Antigen-specific prevention of type 1 diabetes in NOD mice is ameliorated by OX40 agonist treatment

Antigen-specific therapies are possibly the safest approach to prevent type 1 diabetes (T1D). However their clinical translation has yielded poor results and greater efforts need to be put into the development of novel strategies to ameliorate their clinical outcome. OX40 is a costimulatory molecule expressed by T cells after antigen recognition and has been implicated in the control effector but also regulatory T cells (Tregs) function in vivo. The activity of OX40 signal on Tregs function has been controversial. In this context we investigated whether an anti-OX40 agonist antibody treatment can ameliorate antigen-specific immune intervention for the prevention of T1D. We show that treatment of non-obese diabetic (NOD) mice with an OX40 agonistic antibody (OX86) reduced type 1 diabetes (T1D) incidence by inducing both CD4⁺CD25⁺Foxp3⁺ Tregs and CD4⁺Foxp3⁻ T cells expressing the latency-associated peptide (LAP). These OX86-induced CD4⁺Foxp3⁻LAP⁺ T cells also demonstrated suppressive activity in vitro. A significant increase in protection was observed when OX86 was combined with insulin B9:23 (insB9:23) peptide immunizations. Synergy resulted from an expansion of IL-10-expressing insB9:23-reactive Tregs which augmented the proportion of CD4⁺ T cells with in vivo suppressive activity. Consequently, CD4⁺ T cells purified from OX86/insB9:23 combination treatment prevented T1D development when adoptively transferred into recipient mice. These findings suggest that the requirement for OX40 signaling by antigen-induced Tregs can be dominant over its well-documented need for effector memory cell function and may have potentially important implications for improving the clinical translation of antigen-specific prevention of T1D and possibly other autoimmune disorders.     

Prevention of diabetes in NOD mice at a late stage by targeting OX40/OX40 ligand interactions

Autoreactive T cells play a major role in the development of insulin-dependent diabetes mellitus, suggesting that costimulatory molecules that regulate T cell responses might be essential for disease progression. In NOD mice, CD28/B7 and CD40/CD40 ligand (L) interactions control the onset of diabetes from 2 to 4 weeks of age, but blocking these molecules has little effect after this time. Hence, it is possible that other ligand/receptor pairs control a later phase of disease. We now show that OX40 is expressed on CD4 and CD8 T cells several weeks prior to islet destruction, which is initiated around weeks 12-14, and that OX40L is present on dendritic cells in both secondary lymphoid organs and the pancreas from 11 to 13 weeks of age. Blocking OX40L at 6, 9, or 15 weeks after birth had little effect on disease; however, inhibiting OX40/OX40L interactions at week 12, or continuous treatment from week 12 onwards, significantly reduced the incidence of diabetes. Histological examination showed that islet destruction was prevented and insulitis reduced by targeting OX40L. These studies show that OX40/OX40L interactions form a late checkpoint in diabetes development and suggest that these molecules are realistic targets for therapeutic intervention.     

Schistosoma mansoni egg antigens induce Treg that participate in diabetes prevention in NOD mice

Schistosoma mansoni soluble egg antigens (SEA) profoundly regulate the infected host's immune system. We previously showed that SEA prevents type 1 diabetes in NOD mice and that splenocytes from SEA‐treated mice have reduced ability to transfer diabetes to NOD.scid recipients. To further characterize the mechanism of diabetes prevention we examined the cell types involved and showed that CD25⁺ T‐cell depletion of splenocytes from SEA‐treated donors restored their ability to transfer diabetes. Furthermore, SEA treatment increased the number and proportional representation of Foxp3⁺ T cells in the pancreas of NOD mice. We have used in vitro systems to analyze the effect of SEA on the development of NOD Foxp3⁺ T cells. We find that SEA can induce Foxp3 expression in naïve T cells in a TGF‐β‐dependent manner. Foxp3 induction requires the presence of DC, which we also show are modified by SEA to upregulate C‐type lectins, IL‐10 and IL‐2. Our studies show that SEA can have a direct effect on CD4⁺ T cells increasing expression of TGF‐β, integrin β8 and galectins. These effects of SEA on DC and T cells may act in synergy to induce Foxp3⁺ Treg in the NOD mouse.     

Interferon-tau inhibits the development of diabetes in NOD mice

Interferon-alpha (IFN-α) inhibits the development of diabetes in animal models of autoimmune diabetes. However, the mechanism of the action is not fully understood and drug toxicity could limit its potential clinical utility. Interferon-tau (IFN-τ) is another type 1 interferon, which has less toxicity but may have different biologic activity than IFN-α. This study explores the effect of IFN-τ on the diabetic process in non-obese diabetic (NOD) mice. IFN-τ by intraperitoneal, subcutaneous, or oral routes of administration decreased the development of spontaneous diabetes in NOD mice. Islet inflammation was decreased 50%. IFN-τ administration to recipient mice prevented the development of passively transferred and cyclophosphamide accelerated diabetes. IFN-τ treatment also decreased anti-islet effector activity of NOD splenic cells. Immunoregulatory activity of splenic cells was augmented by IFN-τ administration as was the number of splenic CD25⁺CD4⁺ cells. Concanavalin A (Con A)-induced release of IFN-γ was decreased in spleen cells from IFN-τ treated mice. In conclusion, IFN-τ inhibits spontaneous autoimmune diabetes and passively transferred diabetes in the NOD mouse. This diabetes sparing activity may be due to an induction of regulatory cells, possibly CD25⁺CD4⁺ T cells, which in turn inhibit anti-islet effector cell activity and the development of insulitis and diabetes. Due to the lower drug toxicity, IFN-τ could be a better drug candidate than IFN-α for experimental clinical trials.     

Interferon-tau inhibits the development of diabetes in NOD mice

Interferon-alpha (IFN-alpha) inhibits the development of diabetes in animal models of autoimmune diabetes. However, the mechanism of the action is not fully understood and drug toxicity could limit its potential clinical utility. Interferon-tau (IFN-tau) is another type 1 interferon, which has less toxicity but may have different biologic activity than IFN-alpha. This study explores the effect of IFN-tau on the diabetic process in non-obese diabetic (NOD) mice. IFN-tau by intraperitoneal, subcutaneous, or oral routes of administration decreased the development of spontaneous diabetes in NOD mice. Islet inflammation was decreased 50%. IFN-tau administration to recipient mice prevented the development of passively transferred and cyclophosphamide accelerated diabetes. IFN-tau treatment also decreased anti-islet effector activity of NOD splenic cells. Immunoregulatory activity of splenic cells was augmented by IFN-tau administration as was the number of splenic CD25+CD4+ cells. Concanavalin A (Con A)-induced release of IFN-gamma was decreased in spleen cells from IFN-tau treated mice. In conclusion, IFN-tau inhibits spontaneous autoimmune diabetes and passively transferred diabetes in the NOD mouse. This diabetes sparing activity may be due to an induction of regulatory cells, possibly CD25+CD4+ T cells, which in turn inhibit anti-islet effector cell activity and the development of insulitis and diabetes. Due to the lower drug toxicity, IFN-tau could be a better drug candidate than IFN-alpha for experimental clinical trials.     

IL-15 promotes regulatory T cell function and protects against diabetes development in NK-depleted NOD mice

IL-15, an anti-apoptotic cytokine, has been reported to promote the survival and function of NK cells and T cells, including regulatory T cells (Tregs). Here we examined the effect of repeated injections of IL-15 on the development of diabetes in NOD mice. Injection of recombinant murine IL-15, once a day for 2 weeks, neither inhibited nor accelerated diabetes development in untreated NOD mice. However, treatment with IL-15 significantly reduced the incidence and delayed the onset of diabetes in NOD mice that were depleted of NK cells, while NK cell depletion alone had no protection against the disease development. The protective effect in IL-15-treated, NK cell-depleted NOD mice was associated with an increase in immunosuppressive activity of CD4⁺CD25⁺ Tregs. IL-15 also enhanced Foxp3 expression in CD4⁺CD25⁺ cells in an in vitro culture system, and such an effect of IL-15 was abrogated by IL-15-activated NK cells. Inhibition of IL-15-induced Foxp3 expression by IL-15-activated NK cells likely resulted from their IFN-γ production, as recombinant IFN-γ, or the culture supernatant of IL-15-activated wild-type mouse NK cells but not of IL-15-activated IFN-γ-deficient NK cells, mediated a similar inhibition. IFN-γ also diminished the stimulatory effect of IL-15 on Treg function in vitro. These results indicate that IL-15 has the potential to promote Treg function and protect against diabetes development in NOD mice, but such an activity can be eliminated by simultaneous activation of NK cells in IL-15-treated mice.     

Peripherally induced regulatory T cells contribute to the control of autoimmune diabetes in the NOD mouse model

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells and is partly caused by deficiencies in the Foxp3⁺ regulatory T‐cell (Treg) compartment. Conversely, therapies that increase Treg function can prevent autoimmune diabetes in animal models. The majority of Tregs develop in the thymus (tTregs), but a proportion of Foxp3⁺ Tregs is generated in the periphery (pTregs) from Foxp3^?CD4⁺ T‐cell precursors. Whether pTregs play a distinct role in T1D has not yet been explored. We report here that pTregs are a key modifier of disease in the nonobese diabetic (NOD) mouse model for T1D. We generated NOD mice deficient for the Foxp3 enhancer CNS1 involved in pTreg induction. We show that CNS1 knockout decreased the frequency of pTregs and increased the risk of diabetes. Our results show that pTregs fulfill an important non‐redundant function in the prevention of beta cell autoimmunity that causes T1D.     

Increased thymic development of regulatory T cells in NOD mice is functionally dissociated from type I diabetes susceptibility

Regulatory T (Treg) lymphocytes play a central role in the control of autoimmune pathology. Any alteration in Treg‐cell biology in mouse strains used for the study of these disorders therefore raises the question of its direct link with disease susceptibility. Paradoxically, in non‐obese diabetic (NOD) mice increased numbers of Treg cells develop in the thymus. In this report we identify a locus of <7 Mbp that quantitatively controls Treg‐cell development in the thymus of the NOD mouse. This ‘Trd1' region is located centromeric to the H2 complex on chromosome 17 and does not include genes encoding classical MHC molecules. The genomic region identified here contains the Idd16 diabetes susceptibility locus and the use of congenic mouse strains allowed us to investigate the potential link between quantitatively altered thymic Treg cells and diabetes susceptibility. Hybrid mice present similar levels of thymic Treg cells as B6 animals but they developed diabetes with the same kinetics as NOD mice. Therefore, the increased Treg‐cell development in NOD mice controlled by Trd1 is functionally dissociated from the susceptibility of NOD to diabetes.     

Foxp3在小鼠1型糖尿病模型中的表达情况研究

目的:研究Foxp3在小鼠1型糖尿病模型中的表达情况,并探讨其在1型糖尿病发病机制中的作用.方法:利用链脲佐菌素(STZ)致诱小鼠1型糖尿病模型,半定量RT-PCR检测脾脏Foxp3 mRNA的表达,Western blot法检测脾脏Scurfin蛋白的表达,流式细胞术检测CD4+CD25+Treg细胞亚群比例.结果:模型组Foxp3 mRNA及其蛋白产物Scurfin的表达短期内上升,约在7天左右开始下降,到30天左右低于对照组(P＜0.05).7天内,CD4+CD25+Treg细胞亚群比例在模型组与对照组大致相等,而7天后,其比例在模型组逐渐下降(P＜0.05).结论:Foxp3的表达异常,使CD4+CD25+Treg细胞亚群比例下降,以致不能抑制效应性CD4+T细胞的增殖对胰岛的破坏,此机制参与了1型糖尿病的发生.     

系统性红斑狼疮患者外周血调节性T细胞、Foxp3和IL-6的表达

目的探讨系统性红斑狼疮(systemic lupus erythematosus,SLE)患者外周血CD4 CD25 调节性T细胞(regulatory Tcells,Tregs)、Foxp3mRNA和血浆IL-6表达的意义。方法对38例SLE患者和16例正常人采用流式细胞术检测外周血CD4 CD25 Tregs百分率,RT-PCR检测Foxp3mRNA表达,ELISA法检测IL-6水平。结果①SLE活动组和非活动组的CD4 CD25 Tregs水平均显著低于正常对照组(P<0.01);②SLE活动组的Foxp3mRNA表达水平明显低于正常对照组(P<0.05);③SLE活动组和非活动组血浆的IL-6水平均显著高于正常对照组(P<0.01),而且活动组显著高于非活动组(P<0.05);④38例SLE患者的CD4 CD25 Tregs水平与SLEDAI评分呈显著性负相关(P<0.01),Foxp3mRNA水平与CD4 CD25 Tregs呈显著性正相关(P<0.01),血浆IL-6水平与SLEDAI评分之间呈显著性正相关(P<0.01),血浆IL-6水平与CD4 CD25 Tregs/CD4 细胞比值呈显著负相关(P<0.05)。结论CD4 CD25 Tregs和Foxp3以及IL-6可能在SLE的发生和发展中发挥重要作用。     

Anti-suppressor effect of cyclophosphamide on the development of spontaneous diabetes in NOD mice

In the NOD mouse, an autoimmune process beginning by 5 weeks of age with lymphocyte infiltration and destruction of insulin-secreting beta cells leads to overt diabetes which begins to appear by 11 weeks of age. Although there is a high incidence of insulitis by 10 weeks of age (greater than 80%) in both males and females, by 30 weeks of age diabetic symptoms have occurred in 53-80% of females and in 12-40% of males. Intraperitoneal injection of a high dose (200 mg/kg) of cyclophosphamide (CY) consistently induces the onset of diabetes in male and female NOD mice at an age when spontaneous diabetes rarely occurs. Spleen T cells from CY-induced diabetic mice are capable of transferring the disease into irradiated nondiabetic syngeneic recipients. This indicates that the diabetogenic effect of CY is not mediated by direct toxicity on pancreatic beta cells but is mediated by abrogation of a suppressor mechanism which may prevent activation of T cells responsible for the development of diabetes in the NOD mouse. Additionally, CY is only effective in NOD mice and not in F1 hybrids between NOD and other strains of mice. Thus, the potential beta cell aggressor mechanism is not present in these hybrids as it is in homozygous mice, which indicates that it is not under the control of dominant genes.     

Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

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胸腺基质淋巴细胞生成素、OX40和OX40受体在过敏性腹泻小鼠大肠黏膜中表达的关系

目的 建立小鼠过敏性腹泻模型,检测其大肠黏膜中胸腺基质淋巴细胞生成素(TSLP)、OX40、OX40受体(OX40L)的表达和肠系膜淋巴结细胞培养上清中IL-4、IFN-γ的水平,分析其之间的关系,探讨TSLP、OX40、OX40L在过敏性腹泻中的作用.方法 雌性BALB/c小鼠20只随机分成两组即对照组和实验组.采用酶联免疫吸附法(ELISA)检测肠系膜淋巴结细胞培养上清中IL-4和IFN-γ的水平,取大肠组织HE染色观察大肠组织病理改变,SP免疫组织化学技术检测TSLP、OX40、OX40L的表达.结果 ①HE染色结果显示:实验组比对照组小鼠结肠黏膜组织非特异性炎症反应显著,上皮排列不规则,有大量炎性细胞浸润,固有层可见大量嗜酸性粒细胞浸润;②TSLP、OX40、OX40L在过敏性腹泻小鼠大肠黏膜中的表达水平高于对照组的表达水平(t=7.07,t =7.81,t =7.79,P均＜0.01);③Spearman等级相关分析发现TSLP、OX40、0X40L三者间表达强度存在正相关(r=0.889,r=0.932,r=0.943,P均＜0.01),同时三者与肠系膜淋巴结细胞培养上清中IL-4水平呈正相关(r=0.891,r =0.936,r=0.886,P均＜0.05),而与IFN-γ水平呈负相关(r=-0.829,r=-0.881,r=-0.937,P均＜0.05).结论 TSLP、OX40、OX40L三者间表达存在正相关,并诱导了Th1/Th2轴向Th2轴漂移,促进了过敏性腹泻的发生发展.     

补肾宁心方诱导去势鼠Treg扩增并促进成骨细胞PCNA表达

为了探讨补肾宁心方(BSNXD)对去势鼠脾脏内CD4+CD25+Foxp3+Treg数目及特异性细胞因子CTLA-4、TGF-β及IL-10表达的调控,以及BSNXD对成骨细胞(OB)增殖的影响.建立小鼠绝经后骨质疏松(PMO)模型,随机分为Sham组、OVX组、OVX+BSNXD组和OVX+E2组.取各组腰椎和股骨行...     

Characterisation of CD8 monoclonal antibody-induced protection from diabetes in NOD mice

NOD mice can be protected from transferred diabetes for long periods by short-term treatment with CD8 mabs. This protection has previously been shown to be thymus-dependent as thymectomised mice do not show the long-term protection observed in intact mice. In this study we show that the thymus is required only during antibody treatment as its removal thereafter does not affect protection. Recent thymic emigrants (RTEs) are not necessary for long-term tolerance induction and irradiation plays no part as anti-CD8 treatment cannot protect NOD.scid recipients from diabetes development. IL-10 is also shown to play an important role in the anti-CD8 induced protection in intact mice as it is reversed by IL-10R blockade.     

哮喘小鼠CD4~+CD25~+Foxp3~+调节T细胞和Foxp3蛋白的变化及淫羊藿苷干预作用

目的:研究哮喘小鼠CD4+CD25+Foxp3+调节T(Treg)细胞和Foxp3蛋白的变化,探讨淫羊藿苷对哮喘干预机制。方法:将BALB/c小鼠32只随机分为正常对照组、哮喘模型组、淫羊藿苷组、地塞米松阳性对照组,每组8只。哮喘模型制备用卵白蛋白(OVA)致敏大鼠并雾化吸入刺激,治疗组采用相应药物灌胃给药,对照组用等量生理盐水代替。最后一次激发24小时后,采用Buxco肺功能仪有创法检测小鼠气道反应性;HE染色评价观察小鼠肺组织病理形态学改变;流式细胞术检测脾脏CD4+CD25+Foxp3+Treg细胞比例及Foxp3蛋白表达量;免疫印迹法测定肺组织Foxp3蛋白表达量;ELISA法测定血清及肺泡灌洗液(BALF)IL-10水平。结果:与正常对照组比较:模型组气道阻力及气道炎症指数显著增加(P<0.05);脾Treg细胞比例无明显变化(P>0.05);脾Foxp3蛋白表达显著下降(P<0.05),肺组织Foxp3蛋白表达显著升高(P<0.05),外周血IL-10水平显著下降(P<0.05),BALF中IL-10无显著差异(P>0.05);与模型组比较:淫羊藿苷组气道阻力和气道炎症明显减轻(P<0.05);脾Treg细胞比例和脾Foxp3蛋白表达水平无显著变化(P>0.05),但淫羊藿苷可进一步上调哮喘小鼠肺组织Foxp3蛋白表达量(P<0.05),并增加外周血IL-10水平(P<0.05)。结论:哮喘小鼠脾脏CD4+CD25+Foxp3+Treg细胞Foxp3蛋白表达水平下降,淫羊藿苷可显著改善哮喘小鼠气道炎症和气道高反应,可能与其上调肺组织Foxp3蛋白表达和增加外周血IL-10水平相关。     

Graves病患者外周血OX40和OX40L分子表达的研究

探讨OX40和OX40L分子在Graves病患者外周血T淋巴细胞和单核细胞上的表达及其在Graves病发病机制中的可能作用。采用流式细胞仪检测技术对50例初发Graves病患者和30例健康志愿者外周血T细胞上OX40和单核细胞上OX40L的表达水平进行分析,并对28例治疗前后病人淋巴细胞上OX40/OX40L的表达进行了比较。结果:与正常对照组比,初发Graves病患者外周血CD4+T细胞上OX40表达水平明显升高,而CD8+T细胞上OX40表达水平无显著变化;同时,还检测到单核细胞上OX40L表达水平也明显增加。治疗后,FT3(游离三碘甲腺原氨酸)值恢复至健康对照组表达范围的Graves病患者其CD4+T细胞上OX40和单核细胞上OX40L的表达均显著降低至正常水平;而FT3值仍偏高的患者单核细胞上OX40L的表达降低,但未降至健康对照组表达范围,CD4+T细胞上OX40的表达则无显著变化。Graves病患者外周血T细胞和单核细胞上分别存在OX40和OX40L的异常表达,且与治疗效果密切相关,提示OX40/OX40L信号在T细胞与单核细胞相互作用过程中可能有助于自身反应性T细胞的持续活化,从而参与Graves病的免疫发生发展。     

连翘对严重烧伤大鼠外周血Treg及脾脏Foxp3的影响

目的:探讨中药连翘对严重烧伤大鼠的免疫调节作用及其机制.方法:采用大鼠30%体表面积Ⅲ度烧伤模型.实验共分5组,分别为正常对照组(Control),烧伤后8 h组(8PBH),连翘高、中、低剂量组(AFS1、AFS2、AFS3),其中AFS1、AFS2和AFS3组在烧伤前7 d开始灌胃给药,1次/d,分别给予AFS 5 g/kg、2.5 g/kg和1.25 g/kg.各组在烧伤后8 h留取外周血和脾脏组织,流式细胞术测外周血Treg百分数;RT-PCR和免疫组化法测定脾脏组织Foxp3表达.结果:与Control组比较,8PBH组外周血Treg水平显著升高,脾脏组织Foxp3mRNA和蛋白表达明显上调;AFS1、AFS2及APS3组均能显著减轻上述指标变化,并呈剂量依赖关系.结论:连翘具有免疫调节作用,其作用机制与干扰Foxp3基因的表达有关.     

OX40/OX40L interaction induces the expression of CXCR5 and contributes to chronic colitis induced by dextran sulfate sodium in mice

Interactions between APC and T lymphocytes have been implicated as a major factor contributing to inflammatory bowel disease. To test whether OX40/OX40L interaction plays a role in chronic intestinal inflammation, we induced chronic colitis using dextran sulfate sodium and treated the mice with a murine fusion protein (OX40-IgG). Treatment resulted in a dose-dependent and significant reduction of intestinal inflammation (46%) as measured by a histologic score. IL-10 and IL-5 production from mesenteric lymph node cells increased 20-fold and 18-fold, respectively. In colonic tissue, IL-10 mRNA levels increased and the expression of T-bet was decreased to 30%. IL-10 neutralization partly inhibited the beneficial effects of OX40-IgG treatment. Surprisingly, despite the reduction of inflammation we found the number and size of colonic lymphoid follicles increased, with an accumulation of CD4(+) cells in the mantle area. In contrast, the number of CD4(+) cells infiltrating the mucosa was significantly reduced, as was their CXCR5 expression (24-fold). We conclude that OX40/OX40L interaction contributes to the perpetuation of chronic colitis partly by suppressing IL-10 production. Furthermore, our data suggest that the OX40/OX40L-induced CXCR5 expression on CD4(+) cells may be important for the inflammatory process by allowing migration to the germinal center for further differentiation of CD4(+) cells before they infiltrate the chronically inflamed mucosa.