Local Effects of Regulatory T Cells in MUC1 Transgenic Mice Potentiate Growth of MUC1 Expressing Tumor Cells In Vivo

MUC1 transgenic (MUC1.Tg) mice have widely been used as model recipients of cancer immunotherapy with MUC1. Although MUC1.Tg mice have previously been shown to be immunologically tolerant to MUC1, the involvement of regulatory T (Treg) cells in this phenotype remains unclear. Here, we showed that numbers of Treg cells in MUC1-expressing tumors were greater in MUC1.Tg mice than in control C57BL/6 (B6) mice, and that the growth of tumor cells expressing MUC1, but not that of control cells, in MUC1. Tg mice was faster than in B6 mice. The MUC1.Tg mice appeared to develop MUC1-specific peripheral tolerance, as transferred MUC1-specific T cells were unable to function in MUC1.Tg mice but were functional in control B6 mice. The suppressive function of CD4⁺CD25^high cells from MUC1.Tg mice was more potent than that of cells from control B6 mice when Treg cell activity against MUC1-specific T cells was compared in vitro. Therefore, the enhanced growth of MUC1-expressing tumor cells in MUC1.Tg mice is likely due to the presence of MUC1-specific Treg cells.     

CD4+ T Cells Expressing Latency-Associated Peptide and Foxp3 Are an Activated Subgroup of Regulatory T Cells Enriched in Patients with Colorectal Cancer

Latency-associated peptide (LAP) - expressing regulatory T cells (Tregs) are important for immunological self-tolerance and immune homeostasis. In order to investigate the role of LAP in human CD4⁺Foxp3⁺ Tregs, we designed a cross-sectional study that involved 42 colorectal cancer (CRC) patients. The phenotypes, cytokine-release patterns, and suppressive ability of Tregs isolated from peripheral blood and tumor tissues were analyzed. We found that the population of LAP-positive CD4⁺Foxp3⁺ Tregs significantly increased in peripheral blood and cancer tissues of CRC patients as compared to that in the peripheral blood and tissues of healthy subjects. Both LAP⁺ and LAP⁻ Tregs had a similar effector/memory phenotype. However, LAP⁺ Tregs expressed more effector molecules, including tumor necrosis factor receptor II, granzyme B, perforin, Ki67, and CCR5, than their LAP⁻ negative counterparts. The in vitro immunosuppressive activity of LAP⁺ Tregs, exerted via a transforming growth factor-β–mediated mechanism, was more potent than that of LAP⁻ Tregs. Furthermore, the enrichment of LAP⁺ Treg population in peripheral blood mononuclear cells (PBMCs) of CRC patients correlated with cancer metastases. In conclusion, we found that LAP⁺ Foxp3⁺ CD4⁺ Treg cells represented an activated subgroup of Tregs having more potent regulatory activity in CRC patients. The increased frequency of LAP⁺ Tregs in PBMCs of CRC patients suggests their potential role in controlling immune response to cancer and presents LAP as a marker of tumor-specific Tregs in CRC patients.     

Identification of Sestrin3 Involved in the In vitro Resistance of Colorectal Cancer Cells to Irinotecan

Irinotecan, an analogue of camptothecin, is frequently used as a single agent or in combination with other anticancer drugs for the treatment of colorectal cancer. However, the drug resistance of tumors is a major obstacle to successful cancer treatment. In this study, we established that cells acquire chronic resistance to irinotecan. We profiled their differential gene expression using microarray. After gene ontology (GO) and KEGG pathway analysis of the microarray data, we specifically investigated whether Sestrin3 could decrease irinotecan resistance. Our results revealed that Sestrin3 enhanced the anticancer effect of irinotecan in vitro in LoVo cells that had acquired resistance to irinotecan. Irinotecan-resistant LoVo cells showed lower reactive oxygen species (ROS) production than their irinotecan-sensitive parental cells. ROS production was increased by Sestrin3 knockdown in irinotecan-resistant LoVo cells. Our results indicate that Sestrin3 might be a good target to develop therapeutics that can help to overcome resistance to irinotecan.     

The Synergistic Local Immunosuppressive Effects of Neural Stem Cells Expressing Indoleamine 2,3-Dioxygenase (IDO) in an Experimental Autoimmune Encephalomyelitis (EAE) Animal Model

Neurodegenerative diseases provoke robust immunological reactions in the central nervous system (CNS), which further deteriorate the neural tissue damage. We hypothesized that the expression levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that has potent immune suppressive activities, in neural stem cells (NSCs) would have synergistic therapeutic effects against neurodegenerative diseases, since NSCs themselves have low IDO expression. In this study, the synergistic immune suppressive effects of rat fetal NSCs expressing IDO (rfNSCs-IDO) were validated by mixed leukocyte reaction (MLR) in vitro and an experimental autoimmune encephalomyelitis (EAE) animal model in vivo. rfNSCs-IDO showed significantly more suppressive effects on T cell proliferation in the MLR compared to control rfNSCs (rfNSCs-Cont). Importantly, IDO inhibition using 1-methyl-DL-tryptophan (1-MT), an IDO inhibitor, reversed the synergistic effects, confirming IDO-specific effects in rfNSCs-IDO. In the EAE animal model, systemic rfNSCs-IDO injections resulted in significant local immune suppression in the cervical lymph nodes and CNS, evidenced by a reduction in the number of activated T lymphocytes and an increase in regulatory T cell numbers, which induced significantly fewer clinical symptoms and faster recovery. In contrast, rfNSCs-Cont failed to reduce symptoms in the EAE animal models, although they showed local immune suppression, which was significantly less than that in rfNSCs-IDO. Taken together, IDO expression in NSCs synergistically potentiates the immune suppression activities of NSCs and could be applicable for the development of therapeutic modalities against various neurodegenerative diseases.     

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background

The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4⁺ CD25^highFoxp3⁺ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.

Methodology/Principal Findings

CD4⁺CD25⁺ and CD4⁺CD25^neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4⁺CD25^high and CD4⁺CD25^neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4⁺CD25^neg or CD8⁺CD25^neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4⁺CD25⁺ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4⁺CD25^highFoxp3⁺ cells and were resistant to apoptosis, while CD4⁺CD25^neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4⁺CD25^neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway.

Conclusions/Significance

RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.     

Tim-3 Expression Defines Regulatory T Cells in Human Tumors

Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3⁺ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3⁺ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3⁺ CD4 T cells. The majority of tumor-derived Tim-3⁺ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3⁻ CD4 T cell counterparts. In contrast, most Tim-3⁺ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3⁺ CD4 T cells, but not tumor-derived Tim-3⁻ CD4 T cells, significantly suppressed the proliferation of autologous CD8⁺ T cells in vitro. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3⁺Foxp3⁺CD4⁺ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.     

FOXO3a过表达对内皮祖细胞周期相关蛋白的调控

目的:观察FOXO3a(forkhead box O3a)的活性改变对内皮祖细胞(endothelial progenitor cells,EPCs)增殖和细胞周期相关蛋白表达的影响。方法:将携带突变激活FOXO3a基因的腺病毒载体Ad-TM(triple mutant)-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27kip1以及CDK2的表达水平。结果:构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM-FOXO3a组EPCs细胞生长缓慢,集落不明显;Western blot和CCK-8结果显示,Ad-TM-FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27kip1蛋白过表达,CDK2表达下调。结论:FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。     

FOX03a过表达对内皮祖细胞周期相关蛋白的调控

目的：观察FOXO3a（forkhead box O3a）的活性改变对内皮祖细胞（endothelial progenitor cells,EPCs）增殖和细胞周期相关蛋白表达的影响。方法：将携带突变激活FOXO3a基因的腺病毒载体Ad-TM（triple mutant）-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27^kip1以及CDK2的表达水平。结果：构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM—FOXO3a组EPCs细胞生长缓慢,集落不明显;Westem blot和CCK-8结果显示,Ad-TM—FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27^kip1蛋白过表达,CDK2表达下调。结论：FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.     

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an “immune exhaustion”, with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28⁻CD57⁺CD8⁺ T cells between the groups. However, the frequency of Tim-3⁺CD8⁺ and Tim-3⁺CD4⁺ exhausted T cells, but not PD-1⁺ T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1⁺CD8⁺ T cells were directly associated with T cell immune activation in children. The frequency of Tim-3⁺CD8⁺ T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.     

FoxP3+ Regulatory T Cells Attenuate Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3⁺ regulatory T cells (Tregs) are central to gut homeostasis. While Treg proportions are significantly reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their role in experimental NEC induction. Utilizing an established rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35%. The Tregs modulated the immune response as manifested in reduced CD80 expression on antigen presenting cells and decreased T cell activation within the mesenteric lymph nodes. These findings suggest that while Tregs are present in the intestines, their numbers might be insufficient to dampen the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the severity of NEC by limiting the immune response. Strategies to enhance Tregs have a therapeutic potential in controlling the development of NEC.     

Poly(ADP-ribosyl)ation of FOXP3 Protein Mediated by PARP-1 Protein Regulates the Function of Regulatory T Cells

Poly(ADP-ribose) polymerase 1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes but also enhanced suppressive function of regulatory T cells. Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the posttranslational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications for developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.     

Inhibition of PI3K induces Rac Activation and Membrane Ruffling in Proto-Dbl Expressing Cells

Proto-Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is tightly regulated by a combination of mechanisms that involve intra- and intermolecular interaction and N- and C-terminal domain-dependent turnover of the protein. Moreover, the interaction of the PH domain of proto-Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3-kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto-Dbl-induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto-Dbl-induced cell transformation and motility and observed that inhibition of PI3K in proto-Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto-Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.     

Insulin-like Growth Factor-2 Enhances Functions of Antigen (Ag)-specific Regulatory B Cells

Regulatory B cells (Bregs) are important in immune regulation. The factors that regulate Breg functions are less clear. Insulin-like growth factor 2 (IGF2) is capable of inducing hematopoietic stem cell differentiation. This study aimed to investigate the role of IGF2 in the development of Bregs and the enhancement of their function. In this study, the expression of IGF1 receptor (IGF1R) and IGF2R in ovalbumin (OVA)-specific B cells (OVAsBCs) was assessed by real time RT-PCR and Western blotting. The release of interleukin (IL)-10 from OVAsBCs and OVAsBC proliferation were assessed by enzyme-linked immunoassay and proliferation assay. The role of IGF2 in enhancing the function of OVAsBCs was tested with an intestinal allergic inflammation mouse model. The results showed that OVAsBCs expressed high levels of IGF2R. Exposure to both IGF2 and a specific antigen (Ag), OVA, markedly enhanced the expression of IL-10 in OVAsBCs as well as enhanced the IL-10⁺ OVAsBC proliferation. The concurrent exposure to IGF2 and specific Ag markedly induced the IL-10 promoter DNA demethylation via activating the STAT5 pathway. IGF2 also enhanced both the OVAsBC proliferation in vivo and the effect of Ag-specific immunotherapy on inhibiting allergic inflammation in the intestine. We conclude that OVAsBCs express high levels of IGF2R and that IGF2 increases the expression of IL-10 in OVAsBCs and enhances OVAsBC proliferation and the inhibitory effect on allergic inflammation.     

皂苷类成分对血管内皮细胞功能的调节作用研究进展

血管内皮细胞在维持血管生理稳态中发挥了重要的作用,其功能障碍是动脉粥样硬化、冠心病、脑卒中、肿瘤等多种重大疾病发生发展的病理基础,调节血管内皮细胞功能是防治上述疾病的主要途径之一。大量研究表明,皂苷类成分可通过改善血管内皮功能达到治疗疾病的目的。综述了近年来报道的皂苷类成分调节血管内皮功能的研究进展,旨在为皂苷类成分作用机制的阐明和相关重大疾病的防治提供一定参考。     

重组牛痘病毒介导的微小基因表达产物OVA257—264在真核细胞中的抗原呈递效率

应用含重组OVA微小基因及全长基因的牛痘病毒感染能够稳定表达Kb 分子的多种真核细胞 ,通过构象特异的单克隆抗体 2 5 .D 1.16 (anti OVA2 57～ 2 64/Kb)检测细胞表面复合物的形成情况 ,并借助四聚体技术检测了它们对抗原特异性T细胞的诱导能力。结果表明四聚体的构建是成功的 ,进一步用已被重组牛痘病毒感染的L92 9 Kb 细胞作为抗原呈递细胞以诱导特异性细胞毒性T淋巴细胞 (CTL)的产生 ,并对活化后特异性CTL进行检测 ,从胞外IFN γ的分泌情况看 ,诱导后的整个群体细胞发生了活化与增殖 ,H 2Kb OVA四聚体对特异性T细胞进行的结合分析与经典的细胞毒效应相一致。可见 ,重组牛痘病毒介导的微小基因产物OVA2 57～ 2 64不仅能为MHC I类分子的合成提供更有效的肽来源 ,且能更有效诱导特异性CTL的产生。     

Dendritic Cells from Oral Cavity Induce Foxp3+ Regulatory T Cells upon Antigen Stimulation

Evidence is accumulating that dendritic cells (DCs) from the intestines have the capacity to induce Foxp3⁺CD4⁺ regulatory T cells (T-regs) and regulate immunity versus tolerance in the intestines. However, the contribution of DCs to controlling immunity versus tolerance in the oral cavity has not been addressed. Here, we report that DCs from the oral cavity induce Foxp3⁺ T-regs as well as DCs from intestine. We found that oral-cavity-draining cervical lymph nodes contained higher frequencies of Foxp3⁺ T-regs and ROR-γt⁺ CD4⁺T cells than other lymph nodes. The high frequency of Foxp3⁺ T-regs in the oral-cavity-draining cervical lymph nodes was not dependent on the Toll like receptor (TLR) adaptor molecules, Myd88 and TICAM-1 (TRIF). In contrast, the high frequency of ROR-γt⁺ CD4⁺T cells relies on Myd88 and TICAM-1. In vitro data showed that CD11c⁺ DCs from oral-cavity-draining cervical lymph nodes have the capacity to induce Foxp3⁺ T-regs in the presence of antigen. These data suggest that, as well as in the intestinal environment, antigen-presenting DCs may play a vital role in maintaining tolerance by inducing Foxp3⁺ T-regs in the oral cavity.     

Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler’s murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.