Regulation of Diabetogenic Immunity by IL-15-Activated Regulatory CD8 T Cells in Type 1 Diabetes.

Unchecked collaboration between islet-reactive T and B lymphocytes drives type 1 diabetes (T1D). In the healthy setting, CD8 T regulatory cells (Tregs) terminate ongoing T-B interactions. We determined that specific CD8 Tregs from NOD mice lack suppressive function, representing a previously unreported regulatory cell deficit in this T1D-prone strain. NOD mice possess 11-fold fewer Ly-49+ CD8 Tregs than nonautoimmune mice, a deficiency that worsens as NOD mice age toward diabetes and leaves them unable to regulate CD4 T follicular helper cells. As IL-15 is required for Ly-49+ CD8 Treg development, we determined that NOD macrophages inadequately trans-present IL-15. Despite reduced IL-15 trans-presentation, NOD Ly-49+ CD8 Tregs can effectively transduce IL-15-mediated survival signals when they are provided. Following stimulation with an IL-15/IL-15Ra superagonist complex, Ly-49+ CD8 Tregs expanded robustly and became activated to suppress the Ag-specific Ab response. IL-15/IL-15Ra superagonist complex-activated CD8+CD122+ T cells also delayed diabetes transfer, indicating the presence of an underactivated CD8 T cell subset with regulatory capacity against late stage T1D. We identify a new cellular contribution to anti-islet autoimmunity and demonstrate the correction of this regulatory cell deficit. Infusion of IL-15-activated CD8 Tregs may serve as an innovative cellular therapy for the treatment of T1D.

Healthy Donor Polyclonal IgMs Diminish B-Lymphocyte Autoreactivity, Enhance Regulatory T-Cell Generation, and Reverse Type 1 Diabetes in NOD Mice.

Autoimmune diseases such as type 1 diabetes (T1D) arise from unrestrained activation of effector lymphocytes that destroy target tissues. Many efforts have been made to eliminate these effector lymphocytes, but none has produced a long-term cure. An alternative to depletion therapy is to enhance endogenous immune regulation. Among these endogenous alternatives, naturally occurring Igs have been applied for inflammatory disorders but have lacked potency in antigen-specific autoimmunity. We hypothesized that naturally occurring polyclonal IgMs, which represent the majority of circulating, noninduced antibodies but are present only in low levels in therapeutic Ig preparations, possess the most potent capacity to restore immune homeostasis. Treatment of diabetes-prone NOD mice with purified IgM isolated from Swiss Webster (SW) mice (nIgMSW) reversed new-onset diabetes, eliminated autoreactive B lymphocytes, and enhanced regulatory T-cell (Treg) numbers both centrally and peripherally. Conversely, IgM from prediabetic NOD mice could not restore this endogenous regulation, which represents an unrecognized component of T1D pathogenesis. Of note, IgM derived from healthy human donors was similarly able to expand human CD4 Tregs in humanized mice and produced permanent diabetes protection in treated NOD mice. Overall, these studies demonstrate that a potent, endogenous regulatory mechanism, nIgM, is a promising option for reversing autoimmune T1D in humans.

Host Expression of the CD8 Treg/NK Cell Restriction Element Qa-1 is Dispensable for Transplant Tolerance.

Disruption of the non-classical Major Histocompatibility Complex (MHC) Ib molecule Qa-1 impairs CD8 Treg and natural killer (NK) cell function and promotes a lupus-like autoimmune disease. This immune perturbation would be expected to enhance anti-transplant responses and impair tolerance induction, but the effect of Qa-1 deficiency on the transplant response has not been previously reported. Qa-1 deficiency enhanced CD4 TFH and germinal center (GC) B cell numbers in naïve mice and hastened islet allograft rejection. Despite enhanced immunity in B6.Qa-1-/- mice, these mice did not generate an excessive primary CD4 TFH cell response nor an enhanced alloantibody reaction. Both CD8 Tregs and NK cells, which often regulate other cells through host Qa-1 expression, were targets of anti-CD45RB therapy that had not been previously recognized. However, B6.Qa-1-/- mice remained susceptible to anti-CD45RB mediated suppression of the alloantibody response and transplant tolerance induction to mismatched islet allografts. Overall, despite enhanced immunity as demonstrated by augmented CD4 TFH/GC B cell numbers and hastened islet allograft rejection in naïve 12-week old Qa-1 deficient mice, the CD8 Treg/NK cell restriction element Qa-1 does not regulate the primary cellular or humoral alloresponse and is not required for long-term transplant tolerance.

Hematopoietic Stem Cell Mobilization Is Necessary but Not Sufficient for Tolerance in Islet Transplantation.

Overcoming the immune response to establish durable immune tolerance in type 1 diabetes remains a substantial challenge. The ongoing effector immune response involves numerous immune cell types but is ultimately orchestrated and sustained by the hematopoietic stem cell (HSC) niche. We therefore hypothesized that tolerance induction also requires these pluripotent precursors. In this study, we determined that the tolerance-inducing agent anti-CD45RB induces HSC mobilization in nonautoimmune B6 mice but not in diabetes-prone NOD mice. Ablation of HSCs impaired tolerance to allogeneic islet transplants in B6 recipients. Mobilization of HSCs resulted in part from decreasing osteoblast expression of HSC retention factors. Furthermore, HSC mobilization required a functioning sympathetic nervous system; sympathectomy prevented HSC mobilization and completely abrogated tolerance induction. NOD HSCs were held in their niche by excess expression of CXCR4, which, when blocked, led to HSC mobilization and prolonged islet allograft survival. Overall, these findings indicate that the HSC compartment plays an underrecognized role in the establishment and maintenance of immune tolerance, and this role is disrupted in diabetes-prone NOD mice. Understanding the stem cell response to immune therapies in ongoing human clinical studies may help identify and maximize the effect of immune interventions for type 1 diabetes.

Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II.

Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18.

Interleukin (IL)-18 is an important effector of innate and adaptive immunity, but its expression must also be tightly regulated because it can potentiate lethal systemic inflammation and death. Healthy and septic human neonates demonstrate elevated serum concentrations of IL-18 compared with adults. Thus, we determined the contribution of IL-18 to lethality and its mechanism in a murine model of neonatal sepsis. We find that IL-18-null neonatal mice are highly protected from polymicrobial sepsis, whereas replenishing IL-18 increased lethality to sepsis or endotoxemia. Increased lethality depended on IL-1 receptor 1 (IL-1R1) signaling but not adaptive immunity. In genome-wide analyses of blood mRNA from septic human neonates, expression of the IL-17 receptor emerged as a critical regulatory node. Indeed, IL-18 administration in sepsis increased IL-17A production by murine intestinal γδT cells as well as Ly6G(+) myeloid cells, and blocking IL-17A reduced IL-18-potentiated mortality to both neonatal sepsis and endotoxemia. We conclude that IL-17A is a previously unrecognized effector of IL-18-mediated injury in neonatal sepsis and that disruption of the deleterious and tissue-destructive IL-18/IL-1/IL-17A axis represents a novel therapeutic approach to improve outcomes for human neonates with sepsis.

Regulation of B lymphocyte responses to Toll-like receptor ligand binding during diabetes prevention in non-obese diabetic (NOD) mice.

BACKGROUND: Interactions between genetic risk factors and the environment drive type 1 diabetes (T1D). The system of Toll-like receptors (TLR) detects these environmental triggers; however, the target cell that intermediates these interactions to drive T1D remains unknown. METHODS: We investigated the effect of TLR pathway activation (myeloid differentiation primary response 88 [MyD88] vs TIR-domain-containing adapter-inducing interferon-ß [TRIF]) on B cell subsets via flow cytometry, including their activation, survival, proliferation, and cytoskeletal mobilization. The effect of polyinosinic-polycytidylic acid (poly(I:C)) on diabetes development was addressed, including the B cell-dependent activation of diabetes-protective DX5+ cells, using genetic models and adoptive transfer. RESULTS: B lymphocytes from non-obese diabetic (NOD) mice expressed enhanced levels of TLR-responsive proteins. Ex vivo analysis of B lymphocyte subsets demonstrated that TLR3 stimulation via TRIF deletes cells exhibiting a marginal zone phenotype, whereas MyD88-dependent ligands enhance their survival. In vivo, marginal zone B cells were activated by poly(I:C) and were unexpectedly retained in the spleen of NOD mice, in contrast with the mobilization of these cells in non-autoimmune mice, a phenotype we traced to defective actin cytoskeletal dynamics. These activated B cells mediated TLR3-induced diabetes protection. CONCLUSIONS: Immunotherapies must account for both B cell location and activation, and these properties may differ in autoimmune and healthy settings.

Dysregulation of T lymphocyte proliferative responses in autoimmunity.

T cells are critically dependent on cellular proliferation in order to carry out their effector functions. Autoimmune strains are commonly thought to have uncontrolled T cell proliferation; however, in the murine model of autoimmune diabetes, hypo-proliferation of T cells leading to defective AICD was previously uncovered. We now determine whether lupus prone murine strains are similarly hyporesponsive. Upon extensive characterization of T lymphocyte activation, we have observed a common feature of CD4 T cell activation shared among three autoimmune strains-NOD, MRL, and NZBxNZW F1s. When stimulated with a polyclonal mitogen, CD4 T cells demonstrate arrested cell division and diminished dose responsiveness as compared to the non-autoimmune strain C57BL/6, a phenotype we further traced to a reliance on B cell mediated costimulation, which underscores the success of B cell directed immune therapies in preventing T cell mediated tissue injury. In turn, the diminished proliferative capacity of these CD4 T cells lead to a decreased, but activation appropriate, susceptibility to activation induced cell death. A similar decrement in stimulation response was observed in the CD8 compartment of NOD mice; NOD CD8 T cells were distinguished from lupus prone strains by a diminished dose-responsiveness to anti-CD3 mediated stimulation. This distinction may explain the differential pathogenetic pathways activated in diabetes and lupus prone murine strains.

An immunosufficient murine model for the study of human islets.

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti-CD45RB. To assess whether anti-CD45RB-induced transplant tolerance requires B cells, B6 recipients received additional anti-CD20 or B6µMT-/- mice were used. For some anti-CD45RB-treated B6µMT-/- mice, additional anti-CD25 mAb was applied at the early or late stage post-transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti-CD45RB/anti-CD20-treated Foxp3-GFP B6 mice. The results showed that anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 88.9% of xenografts accepted in anti-CD45RB-treated µMT-/- mice. These µMT-/- mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post-transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.

miR-153 regulates SNAP-25, synaptic transmission, and neuronal development.

SNAP-25 is a core component of the trimeric SNARE complex mediating vesicle exocytosis during membrane addition for neuronal growth, neuropeptide/growth factor secretion, and neurotransmitter release during synaptic transmission. Here, we report a novel microRNA mechanism of SNAP-25 regulation controlling motor neuron development, neurosecretion, synaptic activity, and movement in zebrafish. Loss of miR-153 causes overexpression of SNAP-25 and consequent hyperactive movement in early zebrafish embryos. Conversely, overexpression of miR-153 causes SNAP-25 down regulation resulting in near complete paralysis, mimicking the effects of treatment with Botulinum neurotoxin. miR-153-dependent changes in synaptic activity at the neuromuscular junction are consistent with the observed movement defects. Underlying the movement defects, perturbation of miR-153 function causes dramatic developmental changes in motor neuron patterning and branching. Together, our results indicate that precise control of SNAP-25 expression by miR-153 is critically important for proper neuronal patterning as well as neurotransmission.