Kevin S. Goudy

Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk

Lentiviral vectors are attractive tools for liver‐directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase‐defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte‐targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase‐competent vector counterparts. IDLV‐mediated and hepatocyte‐targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte‐targeted IDLVs induced transgene‐specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV‐transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity. Conclusion: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis. (HEPATOLOGY 2011;)

The cellular and molecular mechanisms of immuno-suppression by human type 1 regulatory T cells.

The immuno-regulatory mechanisms of IL-10-producing type 1 regulatory T (Tr1) cells have been widely studied over the years. However, several recent discoveries have shed new light on the cellular and molecular mechanisms that human Tr1 cells use to control immune responses and induce tolerance. In this review we outline the well known and newly discovered regulatory properties of human Tr1 cells and provide an in-depth comparison of the known suppressor mechanisms of Tr1 cells with FOXP3+ Treg. We also highlight the role that Tr1 cells play in promoting and maintaining tolerance in autoimmunity, allergy, and transplantation.

Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity.

Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8+ T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8+STAT5+ T cells with lytic potential infiltrated the skin, even though FOXP3+ Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.

Cellular immune response to cryptic epitopes during therapeutic gene transfer

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8+ T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8+ CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.

Identical β Cell-Specific CD8+ T Cell Clonotypes Typically Reside in Both Peripheral Blood Lymphocyte and Pancreatic Islets

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring β cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8+ T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3α and CDR3β sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. β cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.

Immunotherapy for the prevention and treatment of type 1 diabetes

A major effort has been on-going to develop immunotherapies to prevent and/or treat type 1 diabetes (T1D). This autoimmune disease is characterized by the selective loss of the insulin-producing β cells via the cumulative effects of autoantigen-specific CD4+ and CD8+ T cells, autoantibodies, and activated antigen-presenting cells. To be applicable in a clinical setting, immunotherapies must suppress established β-cell autoimmunity. Preclinical studies and recent clinical findings suggest that antigen-specific and systemic-based strategies can be effective in this regard. However, either approach alone may not be sufficient to block the diabetogenic response and establish long-term protection in the clinic. In this review, we will discuss the importance of both strategies and how a combinatorial approach to treat T1D is appealing.

Liver gene therapy by lentiviral vectors reverses anti-factor IX pre-existing immunity in haemophilic mice.

A major complication of factor replacement therapy for haemophilia is the development of anti‐factor neutralizing antibodies (inhibitors). Here we show that liver gene therapy by lentiviral vectors (LVs) expressing factor IX (FIX) strongly reduces pre‐existing anti‐FIX antibodies and eradicates FIX inhibitors in haemophilia B mice. Concomitantly, plasma FIX levels and clotting activity rose to 50–100% of normal. The treatment was effective in 75% of treated mice. FIX‐specific plasma cells (PCs) and memory B cells were reduced, likely because of memory B‐cell depletion in response to constant exposure to high doses of FIX. Regulatory T cells displaying FIX‐specific suppressive capacity were induced in gene therapy treated mice and controlled FIX‐specific T helper cells. Gene therapy proved safer than a regimen mimicking immune tolerance induction (ITI) by repeated high‐dose FIX protein administration, which induced severe anaphylactoid reactions in inhibitors‐positive haemophilia B mice. Liver gene therapy can thus reverse pre‐existing immunity, induce active tolerance to FIX and establish sustained FIX activity at therapeutic levels. These data position gene therapy as an attractive treatment option for inhibitors‐positive haemophilic patients.

Gene gun-mediated DNA vaccination enhances antigen-specific immunotherapy at a late preclinical stage of type 1 diabetes in nonobese diabetic mice

Type 1 diabetes (T1D) is characterized by the T cell mediated destruction of the insulin-producing beta cells. Antigen-specific immunotherapies are used to selectively tolerize beta cell-specific pathogenic T cells either directly, or indirectly through the induction of immunoregulatory T cells. A key concern of antigen-specific immunotherapy is exacerbating autoimmunity. We compared the T cell reactivity and efficacy induced by plasmid DNA (pDNA) encoding glutamic acid decarboxylase 65 (GAD65) administered via intramuscular versus gene gun vaccination in NOD mice at a late preclinical stage of T1D. Whereas intramuscular injection of pGAD65 promoted a predominant type 1 CD4(+) T cell response and failed to suppress ongoing beta cell autoimmunity, gene gun vaccination preferentially induced IL-4 secreting CD4(+) T cells and significantly delayed the onset of diabetes. These findings demonstrate that gene gun delivery of autoantigen-encoding pDNA preferentially elicits immunoregulatory T cells and offers a safe, effective mode of pDNA vaccination for the treatment of T1D and other autoimmune diseases.

The Type and Frequency of Immunoregulatory CD4+ T-Cells Govern the Efficacy of Antigen-Specific Immunotherapy in Nonobese Diabetic Mice

Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigen-specific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65), interleukin (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65+pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4+ T-cells secreting IL-4, IL-10, or interferon-γ were detected in mice treated with pGAD65+pIL4+pIL10 and pGAD65+pIL10. However, the frequency of FoxP3-expressing CD4+CD25+CD62Lhi T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65+pIL4+pIL10. These immunoregulatory CD4+CD25+ T-cells (CD4+CD25+ Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-β dependent. Furthermore, duration of islet graft protection in pGAD65+pIL4+pIL10-vaccinated diabetic recipients correlated with the persistence of CD4+CD25+ Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4+CD25+ Treg influence antigen-induced suppression of ongoing β-cell autoimmunity in diabetic recipients.

Inducible adeno-associated virus-mediated IL-2 gene therapy prevents autoimmune diabetes

IL-2 and TGF-β1 play key roles in the immunobiology of Foxp3-expressing CD25+CD4+ T cells (Foxp3+Treg). Administration of these cytokines offers an appealing approach to manipulate the Foxp3+Treg pool and treat T cell-mediated autoimmunity such as type 1 diabetes. However, efficacy of cytokine treatment is dependent on the mode of application, and the potent pleiotropic effects of cytokines like IL-2 may lead to severe side effects. In the current study, we used a gene therapy-based approach to assess the efficacy of recombinant adeno-associated virus vectors expressing inducible IL-2 or TGF-β1 transgenes to suppress ongoing β cell autoimmunity in NOD mice. Intramuscular vaccination of recombinant adeno-associated virus to 10-wk-old NOD female mice and a subsequent 3 wk induction of IL-2 was sufficient to prevent diabetes and block the progression of insulitis. Protection correlated with an increased frequency of Foxp3+Treg in the periphery as well as in the draining pancreatic lymph nodes and islets. IL-2 induced a shift in the ratio favoring Foxp3+Treg versus IFN-γ–expressing T cells infiltrating the islets. Induction of IL-2 had no systemic effect on the frequency or activational status of T cells and NK cells. Induction of TGF-β1 had no effect on the Foxp3+Treg pool or the progression of β cell autoimmunity despite induced systemic levels of activated TGF-β1 that were comparable to IL-2. These results demonstrate that inducible IL-2 gene therapy is an effective and safe approach to manipulate Foxp3+Treg and suppress T cell-mediated autoimmunity and that under the conditions employed, IL-2 is more potent than TGF-β1.