IL-10/TGF-β-Treated Dendritic Cells,Pulsed with Insulin,Specifically Reduce the Response to Insulin of CD4+ Effector/Memory T Cells from Type 1 Diabetic Individuals

Introduction  

Diabetogenic autoreactive T cells with effector/memory characteristics are described in type 1 diabetes patients (T1D). Alternatively activated dendritic cells (aaDCs) have been regarded as promising tools for clinical application in autoimmune diseases (ADs), although their ability to induce antigen-specific tolerance in T cells derived from ADs has yet to be determined.     

Bacillus Calmette-Guérin Suppresses Asthmatic Responses via CD4+CD25+ Regulatory T Cells and Dendritic Cells

Purpose

Bacillus Calmette-Guérin (BCG) is known to suppress the asthmatic responses in a murine model of asthma and to induce dendritic cells (DCs) maturation. Mature DCs play a crucial role in the differentiation of regulatory T cells (Tregs), which are known to regulate allergic inflammatory responses. To investigate whether BCG regulates Tregs in a DCs-mediated manner, we analyzed in a murine model of asthma.

Methods

BALB/c mice were injected intraperitoneally with BCG or intravenously with BCG-stimulated DCs and then sensitized and challenged with ovalbumin (OVA). Mice were analysed for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. To identify the mechanisms, IgE, IgG₁ and IgG_2a in the serum were analysed and the CD25₊ Tregs in the mice were depleted with anti-CD25 monoclonal antibody (mAb).

Results

BCG and the transfer of BCG-stimulated DCs both suppressed all aspects of the asthmatic responses, namely, BHR, the production of total IgE and OVA-specific IgE and IgGs, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment reversed these effects.

Conclusions

BCG can attenuate the allergic inflammation in a mouse model of asthma by a Tregs-related mechanism that is mediated by DCs.     

CCR7+ Central and CCR7− Effector Memory CD4+ T Cells in Human Cutaneous Leishmaniasis

Purpose

The profile of central (=T_CM) and effector (=T_EM) memory CD4⁺ T cell subsets and the possible role as surrogate markers of protection is studied in the volunteers with history of cutaneous leishmaniasis (HCL).

Methods

Profile of T cell subsets based on CCR7/CD45RA expressions and phenotypic changes after soluble Leishmania antigen (SLA) stimulation were analyzed. Then, sorted CD4⁺CD45RO^?CD45RA⁺ naïve T, CD4⁺CD45RO⁺CD45RA^?CCR7^? T_EM, CD4⁺CD45RO⁺CD45RA^?CCR7⁺ T_CM subsets were cultured with SLA for proliferation, cytokine production and intracellular cytokine assays.

Results

In the HCL and control volunteers, the mean frequencies of CD4⁺CD45RA⁺CCR7⁺ naïve T cells and CD4⁺CD45RA^?CCR7^? T_EM cells were higher than the other subsets before culture. Frequency of naïve T cells and CD4⁺CD45RA^?CCR7⁺ T_CM cells was significantly decreased (P?=?0.01 for naïve T and P?<?0.05 for T_CM cells) and frequency of T_EM cells was significantly increased after SLA stimulation compared to before culture (P?<?0.001). By CFSE labeling, CD4⁺CD45RO⁺CD45RA^?CCR7⁺ T_CM cells showed more proliferation potential than CD4⁺CD45RO⁺CD45RA^?CCR7^? T_EM cells. Stimulation of the T_EM cells in HCL volunteers induced a significantly higher IFN-γ production (P?=?0.04) with higher number of intracellular IFN-γ positive cells (P?=?0.032) than the same cells from controls. A significantly higher number of T_CM cells produced IL-2 in HCL volunteers compared with controls (P?<?0.05). Most of the intracellular IFN-γ positive T_EM cells were proliferating CFSE-dim populations (P?<?0.05).

Conclusions

A combination of Leishmania-reactive IFN-γ producing CD4⁺CD45RO⁺CD45RA^?CCR7^? T_EM and Leishmania-reactive IL-2 producing CD4⁺CD45RO⁺CD45RA^?CCR7⁺ T_CM are identified in individuals with history of CL which might play a role in protective recall immune response against Leishmania infection.     

Control of the development of CD8αα+ intestinal intraepithelial lymphocytes by TGF-β

The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell-specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4?CD8?TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.     

Interleukin-12-Producing CD103+ CD11b? CD8+ Dendritic Cells Are Responsible for Eliciting Gut Intraepithelial Lymphocyte Response against Encephalitozoon cuniculi

Microsporidia, which belong to the kingdom Fungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b⁻ CD8⁺ DC in the initiation of an antigen-specific IEL in vivo. Effectively, after Encephalitozoon cuniculi infection, CD11b⁻ CD8⁺ DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103⁺ CD11b⁻ CD8⁺ DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103⁺ CD11b⁻ CD8⁺ DC isolated from infected animals in the generation of an IEL response against a live pathogen.     

Human mesenchymal stromal cells enhance the immunomodulatory function of CD8+CD28? regulatory T cells

One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8⁺CD28⁻ Treg cells and found that the MSCs could not only increase the proportion of CD8⁺CD28⁻ T cells, but also enhance CD8⁺CD28⁻T cells'' ability of hampering naive CD4⁺ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4⁺ T cells and inducing the apoptosis of activated CD4⁺ T cells. Mechanistically, the MSCs affected the functions of the CD8⁺CD28⁻ T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8⁺CD28⁺ T cells to CD8⁺CD28⁻ T cells, but did increase the proportion of CD8⁺CD28⁻ T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8⁺CD28⁻ Treg cells, shedding new light on MSCs-mediated immune regulation.     

Differential Sensitivity of Naïve and Memory Subsets of Human CD8+ T Cells to TNF-α-Induced Apoptosis

In this investigation, we have examined the relative sensitivity of human naïve, central memory (T_CM), and two types of effector memory CD8+ T cells (T_EM and T_EMRA) to TNF-α-induced apoptosis. Our data show that naïve and T_CM CD8+ T cells were sensitive, whereas T_EM and T_EMRA CD8+ T cells were relatively resistant to TNF-a-induced apoptosis. The apoptosis profile correlated with the activation of caspase-8 and caspase-3. However, no correlation was observed between relative sensitivity of four CD8 + T cell subsets to apoptosis and the expression of TNFR-I or TNFR-II. T_EM and T_EMRA CD8+ T cells displayed increased phosphorylation of IKKα/β and IκB and increased NF-κB activity as compared to naïve and T_CM CD8+ T cells. Bcl-2, Bcl-x_L and FLIP_L expression was higher and Bax expression was lower in T_EM and T_EMRA CD8+ T cells as compared to naïve and T_CM CD8+ T cells. These data suggest that signaling molecules downstream of TNFRs may be responsible for differential sensitivity among subsets of CD8+ T cells to TNF-α-induced apoptosis.     

TGF-β1诱导CD4+CD25-T细胞分化为CD4+CD25+调节性T细胞

目的 研究TGF-β1诱导CD4 CD25 调节性T细胞分化的能力及其相关机理.方法 ①利用丝裂霉素处理的Balb/C裸鼠脾细胞(同种抗原)刺激C57BL/6小鼠T细胞,同时给予TGF-β1予以干预(按TGF-β1剂量不同设立4组:对照组,低浓度组,中浓度组和高浓度组),共同培养5天后,用流式细胞仪检测CD4 CD25 T细胞比例;同时用RT-PCR检测培养细胞中Foxp3的表达水平.②在第一部分实验基础上,通过MACS分离获取C57BL/6小鼠CD4 CD25-T细胞,用同种抗原刺激后,给予TGF-β1干预,培养5 d后分离CD4 CD25 T细胞,利用混合淋巴细胞培养系统检测其抑制淋巴细胞增殖的能力.结果 T细胞经同种抗原刺激后,在中、高浓度TGF-β1诱导下CD4 CD25 T细胞比例均明显升高,Foxp3的表达水平也相应增加,与对照组相比有显著性差异(P＜0.05).TGF-β1可直接诱导CD4 CD25-T细胞转化为CD4 CD25 T细胞,转化后的CD4 CD25 T细胞可有效抑制淋巴细胞增殖.结论 TGF-β1可诱导CD4 CD25-T细胞转化为CD4 CD25 Treg,促进其表达Foxp3,并能够抑制淋巴细胞增殖.     

Cerebral small-vessel disease protein HTRA1 controls the amount of TGF-β1 via cleavage of proTGF-β1

Cerebral small-vessel disease is a common disorder in elderly populations; however, its molecular basis is not well understood. We recently demonstrated that mutations in the high-temperature requirement A (HTRA) serine peptidase 1 (HTRA1) gene cause a hereditary cerebral small-vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1 belongs to the HTRA protein family, whose members have dual activities as chaperones and serine proteases and also repress transforming growth factor-β (TGF-β) family signaling. We demonstrated that CARASIL-associated mutant HTRA1s decrease protease activity and fail to decrease TGF-β family signaling. However, the precise molecular mechanism for decreasing the signaling remains unknown. Here we show that increased expression of ED-A fibronectin is limited to cerebral small arteries and is not observed in coronary, renal arterial or aortic walls in patients with CARASIL. Using a cell-mixing assay, we found that HTRA1 decreases TGF-β1 signaling triggered by proTGF-β1 in the intracellular space. HTRA1 binds and cleaves the pro-domain of proTGF-β1 in the endoplasmic reticulum (ER), and cleaved proTGF-β1 is degraded by ER-associated degradation. Consequently, the amount of mature TGF-β1 is reduced. These results establish a novel mechanism for regulating the amount of TGF-β1, specifically, the intracellular cleavage of proTGF-β1 in the ER.     

In Vitro Differentiation of Human Monocytes into Dendritic Cells by Peptic–Tryptic Digest of Gliadin Is Independent of Genetic Predisposition and the Presence of Celiac Disease

Introduction  This study was done to further reveal the role of the innate immune system in celiac disease. Methods  Dendritic cells were matured from venous blood of patients with active or treated celiac disease and DQ2–DQ8-positive or negative controls. Dendritic cells were treated with a peptic–tryptic digest of gliadin (500 μg/ml) and their activation was analyzed by fluorescent-activated cell sorting analysis, cytokine secretion, and their ability to elicit T cell proliferation. Results and Discussion  Gliadin upregulated interleukin (IL)-6, IL-8, and IL-12 (p40) secretion in dendritic cells and induced strong expression of the maturation markers human leukocyte antigen (HLA)-DR, CD25, CD83, and CD86 of all subjects irrespective of their genotype or the presence of disease, whereas the digest of bovine serum albumin showed no effect. However, gliadin-stimulated dendritic cells from active celiac showed enhanced stimulation of autologous T cells compared to the other groups. Conclusion  Further research should be aimed at identifying the mechanisms that control inflammation in healthy individuals. Maryam Rakhimova and Birgit Esslinger contributed equally to this work.     

CpG Oligodeoxynucleotide Inhibits Cockroach-Induced Asthma via Induction of IFN-γ+ Th1 Cells or Foxp3+ Regulatory T Cells in the Lung

PurposeCpG oligodeoxynucleotide (CpG-ODN), a TLR9 agonist, activates innate immunity and induces Th1 response. Although the immune modulatory effect of CpG-ODN has been extensively studied, its function in cockroach extract-induced allergic asthma has not been studied. Here, we investigated the inhibitory function of CpG-ODN in cockroach extract-induced asthma in mice with different treatment schemes.MethodsScheme 1: BALB/C mice were intra-nasally co-administered by cockroach extract and CpG-ODN twice a week for 3 weeks; Scheme 2: The mice were intra-nasally pre-treated with CpG-ODN at day 0 and cockroach allergen challenge was performed from day 3 as in scheme 1. Scheme 3: Cockroach allergen challenge was performed as in scheme 1 and CpG-ODN was post-treated at day 21. Then, BAL cell count, flow cytometric analysis of alveolar macrophages, regulatory T cells, and lung tissue histology, Th1 and Th2 cytokines, serum IgE, cockroach specific IgE, IgG1/IgG2a ratio, and airway hyper-responsiveness were evaluated.ResultsMice with repeated intra-nasal exposure to CpG-ODN showed a dramatic decrease in eosinophilic inflammation, goblet cell hyperplasia, and airway hyper-responsiveness with reduction of IL-13, IL-5, and serum IgE, cockroach specific IgE and IgG1/IgG2a ratio. This inhibitory function might be related to the up-regulation of IL-10 and CD4⁺Foxp3⁺ regulatory T cells in the lung. Interestingly, one-time challenge of CpG-ODN either prior or posterior to cockroach extract exposure could modulate airway inflammation and hyper-responsiveness via increase of Th1 response.ConclusionsCollectively, our data suggest that CpG-ODN treatment modulates Th2 inflammation in the lung by induction of regulatory T cells or Th1 response in a cockroach-induced asthma model.     

TGF-β1参与肺癌CD4+CD25+调节性T细胞比例增高的机制初探

CD4^＋CD25^＋调节性T细胞在肿瘤免疫耐受机制中发挥的作用近来备受瞩目,多种肿瘤患者机体内外周血及肿瘤局部均有调节性T细胞的比例增高.TGF-β1是很强的免疫抑制因子,它能影响参与天然免疫和获得性免疫免疫细胞的增殖、活化和分化,抑制免疫调节性细胞因子的产生.在肺癌病变局部,TGF-β1是否在淋巴细胞被诱导分化为CD4^＋CD25^＋调节性T细胞过程发挥了作用,在本研究中试图作一初步探讨.     

IFN-α Acts on T-Cell Receptor-Triggered Human Peripheral Leukocytes to Up-Regulate CCR5 Expression on CD4+ and CD8+ T Cells

Interleukin-12 (IL-12) as well as IL-2 was recently shown to up-regulate CCR5 expression on T-cell receptor (TCR)-triggered human T cells. Because of the functional similarity between interferon-alpha (IFN-) and IL-12, the present study investigated whether IFN- also up-regulates T cell CCR5 expression. CCR5 was marginally detected on T cells from unstimulated human peripheral blood leukocytes (PBLs) and only slightly induced on PBL T cells following stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs). When anti-CD3/anti-CD28-triggered PBLs were exposed to IFN-, T cells expressed high levels of CCR5. The levels of CCR5 expression were comparable to those induced by IL-12. However, when purified T cells instead of unfractionated PBL were stimulated with anti-CD3/CD28 and then exposed to IL-12 or IFN-, CCR5 expression was induced by IL-12 but not by IFN-. IFN- was found to act on anti-CD3/anti-CD28-stimulated PBL to promote their IL-12 production. Moreover, addition of anti-IL-12 mAb to IFN--stimulated cultures of anti-CD3/CD28-pretreated PBL resulted in considerable inhibition of CCR5 expression. Together, these results indicate that IFN- as well as IL-12 up-regulates CCR5 expression on TCR-triggered T cells and that IFN- functions not by acting directly on T cells but via enhancing IL-12 production by PBL.     

Pleural Mesothelial Cells Promote Expansion of IL-17–Producing CD8+ T Cells in Tuberculous Pleural Effusion

IL-17–producing CD8⁺ T lymphocytes (Tc17 cells) have recently been detected in many cancers and autoimmune diseases. However, the possible implication of Tc17 cells in tuberculous pleural effusion remains unclarified. In this study, distribution and phenotypic features of Tc17 cells in both tuberculous pleural effusion (TPE) and peripheral blood from patients with tuberculosis were determined. The effects of proinflammatory cytokines and local accessory cells (pleural mesothelial cells) on Tc17 cell expansion were also explored. We found that TPE contained more Tc17 cells than the blood. Compared with IFN-γ–producing CD8⁺ T cells, Tc17 cells displayed higher expression of chemokine receptors (CCRs) and lower expression of cytotoxic molecules. In particularly, Tc17 cells in TPE exhibited high expression levels of CCR6, which could migrate in response to CCL20. Furthermore, IL-1β, IL-6, IL-23, or their various combinations could promote Tc17 cell expansion from CD8⁺ T cells, whereas the proliferative response of Tc17 cells to above cytokines was lower than that of Th17 cells. Pleural mesothelial cells (PMCs) were able to stimulate Tc17 cell expansion via cell contact in an IL-1β/IL-6/IL-23 independent fashion. Thus this study demonstrates that Tc17 cells marks a subset of non-cytotoxic, CCR6⁺ CD8⁺ T lymphocytes with low proliferative capacity. The overrepresentation of Tc17 cells in TPE may be due to Tc17 cell expansion stimulated by pleural proinflammatory cytokines and to recruitment of Tc17 cells from peripheral blood. Additionally, PMCs may promote the production of IL-17 by CD8⁺ T cells at sites of TPE via cell–cell interactions.     

TGF-β1 and contact mediated suppression by CD4+CD25+CD127− T regulatory cells of patients with self-limiting hepatitis E

Background and aimLiterature on the role of Regulatory T cells (Tregs) in acute viral infections is limited. Having established that the Tregs in self-limiting hepatitis E infection are elevated and functional, this study has focused on characterizing the specificity, phenotypes and identifying the molecules or factors responsible for enhancement of Treg cells and abrogation of Treg-mediated suppression in hepatitis E.MethodsHEV rORF2p specific (a) Treg frequency, subset analysis and expression of surface and intracellular markers on Tregs and CFSE based functional analysis by flow cytometry (b) key cytokines quantification by multiplex (c) suppressive functional assay in the presence of anti-TGF-β₁ or anti-IL-10 or both antibodies or Transwell insert or in combination were performed on samples from 58 acute patients (AVH-E), 45 recovered individuals from hepatitis E and 55 controls.ResultsIn AVH-E, the increased frequencies of Tregs and Teff cells were HEV rORF2p specific and Treg cells were of effector memory phenotype. Higher expressions of HEV rORF2p stimulated CTLA-4, GITR, PD1L, CD103, CD39, TLR2 and TGF-β₁ molecules on Tregs of AVH-E were observed. Tregs produced TGF-β₁ and inhibited the secretion of IFN-γ. Transwell insert and cytokines blocking assays indicated Tregs mediated suppression in AVH-E patients is majorly TGF-β₁ mediated and partly cell-cell contact mediated.ConclusionOverall, we have identified beneficial involvement of HEV specific, functional Tregs and TGF-β₁ as the regulatory molecule responsible for enhancement of Tregs in self-limiting HEV infection. Therefore, use of TGF-β₁ as a possible supplement for boosting Treg response in recovery from severe hepatitis E needs evaluation.     

Therapeutic potential of TGF-β-induced CD4+ Foxp3+ regulatory T cells in autoimmune diseases

Foxp3⁺ T regulatory cell (Treg) subsets play a crucial role in the maintenance of immune homeostasis against self-antigens. The lack or dysfunction of these cells contributes to the pathogenesis and development of many autoimmune diseases. Therefore, manipulation of these cells may provide a novel therapeutic approach to treat autoimmune diseases. In this review, we provide current opinions concerning the classification, developmental, and functional characterization of Treg subsets. Particular emphasis will be focused on the therapeutic role of TGF-β-induced CD4M⁺ Foxp3⁺ cells (iTregs) in established autoimmune disease. Moreover, the similarity and diversity of iTregs and naturally occurring, thymus-derived CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (nTregs) will be discussed, including the finding that the pro-inflammatory cytokine IL-6 can convert nTregs to IL-17-producing cells, whereas iTregs induced by TGF-β are resistant to the effects of this cytokine. Understanding these aspects may help to determine how Tregs can be used in the treatment of autoimmune diseases.     

Chemokine receptor CCR9 suppresses the differentiation of CD4+CD8αα+ intraepithelial T cells in the gut

The chemokine receptor CCR9 equips T cells with the ability to respond to CCL25, a chemokine that is highly expressed in the thymus and the small intestine (SI). Notably, CCR9 is mostly expressed on CD8 but not on CD4 lineage T cells, thus imposing distinct tissue tropism on CD4 and CD8 T cells. The molecular basis and the consequences for such a dichotomy, however, have not been fully examined and explained. Here, we demonstrate that the forced expression of CCR9 interferes with the tissue trafficking and differentiation of CD4 T cells in SI intraepithelial tissues. While CCR9 overexpression did not alter CD4 T cell generation in the thymus, the forced expression of CCR9 was detrimental for the proper tissue distribution of CD4 T cells in the periphery, and strikingly also for their terminal differentiation in the gut epithelium. Specifically, the differentiation of SI epithelial CD4 T cells into immunoregulatory CD4⁺CD8αα⁺ T cells was impaired by overexpression of CCR9 and conversely increased by the genetic deletion of CCR9. Collectively, our results reveal a previously unappreciated role for CCR9 in the tissue homeostasis and effector function of CD4 T cells in the gut.     

Human CD8⁺ T cells drive Th1 responses through the differentiation of TNF/iNOS-producing dendritic cells

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.