Structure-guided design of an invariant natural killer T cell agonist for optimum protection from type 1 diabetes in non-obese diabetic mice.

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure-dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non-obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α-galactosylceramide (α-GalCer) protects them from type 1 diabetes (T1D). However, α-GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure-guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α-GalCer fatty acyl chain derivatives (C8:0-C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α-GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α-GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D.

Stimulation of a shorter duration in the state of anergy by an invariant natural killer T cell agonist enhances its efficiency of protection from type 1 diabetes.

We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.

An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

Decreased renal ischemia-reperfusion injury by IL-16 inactivation.

T-cell-mediated renal injury is a major cause of kidney transplant rejection and renal failure; hence, understanding T-cell migration within the kidney is important for preventing renal injury. Interleukin (IL)-16 is a T-cell chemoattractant produced by leukocytes. Here we measured IL-16 expression in the kidney and its role in renal ischemia-reperfusion injury induced by different conditions in several strains of mice. IL-16 was strongly expressed in distal and proximal straight tubules of the kidney. The IL-16 precursor protein was cleaved to a chemotactic form in cultured tubular epithelial cells. Inactivation of IL-16 by antibody therapy or IL-16 deficiency prevented ischemia-reperfusion injury as shown by reduced levels of serum creatinine or blood urea nitrogen compared to control mice. Further studies indicated that fewer CD4-cells infiltrated the post-ischemic kidneys of IL-16-deficient mice and that the protective effect of IL-16 antibody treatment was lymphocyte-dependent. Our results suggest that IL-16 is a critical factor in the development of inflammation-mediated renal injury and may be a therapeutic target for prevention of ischemia-reperfusion injury of the kidney.

Evidence that a peptide spanning the B-C junction of proinsulin is an early Autoantigen epitope in the pathogenesis of type 1 diabetes.

The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24-33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in female NOD mice. Our findings support the notion that proinsulin p24-33 may be a primary autoantigen epitope in the pathogenesis of T1D in NOD mice.

Regulation of immune responses by natural killer T cells.

Natural killer T (NKT) cells, which comprise a minor population of T cells in primary and secondary lymphoid organs, possess phenotypic characteristics of both NK and T cells. NKT cells respond to various external stimuli by an early burst of cytokines, including IL-4 and IFN-gamma. Thus, a key immunoregulatory role has been attributed to them. Autoimmune diseases, especially type I diabetes (TID), may be caused by dysregulation of the immune system, which leads to hyporesponsiveness of regulatory T helper 2 (Th2) cells and promotion of autoimmune Th1 cells. Furthermore, several lines of evidence exist to support the notion that an NKT cell deficiency in individuals at risk of TID may be causal to TID. As a result, targeting NKT cells using immunotherapeutic agents may prove beneficial in the prevention or recurrence of TID. Indeed, our data demonstrate that stimulation of NKT cells with a specific ligand prevents the onset and recurrence of TID in nonobese diabetic (NOD) mice.

Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes.

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

CD28 co-stimulation restores T cell responsiveness in NOD mice by overcoming deficiencies in Rac-1/p38 mitogen-activated protein kinase signaling and IL-2 and IL-4 gene transcription.

Previously, we reported that T cell hyporesponsiveness induced by TCR ligation is causal to autoimmune diabetes in NOD mice. Neonatal CD28 co-stimulation reverses T cell hyporesponsiveness and protects NOD mice from diabetes by an IL-4-mediated mechanism, indicating that a deficiency in TCR signaling may be overcome by CD28/B7-2 co-stimulation in NOD T cells. To investigate which co-stimulation-induced signaling events mediate this protection, we analyzed the activity of Ras, Rac-1, mitogen-activated protein kinases (MAPK) and several transcription factors in TCR-activated NOD T cells in the presence or absence of CD28 co-stimulation. We show that CD28 co-stimulation restores normal TCR-induced activation of Rac-1 and p38 MAPK in NOD T cells. Deficiencies in TCR-induced nuclear expression of activating protein (AP)-1 binding proteins as well as activation of AP-1 and NF-AT in the IL-2 and IL-4 P1 promoters are also corrected by CD28 co-stimulation. Thus, CD28 co-stimulation reverses NOD T cell hyporesponsiveness by restoring TCR signaling leading to the activation of AP-1 and NF-AT during IL-2 and IL-4 gene transcription. Our findings provide additional evidence that CD28 co-stimulation amplifies signals delivered by the TCR and further explain the mechanism by which CD28 co-stimulation may protect against autoimmune diabetes.

Immunotherapy of spontaneous type 1 diabetes in nonobese diabetic mice by systemic interleukin-4 treatment employing adenovirus vector-mediated gene transfer.

We have previously shown that systemic injection of multiple low doses of recombinant murine interleukin-4 (mIL-4) can prevent type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by activating regulatory T helper (Th) 2 cells in vivo. Here, we have developed a gene transfer approach to the prevention of T1D by testing the therapeutic potential of an adenovirus gene transfer vector engineered to express mIL-4. We found that only two systemic injections of a recombinant adenovirus type 5 vector-expressing mIL-4 (Ad5mIL-4) reduces destructive insulitis and protects NOD mice from the onset of diabetes by eliciting intrapancreatic Th2 cell responses. Host immune responses against the adenovirus vector were detectable; however, the levels of antibody production were insufficient to preclude Ad5mIL-4 treatment as a possible therapeutic agent against T1D. Thus, adenovirus-mediated delivery of IL-4 provides protection of NOD mice from T1D and represents a clinically viable therapeutic approach.

Biolistic-mediated interleukin 4 gene transfer prevents the onset of type 1 diabetes.

We tested the efficacy of biolistic-mediated gene transfer as a noninvasive therapy for type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by expression of murine interleukin 4 (mIL-4) cDNA. Epidermal delivery of 2 microg of DNA yielded transient detection of serum mIL-4, using a conventional cDNA expression vector. A vector stabilized by incorporation of the Epstein-Barr virus (EBV) EBNA1/oriP episomal maintenance replicon produced higher levels of serum mIL-4 that persisted for 12 days after inoculation. Although biolistic inoculation of either vector reduced insulitis and prevented diabetes, the protracted mIL-4 expression afforded by the EBV vector resulted in Th2-type responses in the periphery and pancreas and more significant protection from the onset of diabetes. Our studies demonstrate the efficacy of biolistic gene delivery of stabilized cytokine expression as a viable therapeutic approach to prevent the onset of T1D.

Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes.

We investigated the biological role of CC chemokines in the Th1-mediated pathogenesis of spontaneous type I diabetes in nonobese diabetic (NOD) mice. Whereas an elevated ratio of macrophage inflammatory protein-1alpha (MIP-1alpha):MIP-1beta in the pancreas correlated with destructive insulitis and progression to diabetes in NOD mice, a decreased intrapancreatic MIP-1alpha:MIP-1beta ratio was observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment, which prevents diabetes in NOD mice by polarizing intraislet Th2 responses, decreased CCR5 expression in islets and potentiated a high ratio of MIP-1beta and monocyte chemotactic protein-1 (MCP-1): MIP-1alpha in the pancreas. Furthermore, NOD.MIP-1alpha-/- mice exhibited reduced destructive insulitis and were protected from diabetes. Neutralization of MIP-1alpha with specific Abs following transfer of diabetogenic T cells delayed the onset of diabetes in NOD.Scid recipients. These studies illustrate that the temporal expression of certain CC chemokines, particularly MIP-1alpha, and the CCR5 chemokine receptor in the pancreas is associated with the development of insulitis and spontaneous type I diabetes.

Preferential proliferation and differentiation of double-positive thymocytes into CD8(+) single-positive thymocytes in a novel cell culture medium.

The identification of factors that regulate the proliferation and differentiation of double-positive (DP) into CD4(+) and CD8(+) single-positive (SP) thymocytes has proven difficult due to the inability of DP thymocytes to proliferate, expand, and differentiate into SP thymocytes in available cell culture media. Here we report on the ability of DP thymocytes to differentiate in a novel conditioned medium, termed XLCM, derived from the supernatant of mitogen activated human cord blood mononuclear cells. During a 5-day culture in XLCM in the absence of thymic stromal cells, DP thymocytes from normal mice and MHC double knockout mice (lack SP thymocytes) proliferate, expand, and differentiate into several (alphabetaTCR(+), NK1.1(+)alphabetaTCR(+), and gammadeltaTCR(+)) subsets of CD4(+) and predominantly CD8(+) SP thymocytes. These studies suggest that the use of XLCM may aid in the characterization of factors that regulate the differentiation of DP thymocytes into CD8(+) SP thymocytes.

Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

Activation-induced cell death (AICD) is a mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). Although c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) may be involved in apoptosis in various cell types, the mode of regulation of FasL expression during AICD in T cells by these two MAPKs is incompletely understood. To investigate the regulatory roles of these two MAPKs, we analyzed the kinetics of TCR-induced p38 MAPK and JNK activity and their regulation of FasL expression and AICD. We report that both JNK and p38 MAPK regulate AICD in T cells. Our data suggest a novel model of T cell AICD in which p38 MAPK acts early to initiate FasL expression and the Fas-mediated activation of caspases. Subsequently, caspases stimulate JNK to further upregulate FasL expression. Thus, p38 MAPK and downstream JNK converge to regulate FasL expression at different times after T cell receptor stimulation to elicit maximum AICD.

ZAP-70 is essential for the T cell antigen receptor-induced plasma membrane targeting of SOS and Vav in T cells.

Translocation of the SOS and Vav GDP/GTP exchange factors proximal to Ras and Rac GTPases localized in the plasma membrane glycolipid-enriched microdomains is a pivotal step required for T cell antigen receptor-induced T cell activation. Here we demonstrate that the T cell antigen receptor zeta-chain-associated ZAP-70 kinase and T cell antigen receptor zeta-chain immunoreceptor tyrosine-based activation motifs are essential for the membrane recruitment of SOS and Vav. Plasma membrane targeting of SOS or Vav begins with the assembly of ZAP-70 with Grb-2 and SOS. The subsequent tyrosine phosphorylation of LAT (linker for activation of T cell) by ZAP-70 leads to a shift in equilibrium from the ZAP-70.Grb-2.SOS(Vav) complex to the (Vav)SOS.Grb-2.LAT complex. This shift results in the targeting of SOS and Vav into glycolipid-enriched microdomains and initiation of the Ras and Rac signaling cascades involved in T cell activation, proliferation, and cytokine production.

Insulin B-chain reactive CD4+ regulatory T-cells induced by oral insulin treatment protect from type 1 diabetes by blocking the cytokine secretion and pancreatic infiltration of diabetogenic effector T-cells.

The mechanism of protection from type 1 diabetes conferred by regulatory T-cells induced by oral insulin treatment of NOD mice is not well understood. We demonstrate that oral insulin feeding of NOD mice induces the function of insulin B-chain reactive CD4+ regulatory T-cells, which compete with diabetogenic effector T-cells for the recognition of insulin in NOD.Scid recipient mice. These effector T-cells become deprived of interleukin (IL)-2 and interferon (IFN)-gamma and are unable to expand and migrate to the pancreas. Type 1 diabetes-protective splenic regulatory T-cells secrete relatively little transforming growth factor (TGF)-beta1, suggesting that TGF-beta may not contribute to the inactivation of effector T-cells in NOD.Scid recipients. The observed preferential infiltration of insulin-reactive regulatory T-cells rather than effector T-cells in the pancreas results in a nondestructive insulitis that correlates with an increased intrapancreatic expression of macrophage inflammatory protein-1beta. Thus, oral insulin therapy overcomes a deficiency in regulatory T-cells and protects against type 1 diabetes by inducing insulin B-chain reactive regulatory T-cells to block cytokine secretion and migration of diabetogenic effector T-cells to the pancreas. Our data emphasize that continuous oral insulin feeding over a prolonged period is required to prevent type 1 diabetes.

TCR and CD28 are coupled via ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway.

CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s) by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. Here, we show that TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. Our findings, obtained by using ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which elicits the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules.

Insulin-like growth factors prevent cytokine-mediated cell death in isolated islets of Langerhans from pre-diabetic non-obese diabetic mice.

Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) contribute to the initial stages of the autoimmune destruction of pancreatic beta cells. IL-1beta is released by activated macrophages resident within islets, and its cytotoxic actions include a stimulation of nitric oxide (NO) production and the initiation of apoptosis. Insulin-like growth factors (IGFs)-I and -II prevent apoptosis in non-islet tissues. This study investigated whether IGFs are cytoprotective for isolated islets of Langerhans from non-obese diabetic mice (NOD) mice exposed to cytokines. Pancreatic islets isolated from 5-6-week-old, pre-diabetic female NOD mice were cultured for 48 h before exposure to IL-1beta (1 ng/ml), TNF-alpha (5 ng/ml), IFN-gamma (5 ng/ml) or IGF-I or -II (100 ng/ml) for a further 48 h. The incidence of islet cell apoptosis was increased in the presence of each cytokine, but this was significantly reversed in the presence of IGF-I or -II (IL-1beta control 3.5+/-1.6%, IL-1beta 1 ng/ml 27.1+/-5.8%, IL-1beta+IGF-I 100 ng/ml 4.4+/-2.3%, P<0.05). The majority of apoptotic cells demonstrated immunoreactive glucose transporter 2 (GLUT-2), suggesting that they were beta cells. Islet cell viability was also assessed by trypan blue exclusion. Results suggested that apoptosis was the predominant cause of cell death following exposure to each of the cytokines. Co-incubation with either IGF-I or -II was protective against the cytotoxic effects of IL-1beta and TNF-alpha, but less so against the effect of IFN-gamma. Exposure to cytokines also reduced insulin release, and this was not reversed by incubation with IGFs. Immunohistochemistry showed that IGF-I was present in vivo in islets from pre-diabetic NOD mice which did not demonstrate insulitis, but not in islets with extensive immune infiltration. Similar results were seen for IGF-binding proteins (IGFBPs). These results suggest that IGFs protect pre-diabetic NOD mouse islets from the cytotoxic actions of IL-1beta, TNF-alpha and IFN-gamma by mechanisms which include a reduction in apoptosis.