Negligible role for NK cells and macrophages in delayed xenograft rejection

Abstract Hyperacute rejection (HAR) of a discordant xenograft can be avoided by complement manipulation, but delayed xenograft rejection (DXR) still leads to graft loss. It is generally assumed that macrophages and NK cells play key roles in DXR. In the present study the survival times and cellular infiltrate following guinea pig to rat heart transplantation was analyzed in the course of DXR, following aspecific and specific manipulation of macrophages and NK cells. HAR was overcome by a single injection of cobra venom factor 1 day before heart transplantation. To aspecifically reduce the inflammatory response dominating DXR, dexamethasone (DEXA) was given. Treatment with DEXA markedly reduced infiltration by NK cells, macrophages, and granulocytes. It also led to prolonged graft survival times (median survival of 0.4 days, n = 10, P < 0.05). In the second series of experiments the specific roles of NK cells and macrophages in DXR were further assessed. Monoclonal antibody 3.2.3 was used to selectively deplete NK cells. Liposome‐encapsulated dichloromethylene biphosphonate was given to achieve macrophage depletion. Neither of these specific treatments, alone or combined, led to prolonged graft survival. Immunohistology revealed that at day 2 after transplantation no NK cells or macrophages were present in grafts from the combined treatment group. Only a mild infiltration of granulocytes was observed. Collectively, these results strongly suggest that NK cells and macrophages are not likely to be pivotal cell types in DXR.     

Targeting autoimmune diabetes with gene therapy.

The autoimmune nature of insulin-dependent, or type 1, diabetes targets the beta-cells of the pancreas for destruction and results in a lifelong commitment to insulin replacement therapy. Although the number of formulations and dosing of insulin have become more sophisticated and more efficient in recent years, insulin therapy alone is unable to prevent nephropathy, retinopathy, or vascular and heart disease, which still occur in a large number of patients. Different approaches have been attempted to eliminate the requirement of exogenous insulin administration. Historically, these have included pancreatic and islet transplants, which were later combined with treatments intended to halt the destructive process directed against the islets. Despite significant advances made in all of these areas, each approach faces a hostile immunological response that frequently ends with the loss of the islets. Gene therapy-based approaches add a new dimension to the efforts aimed at specifically blocking the immunological attack against the islets in genetically at-risk individuals (autoimmunity) or the immunological response against transplanted allogeneic islets (rejection). This new technology may have an important role in the therapy and cure of type 1 diabetes.     

Low-dose streptozocin-induced autoimmune diabetes in islet transplantation model.

An islet transplant model was used to gain further insight into the immunologic mechanisms involved in low-dose streptozocin (STZ)-induced diabetes. As shown by others, male C57BL/KsJ mice developed diabetes and insulitis after five daily injections of STZ (40 mg.kg-1.day-1). Syngeneic islet transplants beneath the renal capsule developed insulitis when the islets were transplanted 10-14 days before the daily injections of STZ. In contrast, insulitis of the grafts did not occur when the syngeneic transplants were done 3 days after the five daily injections of STZ. If the donor islets were incubated in vitro with 0.5 mg/ml STZ for 1 h at 37 degrees C and then transplanted after the low-dose STZ administration of the recipients, then a definite insulitis was present in the syngeneic transplants. These findings indicated that this brief exposure to STZ in vitro was sufficient to permit immunologic recognition of the grafts. In vitro STZ-exposed islets transplanted into high-dose STZ-induced diabetic mice also developed insulitis, whereas STZ-exposed islets transplanted into alloxan-induced diabetic mice did not. Donor islets incubated in vitro with STZ and transplanted into unexposed mice did not develop insulitis in the grafts. Thus, preexposure of the recipient to STZ is essential to the development of insulitis within in vitro STZ-exposed islet grafts. This is also specific to islets because in vitro STZ-exposed thyroid tissue transplanted into low-dose STZ recipients failed to exhibit a thyroiditis. These data are consistent with the idea that STZ immunologically alters the beta-cell by inducing an antigenic change.     

Interleukin-13 prevents autoimmune diabetes in NOD mice.

Interleukin (IL)-13 is a cytokine primarily produced by the T-helper (Th)-2 subset of lymphocytes that possesses powerful anti-inflammatory properties. Here, we have evaluated the impact of IL-13 treatment on development of type 1 diabetes in diabetes-prone nonobese diabetic (NOD) mice. Prolonged treatment with recombinant human IL-13 (hIL-13) markedly diminished the incidence of spontaneous type 1 diabetes in the mice. Female NOD mice treated from age 5-16 weeks with hIL-13 also showed significantly milder insulitis than control mice. The preventive action of hIL-13 was associated with a slight but significant change from a type 1 to a type 2 cytokine response. Accordingly, splenic lymphoid cells (SLC) from hIL-13-treated mice secreted less interferon (IFN)-gamma upon ex vivo stimulation with Concanavalin A than controls, and anti-CD3 monoclonal antibody-induced activation of T-cells in vivo resulted in lower blood levels of IFN-gamma and tumor necrosis factor-alpha and augmented blood levels of IL-4 in NOD mice pretreated with hIL-13. hIL-13 treatment also increased the blood levels of IgE and inhibited the transfer of type 1 diabetes by spleen cells from a diabetic donor to irradiated recipients. Taken together, these data add hIL-13 to the list of cytokines capable of downregulating immunoinflammatory diabetogenic pathways in NOD mice, and further support the concept that IL-4-related anti-inflammatory cytokines might have a role in the prevention of type 1 diabetes.     

Fas/Fas ligand interactions play an essential role in the initiation of murine autoimmune diabetes

Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. To elucidate the role of Fas/FasL interactions in NOD diabetes, the effects of neutralizing anti-FasL antibody on autoimmune responses were evaluated. Islet-specific CD8(+) and CD4(+) T-cells expressed FasL upon activation and mediated FasL-dependent cytotoxicity against Fas-expressing target cells in vitro, although their cytotoxicity against islet cells was not blocked by anti-FasL antibody. Moreover, administration of anti-FasL antibody failed to inhibit diabetes in vivo in the CD8(+) T-cell adoptive transfer model. On the other hand, blockade of Fas/FasL interactions significantly inhibited CD4(+) T-cell-dependent diabetes in adoptive transfer models. These results suggest a substantial contribution of Fas/FasL interactions to CD4(+), but not CD8(+), T-cell-mediated destruction of pancreatic beta-cells. When anti-FasL antibody was administered to NOD mice between 5 and 15 weeks of age, the onset of diabetes was slightly delayed but the incidence was not decreased. However, administration of anti-FasL antibody at 2-4 weeks of age completely prevented insulitis and diabetes. These results suggest that Fas/FasL interactions contribute to CD4(+) T-cell-mediated beta-cell destruction and play an essential role in the initiation of autoimmune NOD diabetes.     

输注调节性T细胞延缓非肥胖糖尿病小鼠糖尿病的发生

目的 观察过继输注调节性T细胞(Treg)对非肥胖糖尿病(NOD)小鼠自发性糖尿病发生的影响.方法 分选NOD小鼠天然调节性T细胞(nTreg),并诱导Na(i)ve T细胞转化为CD4+CD25+Foxp3+调节性T细胞(iTreg),分别输注予4周龄的雌性NOD小鼠,观察糖尿病的发生情况,并确定细胞输注后Treg在体内的分布、检测血清中TGF-β1、IL-10及IL-4的表达以探讨Treg的作用机制.结果 对照组小鼠13周龄时即100%自发性发生糖尿病,而输注iTreg和nTreg分别能延缓糖尿病的发生时间至24周龄和25周龄,与对照组比较差异有统计学意义(P＞0.05).Treg细胞输注后主要分布在胸腺,iTreg在体内能促进TGF-β1、IL-10及IL-4表达的上调[血中浓度分别为(543.00±26.51)、(107.67±12.66)、(93.33±12.58)ng/L],与对照组比较差异有统计学意义[(60.67±15.82)、(20.67±6.03)、(30.67±5.51)ng/L,P＜0.05].结论 在体外诱导生成的调节性T细胞可延缓NOD小鼠糖尿病的发生,其机制与上调具有免疫抑制效应的细胞因子相关.     

T-cell promiscuity in autoimmune diabetes

OBJECTIVE—It is well established that the primary mediators of β-cell destruction in type 1 diabetes are T-cells. Nevertheless, the molecular basis for recognition of β-cell–specific epitopes by pathogenic T-cells remains ill defined; we seek to further explore this issue.RESEARCH DESIGN AND METHODS—To determine the properties of β-cell–specific T-cell receptors (TCRs), we characterized the fine specificity, functional and relative binding avidity/affinity, and diabetogenicity of a panel of GAD65-specific CD4⁺ T-cell clones established from unimmunized 4- and 14-week-old NOD female mice.RESULTS—The majority of GAD65-specific CD4⁺ T-cells isolated from 4- and 14-week-old NOD female mice were specific for peptides spanning amino acids 217–236 (p217) and 290–309 (p290). Surprisingly, 31% of the T-cell clones prepared from 14-week-old but not younger NOD mice were stimulated with both p217 and p290. These promiscuous T-cell clones recognized the two epitopes when naturally processed and presented, and this dual specificity was mediated by a single TCR. Furthermore, promiscuous T-cell clones demonstrated increased functional avidity and relative TCR binding affinity, which correlated with enhanced islet infiltration on adoptive transfer compared with that of monospecific T-cell clones.CONCLUSIONS—These results indicate that promiscuous recognition contributes to the development of GAD65-specific CD4⁺ T-cell clones in NOD mice. Furthermore, these findings suggest that T-cell promiscuity reflects a novel form of T-cell avidity maturation.Type 1 diabetes is characterized by the autoimmune-mediated destruction of the insulin-producing β-cells of the islets of Langerhans (1–3). Based on studies in the nonobese diabetic (NOD) mouse, a spontaneous model of type 1 diabetes, the primary effectors of β-cell destruction are CD4⁺ and CD8⁺ T-cells (1,4–6). Early during β-cell autoimmunity, a select panel of autoantigens, including proinsulin, insulin, GAD65, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP), and dystrophia myotonica kinase, are targeted by CD4⁺ and CD8⁺ T-cells in NOD mice (7–12). As the diabetogenic response proceeds, β-cell–specific T-cell reactivity “spreads” in a relatively defined pattern (13,14). Additional autoantigenic determinants are sequentially recognized within a single protein (intramolecularly) and among different antigens (intermolecularly) to effectively amplify the diabetogenic response.The key events involved in the breakdown of self-tolerance within the T-cell compartment and which shape the T-cell receptor (TCR) repertoire of β-cell–specific T-cells remain ill defined. Studies have suggested that defective thymic negative selection contributes to increased production of β-cell–specific T precursors (15–17). Furthermore, the peptide-binding properties of major histocompatibility complex (MHC) class II and class I molecules that are associated with type 1 diabetes susceptibility are thought to shape the TCR repertoire of diabetogenic T-cell effectors (18–22). Properties intrinsic to β-cell–specific TCRs may also contribute to the pathogenicity of T-cell effectors. For instance, avidity maturation promotes the expansion of IGRP-specific CD8⁺ T-cells that in turn display increased TCR avidity/affinity and enhanced pathogenicity (23). One intriguing possibility is that the pathogenicity of an autoreactive T-cell is influenced by the degree of TCR cross-reactivity. In this model, a T-cell expressing a TCR that cross-reacts with multiple β-cell–derived epitopes would be selectively expanded and exhibit increased pathogenicity.Antigen recognition by TCRs is inherently degenerate (24–26). Furthermore, a number of studies have reported cross-reactive T-cell responses between synthetic peptides that exhibit little if any sequence homology with the natural ligand (27–30). Allogeneic recognition by T-cells provides a biologically relevant example of the flexibility associated with TCR recognition (31,32). Goverman and colleagues (33) demonstrated the presence of CD4⁺ T-cells recognizing two nonoverlapping epitopes of myelin basic protein (MBP) in a murine model of experimental autoimmune encephalomyelitis (EAE). This finding suggests that cross-reactive or “promiscuous” T-cell clonotypes may promote tissue-specific autoimmunity. Other studies have reported promiscuous recognition of nonoverlapping epitopes within the same viral or foreign antigen by CD4⁺ and CD8⁺ T-cells (34–36). The molecular basis and functional impact of T-cell promiscuity remains largely undefined.In an effort to determine the properties of β-cell–specific TCRs, we prepared a large panel of GAD65-specific CD4⁺ T-cell clones isolated from unimmunized 4- and 14-week-old NOD female mice. We demonstrate that a significant frequency of these T-cell clones recognize nonoverlapping peptides derived from GAD65, and that this promiscuity correlates with increased pathogenicity.     

Immune regulatory properties of allogeneic adipose-derived mesenchymal stem cells in the treatment of experimental autoimmune diabetes

Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties, suggesting a promising therapeutic application in several autoimmune diseases, but their role in type 1 diabetes (T1D) remains largely unexplored. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSC therapy in T cell-mediated autoimmune diabetes in NOD mice. ADMSC treatment reversed the hyperglycemia of early-onset diabetes in 78% of diabetic NOD mice, and this effect was associated with higher serum insulin, amylin, and glucagon-like peptide 1 levels compared with untreated controls. This improved outcome was associated with downregulation of the CD4(+) Th1-biased immune response and expansion of regulatory T cells (Tregs) in the pancreatic lymph nodes. Within the pancreas, inflammatory cell infiltration and interferon-γ levels were reduced, while insulin, pancreatic duodenal homeobox-1, and active transforming growth factor-β1 expression were increased. In vitro, ADMSCs induced the expansion/proliferation of Tregs in a cell contact-dependent manner mediated by programmed death ligand 1. In summary, ADMSC therapy efficiently ameliorates autoimmune diabetes pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitant with the expansion/proliferation of Tregs, thereby contributing to the maintenance of functional β-cells. Thus, this study may provide a new perspective for the development of ADMSC-based cellular therapies for T1D.     

NK and NKT cells in the rat dental pulp tissues.

OBJECTIVE: The objective of this study was to elucidate the presence of natural killer (NK) and natural killer T (NKT) cells in rat dental pulp. STUDY DESIGN: Male Wistar rats (n = 10) were used. The presence of NK and NKT cells in the coronal and root pulps of the lower first molars was detected immunohistochemically. RESULTS: Infiltration of NK and NKT cells into the normal pulp was observed, and most of these cells were located in the coronal pulp rather than in the root pulp. The population of NKT cells in the pulp was only small compared to the population of NK cells. CONCLUSION: NK and NKT cells are present in normal pulp, indicating an important role for the innate immune system in the pulp tissues.     

Further mapping of the Idd5.1 locus for autoimmune diabetes in NOD mice.

The Idd5 locus for autoimmune diabetes in nonobese diabetic (NOD) mice has been mapped to the proximal half of chromosome 1 and appears to include two loci, Idd5.1 and Idd5.2, Idd5.1 being a candidate homolog of the human IDDM12 locus. Using new recombinant congenic lines, we have reduced the Idd5.1 interval to 5 cM at most, between D1Mit279 and D1Mit19 (not included). This interval now excludes the Casp8 and Cflar (Flip) candidate genes. It still retains Cd28 and Ctla4 and also includes Icos (inducible costimulator). The previously reported differential expression of Ctla4, which is induced at a lower level in NOD than in B6-activated T-cells, was found independent of Idd5.1 itself because Ctla4 expression was induced at a low level in T-cells from Idd5.1-congenic mice. The Idd5.1 locus protected against both spontaneous and cyclophosphamide-induced diabetes, but it did not prevent inflammatory infiltration of the islets of Langerhans. Furthermore, diabetogenic precursor spleen cells from prediabetic NOD and Idd5.1-congenic mice were equally capable of transferring diabetes to immunodeficient NOD.scid/scid recipient mice. The Idd5.1 locus might affect a late event of disease development, subsequent to the onset of insulitis and possibly taking place in the islets of Langerhans.     

On the edge of autoimmunity: T-cell stimulation by steady-state dendritic cells prevents autoimmune diabetes

Targeting of antigens to immature dendritic cells has been shown to result in antigen-specific T-cell tolerance in vivo. In the INS-HA/TCR-HA transgenic mouse model for type 1 diabetes, we tested the potential of the dendritic cell-specific monoclonal antibody DEC-205 conjugated to the hemagglutinin (HA) antigen (DEC-HA) to prevent disease onset. Whereas untreated INS-HA/TCR-HA mice all develop insulitis, and approximately 40% of these mice become diabetic, repeated injection of newborn mice with DEC-HA protected almost all mice from disease development. Histological examination of the pancreata revealed significant reduction of peri-islet infiltrations in DEC-HA-treated mice, and the islet structure remained intact. Moreover, HA-specific CD4+ T-cells from anti-DEC-HA-treated INS-HA/TCR-HA mice exhibited increased expression of Foxp3, cytotoxic T-lymphocyte-associated antigen-4, and the immunosuppressive cytokines interleukin-10 and transforming growth factor-beta. The findings indicate that targeting of the HA antigen to immature dendritic cells in vivo leads to a relative increase of antigen-specific Foxp3+ regulatory T-cells that suppress the development of type 1 diabetes. Our results provide a basis for the development of novel strategies focusing on prevention rather than treatment of autoimmune diseases.     

Cyclophosphamide-induced diabetes in NOD/WEHI mice. Evidence for suppression in spontaneous autoimmune diabetes mellitus

Nonobese diabetic (NOD) mice spontaneously develop a lymphocytic infiltration of pancreatic islets (insulitis) that may progress to overt diabetes. Virtually all NOD/WEHI mice develop insulitis, but very few progress to diabetes. However, cyclophosphamide (CY) can promote the onset of diabetes in NOD mice, including the NOD/WEHI strain. The means by which CY produces diabetes was investigated in NOD/WEHI mice, in which it was hypothesized that active suppression mechanisms prevented the progression from insulitis to diabetes. A study of the time course of insulitis in the islets after CY was given showed that insulitis was initially reduced but rapidly increased over 16 days, and T-lymphocytes were predominant in the lesion. This suggested a compression of the normal time course of the disease seen in NOD mice. CY did not produce diabetes in any of 11 non-NOD strains studied. Fetal isografts in NOD mice given CY several days before were subjected to lymphocytic infiltration and beta-cell destruction. These findings suggested that CY was not directly beta-cell toxic and that altered beta-cells were not essential for beta-cell destruction. This was further demonstrated with subdiabetogenic doses of streptozocin, which significantly damaged beta-cells but did not increase the severity of insulitis or induce diabetes as did CY. Most important, the transfer of mononuclear cells from nondiabetic NOD mice to mice given CY prevented diabetes, which indicated that the likely effect of CY was via immunomodulation, possibly by allowing poised effector cells to act on beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

NK cells in the physiology of the fetomaternal relationships

The NK cells are large granular lymphocytes that are found as approximately 2-15% of the global peripheral lymphocytes. As endometrial granulocytes, they represent 70% of the decidual leukocytes during the first trimester of pregnancy, with a progressive decreasing until term. The complex actions of these numerous and active cells, consist in: 1) placental regulation and control, based on the receptors for the HLA--G and HL--C antigens, that are trophoblast specific and act as universal inhibitors of the NK cells; 2) secretion of a large range of cytokines as IL-1, TNF alpha, IL-8, TGF, IFN gamma, GM-CSF, CSF1 which regulate the trophoblastic proliferation, differentiation and invasion; 3) removal of abnormal trophoblastic cells, which may appear in such a proliferating cell population; 4) infectious protection of the placental unit, due to their unspecific cytotoxic effect. In conclusion, the NK lymphocytes have important implications in the formation of the decidua, as the maternal side of the fetomaternal interface and they are deeply involved in the evolution of normal pregnancy concerning the nutritional, hormonal and immunologic aspects.     

Virus-induced diabetes in autoimmune New Zealand mice

Infection of autoimmune New Zealand mice with the D variant of encephalomyocarditis (EMC) virus results in beta-cell damage and clinical diabetes. The induction of diabetes in parental NZB and NZW strains was independent of sex. However, the susceptibility to virus-induced diabetes in their F1 offspring was sex dependent. This susceptibility was significantly higher in male (NZB X NZW) F1 mice as compared with female F1 mice. Castration of male F1 mice significantly reduced the susceptibility to diabetes. These results suggest that parental NZB and NZW strains have recessive genes at different loci which do not allow sex hormones to influence the susceptibility to diabetes. It is concluded that both the genetic background of the host and sex hormones influence the development of virus-induced diabetes in autoimmune New Zealand mice.     

Distinct monocyte gene-expression profiles in autoimmune diabetes

OBJECTIVE—There is evidence that monocytes of patients with type 1 diabetes show proinflammatory activation and disturbed migration/adhesion, but the evidence is inconsistent. Our hypothesis is that monocytes are distinctly activated/disturbed in different subforms of autoimmune diabetes.RESEARCH DESIGN AND METHODS—We studied patterns of inflammatory gene expression in monocytes of patients with type 1 diabetes (juvenile onset, n = 30; adult onset, n = 30) and latent autoimmune diabetes of the adult (LADA) (n = 30) (controls subjects, n = 49; type 2 diabetic patients, n = 30) using quantitative PCR. We tested 25 selected genes: 12 genes detected in a prestudy via whole-genome analyses plus an additional 13 genes identified as part of a monocyte inflammatory signature previously reported.RESULTS—We identified two distinct monocyte gene expression clusters in autoimmune diabetes. One cluster (comprising 12 proinflammatory cytokine/compound genes with a putative key gene PDE4B) was detected in 60% of LADA and 28% of adult-onset type 1 diabetic patients but in only 10% of juvenile-onset type 1 diabetic patients. A second cluster (comprising 10 chemotaxis, adhesion, motility, and metabolism genes) was detected in 43% of juvenile-onset type 1 diabetic and 33% of LADA patients but in only 9% of adult-onset type 1 diabetic patients.CONCLUSIONS—Subgroups of type 1 diabetic patients show an abnormal monocyte gene expression with two profiles, supporting a concept of heterogeneity in the pathogenesis of autoimmune diabetes only partly overlapping with the presently known diagnostic categories.There is evidence that monocytes of patients with type 1 diabetes are functionally aberrant, showing raised production of interleukin (IL)-1β, IL-6, superoxide anion, and prostaglandin-endoperoxide synthase 2 (PTGS2) (1–3); aberrant generation of antigen-presenting cells (4,5); and abnormal chemotaxis, adhesion, and migratory potential (6). These aberrancies are thought to play a role in the pathogenesis of the disease by disrupting tolerance and aggravating the β-cell cytotoxic potential of infiltrating monocyte-derived dendritic cells and macrophages. However, these aberrant functional findings could not always be reproduced, particularly with regard to the enhanced production of PTGS2 (7) and the poor generation of antigen-presenting cells from monocytes (8). Two issues could be relevant to these discrepancies. First, raised production of proinflammatory monocyte-derived cytokines could be related to hyperglycemia (9). Second, there might be heterogeneity within autoimmune diabetes, such as has been noted previously between adult and juvenile forms of type 1 diabetes on the basis of genetic, immune, and metabolic characteristics (10). This possible heterogeneity in autoimmune diabetes might also become evident in different monocyte activation profiles.To resolve these issues, we focus here on patterns of inflammatory gene expression in monocytes from selected patients distinguished by clinical characteristics and age at diagnosis, as well as from control subjects. Our hypothesis is that monocytes might be distinctly activated and disturbed within the known diagnostic categories of diabetes.Recently, we reported a signature of 18 inflammatory-related genes in monocytes of bipolar patients (11); activated monocytes are thought to play a role in the pathogenesis of bipolar disorder (12,13). Given the reported association between bipolar disorder and autoimmune diabetes (14), and given the possible central role of monocytes in both disorders, we tested this set of 18 proinflammatory monocyte genes in patients with autoimmune diabetes. To these 18 monocyte genes, we added 7 genes identified in a whole-genome expression profile of a set of juvenile-onset type 1 diabetic patients who had been compared with healthy control subjects and type 2 diabetic patients (see supplementary Fig. 1 [available in an online appendix at http://dx.doi.org/10.2337/db08-0496]). Thus, using quantitative RT-PCR (Q-PCR), we validated abnormal expression of 25 monocyte activation genes in latent autoimmune diabetes of the adult (LADA), adult-onset type 1 diabetic and juvenile-onset type 1 diabetic patients, and, as controls, type 2 diabetic patients and healthy subjects.Open in a separate windowFIG. 1.Color-coded correlation matrix illustrating pairwise correlations between the expression levels of the 24 genes aberrantly expressed in patients with various forms of diabetes (相似文献   

Genetic analysis of adult-onset autoimmune diabetes

OBJECTIVE

In contrast with childhood-onset type 1 diabetes, the genetics of autoimmune diabetes in adults are not well understood. We have therefore investigated the genetics of diabetes diagnosed in adults positive for autoantibodies.

RESEARCH DESIGN AND METHODS

GAD autoantibodies (GADAs), insulinoma-associated antigen-2 antibodies (IA-2As), and islet cell autoantibodies were measured at time of diagnosis. Autoantibody-positive diabetic subjects (n = 1,384) and population-based control subjects (n = 2,235) were genotyped at 20 childhood-onset type 1 diabetes loci and FCRL3, GAD2, TCF7L2, and FTO.

RESULTS

PTPN22 (1p13.2), STAT4 (2q32.2), CTLA4 (2q33.2), HLA (6p21), IL2RA (10p15.1), INS (11p15.5), ERBB3 (12q13.2), SH2B3 (12q24.12), and CLEC16A (16p13.13) were convincingly associated with autoimmune diabetes in adults (P ≤ 0.002), with consistent directions of effect as reported for pediatric type 1 diabetes. No evidence of an HLA-DRB1*03/HLA-DRB1*04 (DR3/4) genotype effect was obtained (P = 0.55), but it remained highly predisposing (odds ratio 26.22). DR3/4 was associated with a lower age at diagnosis of disease, as was DR4 (P = 4.67 × 10⁻⁶) but not DR3. DR3 was associated with GADA positivity (P = 6.03 × 10⁻⁶) but absence of IA-2A (P = 3.22 × 10⁻⁷). DR4 was associated with IA-2A positivity (P = 5.45 × 10⁻⁶).

CONCLUSIONS

Our results are consistent with the hypothesis that the genetics of autoimmune diabetes in adults and children are differentiated by only relatively few age-dependent genetic effects. The slower progression toward autoimmune insulin deficiency in adults is probably due to a lower genetic load overall combined with subtle variation in the HLA class II gene associations and autoreactivity.The genetics of type 1 diabetes in children under 17 years of age (which is characterized by autoimmune destruction of the insulin-producing β-cells in the pancreatic islets and insulin deficiency) has been comprehensively studied, with over 50 susceptibility loci reported to date (1) (www.t1dbase.org). Investigation of the genetics of autoimmune diabetes in subjects who develop the disease as adults can elucidate the etiology of this late-onset autoimmunity and could impact its future treatment. However, most studies in adults have been confined to the study of a condition diagnosed as latent autoimmune diabetes in adults (LADA), a classification that is variable (2–4) (www.actionlada.org). In general, these case subjects are aged between 30 and 50 years and noninsulin dependent/not insulin treated at diagnosis and remain so for at least 3–6 months postdiagnosis and must be positive for GAD autoantibodies (GADAs), which are associated with type 1 diabetes (www.actionlada.org). A number of studies have investigated the association of the major histocompatibility complex (MHC) region, specifically the HLA class II genes HLA-DRB1 and HLA-DQB1, with LADA (5–7). They reported an age-at-diagnosis effect for the HLA-DRB1*03/HLA-DRB1*04 (DR3/4) genotype, with the frequency of DR3/4 lower in the older age-groups compared with that in the youngest age-groups, a finding that is also characteristic of pediatric type 1 diabetes. HLA class II susceptibility alone cannot account for type 1 diabetes development (8,9). However, in LADA cases to date, only one non-MHC gene has been shown to be convincingly associated: the −23 HphI INS (rs689) single nucleotide polymorphism (SNP) (10). This SNP is a near perfect proxy for the disease causal variable number tandem repeat (VNTR) polymorphism in the insulin (INS) gene in European ancestry populations. In that study, the 400 case subjects used were diagnosed with type 2 diabetes at an age at diagnosis between 25 and 68 years, were positive for GADA and/or insulinoma-associated antigen-2 autoantibodies (IA-2As), and did not require insulin during the first 3 months following diagnosis. The T allele had an odds ratio (OR) of 0.42 (95% CI 0.31–0.58), which is consistent with reports in childhood-onset type 1 diabetes and is associated with increased immune tolerance to insulin and its precursors (11). The authors concluded that the INS VNTR locus does not distinguish LADA and pediatric type 1 diabetes.In the current study, we have taken a simpler, clinic-based approach to defining adult-onset autoimmune diabetes. To be included in the study, all diabetic case subjects were positive for one or more islet autoantibody, specifically, islet cell antibody (ICA), GADA, IA-2A, or insulin-specific autoantibodies (IAAs), and had glucose levels, oral glucose tolerance test, or HbA_1c at a level diagnostic for diabetes. With these specific criteria for inclusion, the complexities associated with classification of diabetes in adults is avoided (2) and, as advocated by a number of authors in recent commentaries (3,4), the study of autoimmune diabetes may be more informative. Twenty type 1 diabetes–associated loci, selected based on effect size (OR ≥1.15, leading to power ≥0.7 at α = 0.05), potential age-at-diagnosis effects (IL2RA, IL2, and RNLS), and putative associations with thyroid peroxidize autoantibodies (STAT4, UBASH3A, IL2, BACH2, and CTLA4 [12,13]) were tested for association with autoimmune diabetes diagnosed in adults. The obesity gene, FTO, has been shown to be associated with LADA (14) and so was also tested for association in the autoimmune diabetic case subjects, as was the gene TCF7L2, which has the strongest effect of any locus in type 2 diabetes (15). Finally, FCRL3 is thought to be a general autoimmunity locus because of its association with several autoimmune diseases (16–19), so both FCRL3 and the gene GAD2 encoding the GAD antigen itself were also tested for association in these 1,212 adult-onset autoimmune diabetic case subjects. Hence, we have tested more loci in more adult-onset autoimmune diabetic case subjects than any other study has reported on to date.     

Anti-islet autoantibodies trigger autoimmune diabetes in the presence of an increased frequency of islet-reactive CD4 T cells

OBJECTIVE

To define cellular mechanisms by which B cells promote type 1 diabetes.

RESEARCH DESIGN AND METHODS

The study measured islet-specific CD4 T cell regulation in T-cell receptor transgenic mice with elevated frequencies of CD4 T cells recognizing hen egg lysozyme (HEL) autoantigen expressed in islet β-cells and thymic epithelium under control of the insulin-gene promoter. The effects of a mutation in Roquin that dysregulates T follicular helper (Tfh) cells to promote B-cell activation and anti-islet autoantibodies were studied, as were the effects of HEL antigen–presenting B cells and passively transferred or maternally transmitted anti-islet HEL antibodies.

RESULTS

Mouse anti-islet IgG antibodies—either formed as a consequence of excessive Tfh activity, maternally transmitted, or passively transferred—caused a breakdown of tolerance in islet-reactive CD4⁺ cells and fast progression to diabetes. Progression to diabetes was ameliorated in the absence of B cells or when the B cells could not secrete islet-specific IgG. Anti-islet antibodies increased the survival of proliferating islet-reactive CD4⁺ T cells. FcγR blockade delayed and reduced the incidence of autoimmune diabetes.

CONCLUSIONS

B cells can promote type 1 diabetes by secreting anti-islet autoantibodies that act in an FcγR-mediated manner to enhance the expansion of islet-reactive CD4 T cells and cooperate with inherited defects in thymic and peripheral CD4 T–cell tolerance. Cooperation between inherited variants affecting CD4 T–cell tolerance and anti-islet autoantibodies should be examined in epidemiological studies and in studies examining the efficacy of B-cell depletion.Type 1 diabetes is an autoimmune disease caused by islet-reactive T cells that destroy insulin-producing β-cells. T-cell tolerance mechanisms normally prevent autoimmune diabetes in mice and humans (1,2), and T cell–directed treatment with anti-CD3 monoclonal antibodies slows the loss of insulin production in the 1st year after diabetes diagnosis (3). With the focus on T cells, the role of antibodies and B cells in type 1 diabetes etiopathogenesis is often neglected. A single clinical case of type 1 diabetes in a person with X-linked agammaglobulinemia indicates that type 1 diabetes can develop in the absence of antibodies and B cells (4). However, antibodies to insulin and other islet antigens are predictive of subsequent type 1 diabetes (5), and B cells are required for diabetes development in the nonobese diabetic (NOD) mouse model of type 1 diabetes (6–8). A recent clinical trial of B-cell depletion with anti-CD20 in humans with newly diagnosed type 1 diabetes demonstrated a significant delay in further loss of insulin synthesis and established a role for B cells (9), but how B cells contribute to type 1 diabetes pathology remains unclear. Better understanding of mechanisms by which B cells can promote type 1 diabetes may improve the efficacy of interventions that target B cells.B cells may contribute to type 1 diabetes pathology by capturing and presenting autoantigens to islet-reactive CD4+ T cells (10–12). The effect on CD4 T cells is particularly relevant because B cells present captured antigens to CD4 cells via major histocompatibility complex (MHC) II molecules, and type 1 diabetes is strongly associated with particular HLA-DR haplotypes. However, an alternative possibility is that B cells contribute to type 1 diabetes pathology by secreting autoantibodies. In humans, the high predictive value of anti-islet autoantibodies for progression to type 1 diabetes is well documented (13–17). However, it remains unresolved whether or not autoantibodies are simply a biomarker of breakdown in T-cell tolerance (5). Indirect evidence against a pathogenic role for autoantibodies comes from the decreased incidence of type 1 diabetes in offspring of diabetic mothers compared with diabetic fathers, despite transmission of maternal anti-islet autoantibodies (18–20). Paradoxical protection from diabetes in children with maternally derived islet-autoantibodies has been reported (21), although this protection was not observed in children with the high-risk HLA-DR3/DR4-DQ8 genotype. This observation could indicate that the effects of anti-islet antibodies are influenced by underlying heterogeneity in the efficiency of CD4 T–cell tolerance mechanisms, which are affected by variability in MHC II antigen presentation.Only a few experimental studies have investigated whether or not secreted autoantibodies influence type 1 diabetes progression, and none have examined their influence on cellular mechanisms of tolerance in islet-reactive CD4 T cells. In NOD mice, diabetes was reduced when maternal transmission of antibodies from NOD mothers was prevented (22,23). It is not yet known whether these maternal effects reflect transmission of anti-islet autoantibodies, antibodies against microbial flora, or changes in maternal transmission of microbial commensals themselves. In one study, type 1 diabetes incidence in litters from NOD mothers clustered according to the titer of anti-insulin antibodies in the mother’s serum (24); however, in another study there was no correlation (25). Supporting a pathogenic role for autoantibodies, NOD mice lacking different activating Fcγ receptors were shown to be protected from diabetes (26). Also, passive transfer of rabbit or mouse antibodies against islet-expressed ovalbumin (OVA) has been shown to enhance activation of islet-reactive CD8+ T cells and break tolerance (27). Hence, experimental studies are needed to directly investigate the effects of anti-islet autoantibodies on islet-specific CD4 T cells and how these may interact with inherited defects in islet-specific CD4 T–cell tolerance. Here we find that anti-islet autoantibodies are potent cofactors in type 1 diabetes progression and not simply markers of breakdown in CD4+ T–cell tolerance.     

Prevention of autoimmune diabetes with nonactivating anti-CD3 monoclonal antibody.

Autoreactive T cells mediate diabetes in animal models of insulin-dependent diabetes mellitus (IDDM) and are believed to cause the disease in humans. Therefore, immunotherapies directed against T cells are of particular interest for the treatment of IDDM. One candidate for such immunotherapy is anti-CD3 monoclonal antibodies (MoAbs), but clinical side effects are common with anti-CD3 treatment due to the ability of these MoAbs to activate T cells in vivo. However, F(ab')2 fragments of anti-CD3 are nonactivating and immunosuppressive. We evaluated the effects of whole anti-CD3 MoAb and F(ab')2 fragments in the setting of experimental autoimmune diabetes. Treatment with whole MoAb or F(ab')2 fragments significantly reduced the hyperglycemia induced with multiple low dosages of streptozocin (MDSDM; 232 +/- 23 mg/dl, P less than 0.01 and 235 +/- 16 mg/dl, P less than 0.01 vs. 325 +/- 25 mg/dl, respectively) in male CD1 mice. Both whole MoAb and F(ab')2 fragments suppressed the development of insulitis (P less than 0.001). Treatment with whole MoAb resulted in marked weight loss (10.4 +/- 1.5% of total body wt), and the mice appeared ill and listless, whereas, mice treated with F(ab')2 fragments gained weight (4.9 +/- 5.5% of total body wt) and appeared healthy. Treatment with whole MoAb caused activation of T cells in vivo as reflected by proliferation of freshly isolated spleen cells to recombinant interleukin-2. Depletion of T cells with whole MoAb was more pronounced than with F(ab')2 fragments, and T-cell receptor (TCR) reexpression on remaining cells occurred with F(ab')2 fragments within 48 h after F(ab')2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)