Class II positive human dermal fibroblasts restimulate cloned allospecific T cells but fail to stimulate primary allogeneic lymphoproliferation

Recently, a number of laboratories have shown that gamma interferon (IFN-gamma) is a potent modulator of HLA class II antigen expression in a variety of cell types ranging from classical antigen presenting cells to those not expected to participate in physiological antigen presentation such as fibroblasts. In order to examine the role of HLA class II expressing fibroblasts in antigen presentation, we established dermal fibroblast (FIB) strains from five HLA typed donors. After optimal preculture with IFN-gamma, class II positive FIB were fully competent to restimulate proliferative responses of two DR specific T cell clones and one DP specific T cell line. However, they failed to elicit strong primary allogeneic proliferation from fully DR mismatched fresh PBMC. This failure was not due to a direct suppressive effect of FIB and could not be corrected by exogenous IL1 or by factors contained in conventional mixed leukocyte culture supernatants.     

CD4+Foxp3+调节性T细胞表达升高提示免疫应答而非移植耐受

目的 探讨诱导CD4+Foxp3+调节性T细胞(regulatory T cells,Tregs)表达的机制及其在移植耐受中可能的作用.方法 构建新生移植耐受小鼠动物模型,分析移植耐受与同种异体反应性T细胞、Tregs表达及嵌合体的关系;运用过继转移实验、EGFP转基因小鼠,研究移植排斥过程中Tregs表达及抗原特异性.结果 新生耐受小鼠形成嵌合体,同种异体反应性T细胞被克隆清除,但Tregs表达与初始小鼠相同;同种异体反应性T细胞识别抗原诱导免疫反应,Tregs在移植排斥反应中表达升高,且不仅同种异体反应性Tregs表达升高,非同种异体反应性Tregs表达也升高.结论 Tregs在移植排斥中非特异性诱导产生,可能为一种负反馈免疫调控机制.     

Melatonin provides signal 3 to unprimed CD4(+) T cells but failed to stimulate LPS primed B cells

Growing evidence has supported the conclusion that melatonin, a pineal hormone, modulates the immune function. In our previous study, we evaluated in vivo the potential role of melatonin in the regulation of the antigen specific T and B cells. In the present study, we observe that melatonin down-regulated the expression of the co-stimulatory molecule B7-1 but not B7-2 on macrophages. Further, melatonin encouraged the proliferation of anti-CD3 antibody activated CD4(+) T cells only in the presence of antigen-presenting cells and promoted the production of Th2-like cytokines. Furthermore, it failed to influence the activity of B cells in a T-independent manner. Melatonin suppressed the release of TNF-alpha by LPS or IFN-gamma activated macrophages but failed to inhibit nitric oxide (NO) release. Thus the study shows that melatonin can engineer the growth of unprimed CD4(+) T cells if both the signals are provided by antigen-presenting cells. However, it could not regulate the function of B cells.     

调节性T细胞在大鼠小肠移植急性排斥反应中的作用

目的 分析调节性T细胞在大鼠急性排斥反应中的作用。方法 采用免疫组化SABC染色法，测定BN→LEW大鼠小肠移植急性排斥反应时，外周血及移植肠浸润淋巴细胞中调节性T细胞：CD4^ ,CD8^ ,CD25^ T淋巴细胞及相关细胞因子IL－4和IFN－γ的表达，并与同基因大鼠间小肠移植（BN→BN）作比较。结果 外周血淋巴细胞分析显示，大鼠小肠移植急性排斥反应时，以CD4^ ,CD25^ ,T细胞为主，CD8^ 淋巴细胞只占少部分；分泌IL－4的细胞在术后4，7，14d分别只占14．3％，16．2％和16．9％。移植肠基底浸润的淋巴细胞以CD4^ ,CD25^＋和分泌IFN－γ的淋巴细胞为主。结论 在大鼠同种小肠移植中，急性排斥反应与CD25^ ,CD4^ T细胞及Th1相关细胞因子IFN－γ的表达增加相关。而CD8^＋T淋巴细胞和Th2相关细胞因子IL－4可能具有保护作用。     

Synthetic peptides corresponding to the F protein of RSV stimulate murine B and T cells but fail to confer protection

Summary We have previously located a major neutralization site of the fusion protein of respiratory syncytial virus (RSV) in the polypeptide region extending from amino acids Ile221 to Glu232. In this report, 8 peptides corresponding to the six major hydrophilic regions of the F₁ subunit were selected to analyse their immunogenic and protective capacities as well as their ability to block the high neutralization activities of 4 monoclonal antibodies (MAbs). Only 5 of the 8 peptides tested induced specific antibodies while all induced an in vitro interleukin-2 response of splenocytes from immunized mice. Peptide 3 (Ile221-Phe237) was able to elicit neutralizing antibodies, confirming our previous hypothesis concerning the location of a neutralization site. However, immunization with the latter did not induce significant reduction of virus in lungs of BALB/c mice upon challenge, probably due to an inadequate level of circulating neutralizing antibodies. Interestingly, peptides 2 (Asn216-Glu232), 3 (Ile221-Phe237), and 5 (Ser275-Ile288) blocked in vitro neutralization by four different F₁ specific MAbs. A hypothesis is proposed to explain these results.     

The effects of glucocorticoid therapy on the inflammatory and dendritic cells in muscular dystrophies

Various clinical trials have documented the therapeutic benefit of glucocorticoids (GCs) in enhancing muscle strength and slowing disease progression of Duchenne and Becker muscular dystrophies (DMD/BMD). We hypothesized that GCs may have relevance to the differential anti-inflammatory effect on mononuclear inflammatory cells (MICs) and Dendritic cells (DCs) infiltrating the dystrophic muscles. In this prospective study, two muscle biopsies were obtained (before and after 6-month prednisone therapy) from 30 patients with dystrophies (DMD = 18; BMD = 6; and limb girdle muscular dystrophies (LGMD) = 6). MICs and DCs infiltrating the muscles were examined using mouse monoclonal antibodies and immunoperoxidase staining methods. Muscle strength was evaluated monthly by manual testing, motor ability and timed tests. Prednisone therapy was associated with: (i) functional improvement of overall motor disability, in upper limbs of DMD (P < 0.001) and BMD (P < 0.01) and lower limbs of DMD (P < 0.001) and BMD (P < 0.05); (ii) histological improvement such as fibre size variation (DMD, P < 0.01; BMD, P < 0.05), internalization of nuclei (DMD, P < 0.05), degeneration and necrosis (DMD and BMD, P < 0.01), regeneration (DMD, P < 0.001; BMD, P < 0.01) and endomysial connective tissue proliferation (DMD, P < 0.01; BMD, P < 0.05) and (iii) reduction of total MICs (P < 0.01) and DCs (P < 0.01). There was a positive correlation between the degree of improvement in overall motor disability and reduction of DCs numbers (In upper limbs; r = 0.638, P < 0.01 for DMD and r = 0.725, P < 0.01 for BMD, in Lower limbs; r = 0.547, P < 0.05 for DMD and r = 0.576, P < 0.05 for BMD). Such improvements and changes of MICs/DCs were absent in LGMD. In DMD/BMD, prednisone therapeutic effect was associated with reduced MICs and DCs numbers. Whether this therapeutic effect reflects targeting of the deleterious immune response produced by these cells mandates further investigations.     

外显子跳跃治疗Duchenne型肌营养不良症的研究进展

Duchenne型肌营养不良症是一种致死性肌肉疾病,抗肌萎缩蛋白基因缺陷是导致本病的原因,目前本病尚无特效的疗法.反义寡核苷酸(antisense oligonucleotides,AOs)诱导的外显子跳跃作为一种新的治疗手段具有良好的应用前景.本文主要从外显子跳跃治疗的原理、基础研究及临床研究进行综述.     

Switch from perforin-expressing to perforin-deficient CD8+ T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo

Although CD8⁺ cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8⁺ CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8⁺ T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8⁺ cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8⁺ CTL have two killing strategies.     

DMD的基因、细胞和药理学治疗研究进展

杜兴氏肌营养不良（Duchenne muscular dystrophy，DMD）是一种X连锁隐性遗传的进行性肌萎缩疾病，主要由抗肌萎缩蛋白基因缺陷引起。抗肌萎缩蛋白是一种肌纤维膜结构蛋白，在维持肌细胞膜结构完整性中起重要作用。目前此疾病的治疗手段主要有基因治疗、细胞治疗和药理学治疗。针对疾病的根本原因，基因治疗和细胞治疗通过传递正常基因或修正突变基因以及移植正常细胞，如干细胞来进行治疗；药理学治疗则主要针对抗肌萎缩蛋白基因缺陷引起的下游事件，运用各种药物来减缓DMD病理进程．从而改善患者的生活质量．延长寿命。主要概述DMD治疗的最新讲展．并分析目前尚存存的问颢．     

Effective induction of immune tolerance by portal venous infusion with IL-10 gene-modified immature dendritic cells leading to prolongation of allograft survival

Dendritic cells (DC) not only initiate T cell responses, but are also involved in the induction of tolerance. The functional properties of DC are strictly dependent on their state of maturation. It has been shown that immature DC can induce immune tolerance and prolong allograft survival. Interleukin-10 (IL-10) is an important immunosuppressive cytokine which inhibits maturation and function of DC. In order to improve the tolerogenicity of DC, we and others showed that adenovirus vectors can effectively mediate IL-10 genetic modification of DC, and IL-10 genetic modification can inhibit MHC II, B7.2, and CD40 expression, IL-12 secretion and the T cell stimulatory capacity of DC. The primary aim of this study is to examine the in vivo effects of this approach on allograft survival in a murine cardiac allograft transplantation model. To our surprise, we observed that infusion of immature DC genetically modified to express IL-10 (DC-IL-10) via the tail vein could not prolong allograft survival in the recipients, but shortened their survival. More interestingly, portal venous infusion of DC-IL-10 markedly prolonged allograft survival. The diverse effects of DC-IL-10 infusion through different routes may be due to the different immune responses to alloantigens in recipients that received DC-IL-10 via either the portal or the tail vein. Decreased cytotoxicity, polarization of Th2 response, poor T cell stimulating activity of liver DC and enhanced incidence of donor DC in the recipients may contribute to the more efficient prolongation of allograft survival observed after portal venous infusion of DC-IL-10. These results suggest that portal venous infusion may be an effective approach for immature DC to induce immune tolerance or hyporesponsiveness against donor antigens, and prolong allograft survival.Abbreviations APC Antigen-presenting cells - CTL Cytotoxic T lymphocytes - DC Dendritic cells - DC-IL-10 IL-10 gene-modified immature dendritic cells - iDC Immature dendritic cells - IL-10 Interleukin-10 - MLR Mixed leukocyte reaction - MOI Multiplicity of infection     

Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival

Intraperitoneal peptide injection of TCR-transgenic mide or expression of antigen in hepatocytes leads to an accumulation in the liver of specific apoptotic CD8+ T cells expressing activation markers. To determine whether liver cells are capable of directly activating naive CD8+ T cells, we have studied the ability of purified hepatocytes to activate TCR-transgenic CD8+ T cells in vitro. We show that hepatocytes which do not express CD80 and CD86 co-stimulatory molecules are able to induce activation and effective proliferation of specific naive CD8+ T cells in the absence of exogenously added cytokines, a property only shared by professional antigen-presenting cells (APC). Specific T cell proliferation induced by hepatocytes was comparable in magnitude to that seen in response to dendritic cells and was independent of CD4+ T cell help or bystander professional APC co-stimulation. During the first 3 days, the same number of divisions was observed in co-cultures of CD8+ T cells with either hepatocytes or splenocytes. Both APC populations induced expression of early T cell activation markers and specific cytotoxic T lymphocyte (CTL) activity. However, in contrast to T cells activated by splenocytes, T cells activated by hepatocytes lost their cytolytic function after 3 days of co-culture. This correlated with death of activated T cells, suggesting that despite efficient activation, proliferation and transient CTL function, T cells activated by hepatocytes did not survive. Death could be prevented by adding antigen-expressing splenocytes or exogenous IL-2 to the co-culture, indicating that hepatocytes are not involved in direct killing of CD8+ T cells but rather fail to promote survival. Dying cells acquired a CD8low TCRlow B220+ phenotype similar to the one described for apoptotic intrahepatic T cells, suggesting an alternative model to account for the origin of these cells in the liver. The importance of these findings for the understanding of peripheral tolerance and the ability of liver grafts to be accepted is discussed.     

Metastasis-suppressed C8161 melanoma cells arrest in lung but fail to proliferate

The incidence of melanoma continues to increase at a rapid rate. As for most cancers, it is melanoma metastases, rather than the primary malignancy, that is the principal cause of death. We previously showed that the introduction of a normal copy of chromosome 6 into the metastatic human melanoma cell line C8161 suppresses metastasis at a step subsequent to tumor cells entering the bloodstream. To better define the step(s) in metastasis blocked by the addition of chromosome 6 we engineered cells that constitutively express green fluorescent protein (GFP). When these tagged, chromosome 6 hybrid cells were injected intravenously into athymic mice, grossly detectable metastases did not form. However, fluorescence microscopy revealed micro-metastases (single cells or clusters of <10 cells) in the lungs, suggesting that these cells lodged in the lungs but failed to proliferate. Cells isolated from lung up to 60 days post-injection grew in culture and/or formed tumors when injected into the skin, indicating that they were still viable, but dormant. This result implies that the gene(s) on chromosome 6 interfere specifically with growth regulatory response in the lung, but not in the skin. Thus, the gene(s) responsible for metastasis suppression represents a new class of metastasis inhibitors acting at the final stages of the metastatic cascade - that is, affecting the ability of the cells to survive and proliferate at a specific secondary site. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

Involvement of Hofbauer cells and maternal T cells in villitis of unknown aetiology

Aims:  The nature of villitis of unknown aetiology (VUE) is intriguing in terms of its aetiology, origin of inflammatory cells and immunophenotype of T cells involved. The aim was to determine the origin of macrophages and the immunophenotype of T lymphocytes in VUE associated with various complications of pregnancy.
Methods and results:  Placentas with VUE ( n  = 45) were studied by chromogenic in-situ hybridization (CISH) for Y chromosome (DYZ1) and immunohistochemistry for CD14, CD68, Ki67 ( n  = 10; all from male neonates) and a panel of T-cell antigens (CD3, CD4 and CD8) ( n  = 35). All of the placentas from male neonates showed CISH+ signals from Y chromosomes in the majority of macrophages, but not in lymphocytes, indicating that the macrophages were of fetal origin. Many macrophages of the affected chorionic villi were Ki67+, suggesting that they are hyperplastic Hofbauer cells. Among the lymphocytes, CD8+ T cells outnumbered CD4+ T cells in all placentas with different obstetrical conditions.
Conclusions:  We define primary components of VUE as maternal CD8+ T cells and hyperplastic Hofbauer cells. We propose that VUE is a unique inflammatory reaction where the leucocytes from two hosts are key partners, analogous to either allograft rejection or graft-versus-host disease.     

CD8+ regulatory T cells are responsible for GAD-IgG gene-transferred tolerance induction in NOD mice

Our previous studies demonstrated that lipopolysaccharide (LPS)-stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non-obese diabetic (NOD) mice from diabetes by inducing GAD-specific tolerance, and also that there are increased numbers of CD4(+) regulatory T cells (Tregs) in GAD-IgG-treated NOD mice. However, little is known about the role of CD8(+) Tregs in GAD-IgG gene-transferred tolerance induction in NOD mice. Here, we found that GAD-IgG-transduced splenocytes induced an increase in the number of CD8(+) Foxp3(+) Tregs in vitro. Using a T-cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8(-) or CD8(-) CD25(-) GAD-IgG-transduced splenocytes showed a decrease in the percentage of CD8(+) Foxp3(+) T cells, a high incidence of diabetes, serious insulitis, GAD-specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8(+) Tregs, which were induced in vitro by GAD-IgG-transduced splenocytes, were also responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.     

Autologous apoptotic T cells interact with dendritic cells,but do not affect their surface phenotype or their ability to induce recall immune responses

Dendritic cells (DCs) play an important role in determining immunogenicity and the subsequent immune response. They may also have a role in maintaining peripheral tolerance to self-antigens by initiating an immune response only in the context of danger signals released from cells during stress, damage or death. These signals may originate from surrounding T cells as well as from other cells. Therefore, in this study the effect of autologous T cell injury on DC morphology and function has been investigated. Co-incubation of apoptotic or necrotic T cells with immature DCs altered their morphology towards a more mature appearance, with more cells showing activation as judged by spreading and formation of arborizing long processes. The apoptotic autologous T cells were rarely phagocytosed by immature DCs, compared to macrophages. The DC surface phenotype was not affected by the co-incubation with autologous injured T cells. The ability of DCs to elicit a secondary immune response was not altered by exposure to autologous injured T cells. These findings suggest that DC can continue to function in T cell activation, rather than in tolerogenic mode, even in the presence of large numbers of dying autologous T cells, such as may be present in the aftermath of an acute antiviral response.     

人源载体介导的minidystrophin-EGFP融合基因在Cos-7细胞中的表达

目的 构建微小肌营养不良蛋白（minidystrophin）和增强绿色荧光蛋白（enhaneed green fluorese protein，EGFP）融合基因的人源载体，观察该载体在Cos-7细胞中的表达。方法 以正常人肌营养不良基因cDNA（GenBank NM_004006）为模板，通过PCR克隆的方法构建minidystrophin基因，融合EGFP基因后连接到人源载体pHmeo，大量提取荸组质粒，转染Cos-7细胞，通过逆转录聚合酶链反应、荧光显微镜观察等方法检测该载体在细胞内的表达。结果 成功构建pHmDysG载体，转染Cos-7后，逆转录聚合酶链反应可扩增出735bp特异条带，荧光显微镜观察可见表达蛋白分布于细胞膜上。结论 prim载体介导的minidystrophin基因可以在真核细胞表达，并被有效地转运至细胞膜，可望用于杜氏肌营养不良基因治疗的研究。     

DMD and BMD in the same family due to two distinct mutations

We report on a family with a boy affected by Duchenne muscular dystrophy (DMD) and an asymptomatic cousin with a Beckertype dystrophin abnormality, diagnosed by chance. Dystrophin gene analysis showed that these conditions were caused by two distinct deletions with breakpoints in different exons. In Xp21 families, DNA analysis and dystrophin testing of asymptomatic males with high CK plasma levels might detect different dystrophin mutations in separate haplotypes as in our family, although we stress there should be clear clinical or familial indications for such testing. © 1995 Wiley-Liss, Inc.     

Approaches for immunological tolerance induction to stem cell-derived cell replacement therapies

The shortage of donors for organ transplantation and also to treat degenerative diseases has led to the development of the new field of regenerative medicine. One aim of this field, in addition to in vivo induction of endogenous tissue regeneration, is to utilize stem cells as a supplementary source of cells to repair or replace tissues or organs that have ceased to function owing to ageing or autoimmunity. Embryonic stem cells hold promise in this respect because of their developmental capacity to generate all tissues within the body. More recently, the discovery of induced pluripotent stem cells, somatic cells reprogrammed to a primitive embryonic-like state by the introduction of pluripotency factors, may also act as an important cell source for cell replacement therapy. However, before cell replacement therapy can become a reality, one must consider how to overcome the potential transplant rejection of stem cell-derived products. There are several potential ways to circumvent the hurdles presented by the immune system in this setting, not least the induction of immunological tolerance in the host. In this review, we consider this and other approaches for engendering acceptance of stem cell-derived tissues.