Characterization and Immunomodulation of Regulatory T cells in Type 1 Diabetes

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disorder characterized by the complete destruction of the insulin-producing pancreatic β cells. The result of β cell loss leads to life-long insulin injections in order to maintain blood glucose levels, and is associated with both micro-vascular and macro-vascular diseases. Disease onset is related to a genetic predisposition, environmental factors, and a cell-mediated immunophenotype. Previous studies using the non-obese diabetic mouse (NOD) suggest that T1D can be prevented by the administration of immunoregulatory proteins that induce/expand both antigen specific and antigen non-specific regulatory cells. The aims of the studies described within are to; i.) test the hypothesis that antigen-specific therapies can prevent diabetes, and ii) to understand the cause(s) of immunoregulatory T cell deficiency in diabetes development. Results from our first study demonstrate that gene therapy with pDNA encoding the β cell protein gluatamic acid decarboxyasle 65 (GAD65) via gene gun significantly prevents diabetes onset in the NOD mouse. Diabetes protection was attributed to the induction of interleukin-4 secreting immunoregulatory T cells. We also discovered that delivery of the same construct via intra-muscularly injection exacerbated diabetes by the preferentially induction of pathogenic type 1 T effector cells. Therefore, our findings show that the route of delivery of pDNA encoding autoantigens is integral in shaping the type of effector cell response. Our second study illustrates that T1D can be regulated by the differential expression of the il2 gene located in the Idd3 locus of the NOD mouse genome. We found that reduced IL-2 production by NOD CD4+ T cells resulted in ~two-fold less induction of FoxP3-expressing regulatory T cells that are critical for regulating autoimmunity. Furthermore, we described a new role for IL-21 whereby IL-21 negatively regulates IL-2 production by CD4+ T cells, thus, perpetuating the inhibition of regulatory T cells. Most importantly, we successfully devised an IL-2 therapy regimen for NOD mice that overcomes the regulatory T cell deficiency which could lead to a new therapeutic approach to prevent and/or treat T1D in humans

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

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 β cells. Antigen-specific immunotherapies are used to selectively tolerize β 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 β 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

Manipulating Immune Tolerance with Micro-RNA Regulated Gene Therapy

The success of in vivo gene therapy greatly depends on the ability to control the immune response toward the therapeutic transgene. Over the last decade several vector-based and pharmacological approaches have been explored to control the immune-mediated clearance of transgene-expressing cells after viral delivery. One important outcome from these studies is the concept that expression of a transgene in tolerance-promoting organs, such as the liver and tolerogenic antigen-presenting cells, can help safeguard transgene-expressing cells from immune-mediated clearance. Gene therapists are now manipulating vectors to target naturally occurring tolerogenic properties of the body by: (i) incorporating tissue/cell specific promoters for targeted expression, (ii) using viral-capsid engineering to alter tropism and avoid pre-existing immunity, and (iii) regulating cell and activation dependent expression by including micro-RNA (miR) targets into expression cassettes. The combination of these three layers of vector regulation greatly enhances the targeting of tolerogenic cells and limits off-target expression of the transgene, which can lead to the induction of transgene-specific pathogenic effector T cells. In this review, we discuss the application of using miR transgene regulation to generate tolerogenic responses and speculate on possible mechanisms used by the liver to induce the transgene-specific regulatory T cells

Reduced IL-2 expression in NOD mice leads to a temporal increase in CD62LloFoxP3+CD4+ T cells with limited suppressor activity

IL-2 plays a critical role in the induction and maintenance of FoxP3-expressing regulatory T cells (FoxP3+Treg). Reduced expression of IL-2 is linked to T cell-mediated autoimmune diseases such as Type 1 diabetes (T1D), in which an imbalance between FoxP3+Treg and pathogenic T effectors exists. We investigated the contribution of IL-2 to dysregulation of FoxP3+Treg by comparing wildtype NOD mice with animals congenic for a C57BL/6-derived disease resistant Il2 allele and in which T cell secretion of IL-2 is increased (NOD.B6Idd3). Whereas NOD mice exhibited a progressive decline in the frequency of CD62LHIFoxP3+Treg due to an increase in CD62LLOFoxP3+Treg, CD62LHIFoxP3+Treg were maintained in the pancreatic lymph nodes and islets of NOD.B6Idd3 mice. Notably, the frequency of proliferating CD62LHIFoxP3+Treg was elevated in the islets of NOD.B6Idd3 versus NOD mice. Increasing levels of IL-2 in vivo also resulted in larger numbers of CD62LHIFoxP3+Treg in NOD mice. These results demonstrate that IL-2 influences the suppressor activity of the FoxP3+Treg pool by regulating the balance between CD62LLO and CD62LHI FoxP3+Treg. In NOD mice reduced IL-2 expression leads to an increase in nonsuppressive CD62LLOFoxP3+Treg, which in turn correlates with a pool of CD62LHIFoxP3+Treg with limited proliferation

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;

Acute Cardiovascular and Metabolic Responses to Three Modes of Treadmill Exercise in Older Adults with Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative condition characterized by muscle tremors, rigidity and dyskinesis leading to balance and gait abnormalities that could alter physiologic responses during exercise. Locomotion on an aquatic treadmill (ATM) or anti-gravity treadmill (AGTM) may be a safe alternative to exercise on a traditional land treadmill (LTM) in those with PD. PUPROSE: To determine the acute cardiovascular and metabolic responses to three different modes of treadmill exercise in older adults diagnosed with Parkinson’s disease. METHODS: Eight adults diagnosed with PD (68 ± 3 years of age) completed one exercise session on an LTM, one session on an ATM, and one session on an AGTM at 50% body weight. Participants walked from 1 to 3 mph in 0.5 mph increments at 0% grade during each exercise session. Heart rate (HR), energy expenditure (EE), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured at rest and during steady-state exercise at each speed on each treadmill. Rate of perceived exertion was also measured during steady-state exercise. Rate pressure product (RPP) was calculated. RESULTS: All variables, with the exception of DBP, increased as speed increased across all treadmill modes (p \u3c 0.001). Between treadmill modes across all speeds, EE was statistically different (p = 0.025). There was a significant interaction effect for mode and speed for HR (p \u3c 0.001) and RPP (p = 0.003). At all speeds except 1.5 mph, HR was higher on the LTM versus the AGTM (p \u3c 0.05). CONCLUSION: Exercising on an ATM or an AGTM elicits similar physiologic responses to exercise on an LTM in adults with P

Changes in Balance, Gait and Motor Skills Following Treadmill Exercise in Adults with Parkinson’s Disease

Locomotion on an aquatic treadmill or anti-gravity treadmill may be a safe and effective alternative to exercise on a traditional land treadmill in those with Parkinson’s disease as the removal of body weight in these environments may allow the participant to exercise with less concern of falling at higher speeds before reaching volitional fatigue. PURPOSE: To determine the training effects of three different treadmill modalities on dynamic balance, gait, and fine motor control in older adults diagnosed with Parkinson’s disease. METHODS: Ten adults diagnosed with Parkinson’s disease (70 ± 5 years of age) completed 8 exercise sessions (4 weeks, 2x/week) each separately on a land treadmill, aquatic treadmill, and anti-gravity treadmill at 50% body weight. Two weeks separated each intervention and the order was randomized. A 4-week control period occurred at the start of the study in which no treadmill exercise was performed. Each exercise session included a 2-minute warm-up and 30 minutes at a moderate intensity. Before and after each intervention, balance, gait and fine motor control were measured. Dynamic balance and gait were assessed using a Timed-Up-and-Go test and Performance Oriented Mobility Assessment (POMA). Fine motor control was assessed with the Purdue Pegboard Test. RESULTS: The gait assessment of the POMA was significant across all time points (p = 0.028). All other variables were statistically similar (p \u3e 0.05) across all time points. CONCLUSION: Exercising on a traditional land treadmill, aquatic treadmill, or anti-gravity treadmill for 60 min/week for 4 weeks at a moderate intensity did not alter balance, gait or fine motor control in adults with Parkinson’s disease