infections. In addition to their potent antiviral effects, these cytokines mediate a number of immunoregulatory functions and can promote IFN-γ expression in T cells. However, during viral infections of mice IFN-γ production is not always observed at the same time as systemic IFN-α/β production and when, elicited at these times, is IFN-α/β–independent. We demonstrate that type 1 interferons not only fail to induce, but also act to inhibit, IFN-γ expression by both NK and T cells. The mechanism of inhibition is dependent upon the IFN-α/β receptor and the signal transducer and activator of transcription 1 (STAT1). In the absence of STAT1, not only are the IFN-α/β–mediated inhibitory effects completely abrogated, but the cytokines themselves can induce IFN-γ expression. These results indicate that endogenous biochemical pathways are in place to negatively regulate NK and T cell IFN-γ expression elicited by IFN-α/β or other stimuli, at times of innate responses to viral infections. They also show that type 1 interferon signaling can occur through STAT1-dependent and independent mechanisms and suggest that efficient induction of IFN-γ expression by IFN-α/β requires STAT1 regulation. Such immunoregulatory pathways may be critical for shaping the endogenous innate and virus-specific adaptive immune responses to viral infections.

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity.

Tregitope update: Mechanism of action parallels IVIg

In the course of screening immunoglobulin G (IgG) sequences for T cell epitopes, we identified novel Treg epitope peptides, now called Tregitopes, contained in the highly conserved framework regions of Fab and Fc. Tregitopes may provide one explanation for the expansion and stimulation of Treg cells following intravenous immunoglobulin (IVIg) therapy. Their distinguishing characteristics include in silico signatures that suggest high-affinity binding to multiple human HLA class II DR and conservation across IgG isotypes and mammalian species with only minor amino acid modifications. Tregitopes induce expansion of CD4(+)/CD25(hi)/FoxP3(+) T cells and suppress immune responses to co-incubated antigens in vitro. By comparing the human IgG Tregitopes (hTregitopes 167 and 289, located in the IgG CH1 and CH2 domains) and Fab to murine sequences, we identified class II-restricted murine Tregitope homologs (mTregitopes). In vivo, mTregitopes suppress inflammation and reproducibly induce Tregs to expand. In vitro studies suggest that the Tregitope mechanism of action is to induce Tregs to respond, leading to production of regulatory signals, followed by modulation of dendritic cell phenotype. The identification of Treg epitopes in IgG suggests that additional Tregitopes may also be present in other autologous proteins; methods for identifying and validating such peptides are described here. The discovery of Tregitopes in IgG and other autologous proteins may lead to the development of new insights as to the role of Tregs in autoimmune diseases.

In Vitro and In Vivo Studies of IgG-derived Treg Epitopes (Tregitopes): A Promising New Tool for Tolerance Induction and Treatment of Autoimmunity

Tregitopes are regulatory T cell epitopes derived from immunoglobulin G (IgG) that stimulate CD25+ FoxP3+ T cells to expand. In conjunction with these Tregs, Tregitopes can prevent, treat, and even cure autoimmune disease in mouse models, suppress allo-specific responses in murine transplant models, inhibit CD8+ T cell responses to recombinant adeno-associated virus (AAV) gene transfer vectors, and induce adaptive Tregs in DO11.10 mice. In this review of recent Tregitope studies, we summarize their effects in vitro and describe recent comparisons between intravenous IgG (IVIG) and Tregitopes in standard in vivo immune tolerance models. Further investigations of the mechanism of action of Tregitopes in the preclinical models described here will lead to clinical trials where Tregitopes may have the potential to alter the treatment of autoimmune disease, transplantation, and allergy, and to improve the efficiency of gene and protein replacement therapies.

Early cytokine responses to viral infections and their roles in shaping endogenous cellular immunity.

Immune responses to different infectious agents have overlapping constituents. However, certain of these are induced to higher levels and/or play more important roles in defense under conditions of infections with particular agents. In the case of many viral infections, immune responses important in protection include the unique or uniquely prominent systemic type 1 interferons (IFN), i.e., IFN-α/β, activation and function of natural killer (NK) cells, and activation and function of CD8+ T cells. The studies presented here extend characterization of endogenous innate cytokine responses elicited at early times during viral infections and dissection of immunoregulatory roles for these cytokines in promoting cell-mediated immunity.

Stat3 cleavage by caspases: impact on full-length Stat3 expression, fragment formation, and transcriptional activity.

Stat3 and its isoforms belong to a family of cytoplasmic transcription factors that affect the synthesis of various proteins. Caspases are cysteinyl-aspartate proteases that function under apoptotic and non-apoptotic conditions. We now report that, in addition to transcriptional splicing, Stat3 fragmentation can be mediated by caspases. Caspase activation in DU145 cells was achieved by staurosporine (STS) exposure, and Western analysis revealed a reduction in full-length Stat3 (fl-Stat3) expression that was caspase-mediated. This proteolytic relationship was further studied by exposing purified Stat3 protein to a mixture of active caspases under cell-free conditions. This demonstrated that caspases directly cleaved Stat3 and Stat3 cleavage was accompanied by the apparent formation of cleavage fragment(s). Stat3 cleavage fragments, reflecting multiple caspase cleavage sites, also were observed in vitro following STS exposure in DU145 cells and in HEK293T cells transfected to express Stat3 truncation mutants. The impact of cleavage on Stat3 transcriptional activity next was assessed and revealed that cleavage of fl-Stat3 was accompanied by reductions in Stat3-DNA binding, Stat3-driven reporter protein (luciferase) activity, and the expression of selected Stat3-dependent genes. Further, reduced Stat3 expression correlated with increased sensitivity to apoptotic stimuli. In concomitant experiments, reporter activity was assessed in Stat3 truncation mutant-expressing HEK293T cells and revealed that, under non-apoptotic conditions, expression of different Stat3 fragments induced differential effects on Stat3-driven luciferase activity. These findings demonstrate that fl-Stat3 undergoes proteolytic processing by caspases that reduces its expression and leads to the formation of cleavage fragments that may modulate Stat3 transcriptional activity.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

Beyond humanization and de-immunization: tolerization as a method for reducing the immunogenicity of biologics.

Immune responses to some monoclonal antibodies (mAbs) and biologic proteins interfere with their efficacy due to the development of anti-drug antibodies (ADA). In the case of mAbs, most ADA target ‘foreign’ sequences present in the complementarity determining regions (CDRs). Humanization of the mAb sequence is one approach that has been used to render biologics less foreign to the human immune system. However, fully human mAbs can also drive immunogenicity. De-immunization (removing epitopes) has been used to reduce biologic protein immunogenicity. Here, we discuss a third approach to reducing the immunogenicity of biologics: introduction of Treg epitopes that stimulate Treg function and induce tolerance to the biologic protein. Supplementing humanization (replacing xeno-sequences with human) and de-immunization (reducing T effector epitopes) with tolerization (introducing Treg epitopes) where feasible, as a means of improving biologics ‘quality by design’, may lead to the development of ever more clinically effective, but less immunogenic, biologics.

Teaching tolerance: New approaches to enzyme replacement therapy for Pompe disease.

Babies born with Pompe disease require life-long treatment with enzyme-replacement therapy (ERT). Despite the human origin of the therapy, recombinant human lysosomal acid α glucosidase (GAA, rhGAA), ERT unfortunately leads to the development of high titers of anti-rhGAA antibody, decreased effectiveness of ERT, and a fatal outcome for a significant number of children who have Pompe disease. The severity of disease, anti-drug antibody (ADA) development, and the consequences thereof are directly related to the degree of the enzyme deficiency. Babies born with a complete deficiency GAA are said to have cross-reactive immunologic material (CRIM)–negative Pompe disease and are highly likely to develop GAA ADA. Less frequently, GAA ADA develop in CRIM-positive individuals. Currently, GAA-ADA sero-positive babies are treated with a combination of immunosuppressive drugs to induce immunological tolerance to ERT, but the long-term effect of these regimens is unknown. Alternative approaches that might redirect the immune response toward antigen-specific tolerance without immunosuppressive agents are needed. Methods leading to the induction of antigen-specific regulatory T cells (Tregs), using peptides such as Tregitopes (T regulatory cell epitopes) are under consideration for the future treatment of CRIM-negative Pompe disease. Tregitopes are natural T cell epitopes derived from immunoglobulin G (IgG) that cause the expansion and activation of regulatory T cells (Treg). Teaching the immune system to tolerate GAA by co-delivering GAA with Tregitope peptides might dramatically improve the lives of CRIM-negative babies and could be applied to other enzyme replacement therapies to which ADA have been induced.

Tregitope Peptides: The Active Pharmaceutical Ingredient of IVIG?

Five years ago, we reported the identification and characterization of several regulatory T-cell epitopes (now called Tregitopes) that were discovered in the heavy and light chains of IgG (De Groot et al. Blood, 2008). When added ex vivo to human PBMCs, these Tregitopes activated regulatory T cells (Tregs), increased expression of the transcription factor FoxP3, and induced IL-10 expression in CD4+ T cells. We have now shown that coadministration of the Tregitopes in vivo, in a number of different murine models of autoimmune disease, can suppress immune responses to antigen in an antigen-specific manner, and that this response is mediated by Tregs. In addition we have shown that, although these are generally promiscuous epitopes, the activity of individual Tregitope peptides is restricted by HLA. In this brief report, we provide an overview of the effects of Tregitopes in vivo, discuss potential applications, and suggest that Tregitopes may represent one of the “active pharmaceutical ingredients” of IVIg. Tregitope applications may include any of the autoimmune diseases that are currently treated almost exclusively with intravenous immunoglobulin G (IVIG), such as Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) and Multifocal Motor Neuropathy (MMN), as well as gene therapy and allergy where Tregitopes may provide a means of inducing antigen-specific tolerance.