David V. Serreze

Identification of the β cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic β cells. CD8+ T cells are essential for this β cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.

Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4+ CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.

Major histocompatibility complex class I-deficient NOD-B2mnull mice are diabetes and insulitis resistant

Specific allelic combinations within the class II region of the major histocompatibility complex (MHC) represent a major genetic component for susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in humans. We produced and used a stock of NOD/Lt mice congenic for a functionally inactivated β2-microglobulin (B2mnull) locus to assess whether there was an absolute requirement for MHC class I expression and/or CD8+ T-cells in diabetogenesis. These NOD-B2mnull mice do not express cell surface MHC class I molecules or produce detectable levels of CD8+ T-cells and are diabetes and insulitis resistant. Previous results from transgenic mouse models indicated that intracellular accumulation of MHC class I molecules negatively affects pancreatic β-cell function and can result in the development of nonautoimmune insulin-dependent diabetes mellitus (IDDM). MHC class I molecules have been shown to accumulate intracellularly in the presence of a disrupted B2m locus, but this mutation does not negatively affect plasma insulin levels in either NOD/Lt mice or in those of a mixed 129 and C57BL/6 genetic background. Interestingly, 14% of the male mice in this mixed background did develop hyperinsulinemia (> 1,500 pM) independent of the disrupted B2m locus, suggesting that these mice could conceivably develop insulin-resistant diabetes. However, none of these mice became diabetic at up to 22 months of age. Thus, elimination of cell surface MHC class I expression with a disrupted B2m gene blocks autoimmune diabetes in NOD/Lt mice, without engendering a separate, distinct form of glucose intolerance.

TRPV1+ Sensory Neurons Control β Cell Stress and Islet Inflammation in Autoimmune Diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.

Immunostimulation circumvents diabetes in NODLt mice

Diabetes susceptibility in non-obese diabetic (NOD) mice may entail faulty activation of immunoregulatory cells resulting from cytokine deficiencies. Depletion of T cells prevents disease onset in these mice. Since we had previously shown that IL-2 treatment in vivo restored the ability of NOD/Lt mice to produce self-restricted suppressor T cells (Ts) in a syngeneic mixed lymphocyte reaction (SMLR), we investigated the possibility that diabetes could be circumvented by treatment with immunostimulatory agents that increase cytokine production. By 20 weeks of age, 75% of vehicle-treated NOD/Lt female controls had become glycosuric, while glycosuria developed in only 17% of NOD/Lt females injected with human recombinant interleukin-2 (rIL-2, 250 U twice weekly) beginning at 6 weeks of age. Treatment of mice with Poly [I:C] alone [50 micrograms twice weekly, an inducer of Interferon (IFN) alpha/beta] or in conjunction with rIL-2 was even more effective, completely preventing glycosuria for 20 weeks. However, therapeutic effects required continuous administration of the immunostimulants since pancreatic insulin content declined and severity of insulitis increased following cessation of treatment. IL-2 treatment increased transcription of interleukin-1 (IL-1) mRNA in peritoneal macrophages and increased lipopolysaccharide (LPS)-stimulated IL-1 secretion in comparison to controls. In the presence of stimulators from IL-2-treated mice, T lymphocytes isolated from both controls and IL-2-treated NOD/Lt mice proliferated in a SMLR and acquired Ts function. Peritoneal macrophages from Poly [I:C]-treated mice exhibited increased IFN alpha gene transcription and LPS-stimulated IL-1 secretion. T cells isolated from Poly [I:C]-treated mice were capable of suppressing NOD-Lt T cell responses to alloantigens in a mixed lymphocyte culture without prior activation in a SMLR. Thus, Poly [I:C] treatment may recruit a different population of regulatory cells than those elicited by treatment with IL-2. However, the mechanisms by which autoreactive T-cell clones may be regulated by these two treatments in NOD/Lt mice may be synergistic. These results indicate that in addition to T-cell depletion protocols, diabetes in NOD mice can be prevented by treatment with immunostimulatory agents.

Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic β-cell autoreactive T cells from a Th1 (IFN-γ) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-γ, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-γ to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-γ. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among β-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Identification of a CD8 T Cell That Can Independently Mediate Autoimmune Diabetes Development in the Complete Absence of CD4 T Cell Helper Functions

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic β cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic β cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4αβ Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted β cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

NOR/Lt Mice: MHC-Matched Diabetes-Resistant Control Strain for NOD Mice

NOR/Lt is an insulitis-resistant and diabetes-free strain produced from an isolated genetic contamination within an NOD/Lt pedigree line. The albino coat-color phenotype, strain-specific endogenous retroviral profile, and skin graft tests indicated an NOD/Lt × C57BL/KsJ outcross-backcross segregant as the source of the contaminating genome. Analysis of 53 polymorphic DNA, biochemical, and immunologic markers distinguishing NOD/Lt from C57BL/KsJ revealed that 4 chromosomes (chromosomes 2, 4, 11, and 12) in NOR/Lt contained C57BL/KsJ-derived genes. The remaining markers on 14 chromosomes, including the diabetogenic H-2g7 complex on chromosome 17, were of NOD origin. Although completely resistant to cyclophosphamide-induced diabetes, NOR/Lt mice exhibited the same peripheral T-lymphocyte accumulation characteristic of NOD/Lt. Similarly, NOR/Lt peritoneal macrophages exhibited depressed interleukin-1 secretion characteristic of NOD/Lt. In addition to their diabetes resistance, NOR/Lt mice were distinguished from NOD/Lt by exhibiting more robust suppressor T-lymphocyte function. Outcross of NOR/Lt with NOD/Lt to generate heterozygosity at those chromosomal segments, defined by C57BL/KsJ markers in NOR/Lt parentals, did not produce insulitis or diabetes in F1 females. However, these F1 females were sensitive to cyclophosphamide-induced diabetes. In summary, the NOR/Lt strain is an MHC-matched diabetes-resistant control strain for NOD/Lt. Moreover, NOR/Lt will help identify the location and function of a non-MHC gene or genes capable of conferring resistance against insulitis and diabetes.

Crosses of NOD Mice With the Related NON Strain: A Polygenic Model for IDDM

Chromosome locations of non-major histocompatibility complex (MHC) genes contributing to insulin-dependent diabetes mellitus (IDDM) in mice have been determined by outcrossing NOD mice to other inbred strains congenic for the NOD MHC haplotype (H2g7). At least nine non-MHC IDDM susceptibility genes (Idd) were previously identified at first backcross (BC1) after outcross of NOD to C57BL/10.H2g7 congenic mice (B10.H2g7). We investigated whether the same set of Idd loci segregated with IDDM susceptibility after outcross of NOD to NON.H2g7 congenic mice. Since the outcrosses to NON.H2g7 and B10.H2g7 were performed in the same vivarium, direct comparisons were made of the chromosomal locations and relative strengths of Idd alleles in diabetic progeny from the two different outcrosses. In comparison with the NOD x B10.H2g7 outcross, the NOD x NON.H2g7 outcross produced significantly higher IDDM frequencies in F1, F2, and BC1 generations. The high F2 diabetes frequency allowed evaluation of the effects of homozygous expression of both the susceptibility and the resistance allele at Idd loci. This analysis demonstrated that no single non-MHC Idd locus was essential for the onset of diabetes in this cross. After outcross to NON.H2g7, Idd4 (chromosome [Chr] 11), Idd5 (Chr 1), and Idd8 (Chr 14) did not segregate with IDDM in either the BC1 or the F2 generation. Diabetogenic NOD-derived alleles at Idd2 (Chr 9), Idd3 (Chr 3), and Idd10 (Chr 3) were segregating in the BC1. An NON-derived allele contributing to susceptibility on Chr 7 (Idd7) was also detected. Dominant traits, detectable only in the F2 cross, were encoded by Chr 4 (Idd9) and two newly mapped loci on Chr 13 (Idd14) and 5 (Idd15). A third dominant trait was encoded by Chr 6 (possibly Idd6), but here, in contrast to Idd9, Idd14, and Idd15, the NON allele was diabetogenic. Stepwise logistic regression analysis of the BC1 and F2 data confirmed that the ability to identify certainty of the non-MHC Idd loci was contingent on the extent of homozygosity for NOD background genes. This study shows that the diabetogenic phenotype can be achieved through the actions of variable combinations of MHC-unlinked genes and a diabetogenic MHC haplotype.

Activated NKT cells inhibit autoimmune diabetes through tolerogenic recruitment of dendritic cells to pancreatic lymph nodes

NKT cell activation by α-galactosylceramide (α-GalCer) inhibits autoimmune diabetes in NOD mice, in part by inducing recruitment to pancreatic lymph nodes (PLNs) of mature dendritic cells (DCs) with disease-protective effects. However, how activated NKT cells promote DC maturation, and what downstream effect this has on diabetogenic T cells was unknown. Activated NKT cells were found to produce a soluble factor(s) inducing DC maturation. Initially, there was a preferential accumulation of mature DCs in the PLNs of α-GalCer-treated NOD mice, followed by a substantial increase in T cells. Adoptive transfer of a diabetogenic CD8 T cell population (AI4) induced a high rate of disease (75%) in PBS-treated NOD recipients, but not in those pretreated with α-GalCer (8%). Significantly, more AI4 T cells accumulated in PLNs of α-GalCer than PBS-treated recipients, while no differences were found in mesenteric lymph nodes from each group. Compared with those in mesenteric lymph nodes, AI4 T cells entering PLNs underwent greater levels of apoptosis, and the survivors became functionally anergic. NKT cell activation enhanced this process. Hence, activated NKT cells elicit diabetes protection in NOD mice by producing a soluble factor(s) that induces DC maturation and accumulation in PLNs, where they subsequently recruit and tolerize pathogenic T cells.