Diminished myelin-specific T cell activation associated with increase in CTLA4 and Fas molecules in multiple sclerosis patients treated with IFN-beta.

Multiple sclerosis (MS) is a chronic inflammatory disease of the white matter of the central nervous system (CNS) characterized by focal areas of demyelination. Interferon-beta (IFN-beta) provides an effective treatment that lessens the frequency and severity of exacerbations in relapsing-remitting multiple sclerosis (RRMS), but the mechanisms by which IFN-beta is efficient remain uncertain. The data presented here demonstrate that IFN-beta impairs the proliferative response to myelin basic protein (MBP) and myelin, as well as increasing the expression of the CTLA4 intracellular molecule. Moreover, this treatment increases the expression of surface Fas molecules and of the soluble form of these molecules. Our hypothesis is that the increase in Fas and CTLA4 molecules in MS patients may lead to lymphocyte apoptosis, which suggests possible mechanisms underlying the therapeutic response to IFN-beta.     

Impact of PRRSV on activation and viability of antigen presenting cells

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine industry. The causal agent, PRRS-virus (PRRSV), is able to evade the host immune response and survive in the organism causing transient infections. Despite all scientific efforts, there are still some gaps in the knowledge of the pathogenesis of this disease. Antigen presenting cells (APCs), as initiators of the immune response, are located in the first line of defense against microorganisms, and are responsible for antigen recognition, processing and presentation. Dendritic cells (DCs) are the main type of APC involved in antigen presentation and they are susceptible to PRRSV infection. Thus, PRRSV replication in DCs may trigger off different mechanisms to impair the onset of a host effective immune response against the virus. On the one side, PRRSV may impair the basic functions of DCs by regulating the expression of major histocompatibility complex class II and CD80/86. Other strategy followed by the virus is the induction of cell death of APCs by apoptosis, necrosis or both of them. The impairment and/or cell death of APCs could lead to a failure in the onset of an efficient immune response, as long as cells could not properly activate T cells. Future aspects to take into account are also discussed in this review.     

Insulin-specific T cell help to TNP-specific B cells requires activation via the antigen T cell receptor

In the present study, the production of large numbers of insulin (Ins)-specific, H-2-restricted T helper (Th) cell clones is described. Among 148 clones analyzed, 121 clones had Th cell function, and these were divided into 75 Ins-specific Th cell clones and 52 autoreactive clones. The 148 clones were isolated from Ins-specific T cell lines produced by in vitro stimulation of T lymphocytes from (b X d)F1 mice immunized with Ins 7, 14, 32 or 56 days before. The following characteristics were tested with regard to the Th cell clones: restriction specificity and antigen requirement for optimal help or interleukin 2 (IL2) production. No differences in these characteristics were found among clones originating from day 7, 14, 32 or 56 T cell lines. A preference for H-2b as restriction element and an antigen concentration of about 0.01 microgram trinitrophenylated (TNP)-Ins/ml for optimal help were general traits. Optimal IL2 release is not yet obtained with 100 micrograms TNP-Ins/ml. Thus, the antigen requirement for optimal help and IL2 release differs by a factor 10(4) at least. Certain twice-cloned Th cell lines were tested for IL2 production when stimulated with anti-Thy-1 monoclonal antibodies (mAb). All clones analyzed were stimulated by mAb H155 but not with mAb H140 nor with mAb against Lyt-1, L3T4, LFA-1 or H-2K/D molecules. Therefore, we determined whether TNP-conjugated H155 mAb would mediate Th cell-B cell collaboration as well as TNP-Ins. The results with nine different Th cell clones and six different TNP-conjugated mAb used in a 10(7)-fold concentration range showed that Th cell clones have to be triggered via the T cell receptor for expression of helper function to B cells. Thus, though IL2 gene activation, synthesis or release apparently can be activated via at least two pathways: T cell receptor or Thy-1, it seems that activation of the genes responsible for synthesis and release of the helper factors, which ensure antigen-specific B cell proliferation and differentiation, needs Th cell-B contact mediated via the antigen-specific T cell receptor.     

Particle shape dependence of CD8+ T cell activation by artificial antigen presenting cells

Previous work developing particle-based acellular, artificial antigen presenting cells (aAPCs) has focused exclusively on spherical platforms. To explore the role of shape, we generated ellipsoidal PLGA microparticles with varying aspect ratios (ARs) and synthesized aAPCs from them. The ellipsoidal biomimetic aAPCs with high-AR showed significantly enhanced in vitro and in vivo activity above spherical aAPCs with particle volume and antigen content held constant. Confocal imaging indicates that CD8+ T cells preferentially migrate to and are activated by interaction with the long axis of the aAPC. Importantly, enhanced activity of high-AR aAPCs was seen in a mouse melanoma model, with high-AR aAPCs improving melanoma survival compared to non-cognate aAPCs (p = 0.004) and cognate spherical aAPCs (p = 0.05). These findings indicate that particle geometry is a critical design criterion in the generation of aAPCs, and may offer insight into the essential role of geometry in the interaction between CD8+ T cells and biological APCs.     

Direct helper T cell-induced B cell differentiation involves interaction between T cell antigen CD28 and B cell activation antigen B7.

Cognate interactions between major histocompatibility complex class II antigen (Ag)-reactive CD4+ T helper (Th) and Ag-presenting B cells induce first the activation of B cells and their subsequent differentiation into Ig-secreting cells (IgSC). The Th cell-associated homodimeric glycoprotein CD28 has been implicated as an important regulator of Th activation. Recently, B cell-associated early activation Ag B7 has been identified as a ligand for the CD28 molecule. In this study, we have examined using monoclonal antibodies (mAb) the roles of CD28 and B7 molecules during the Th-B cell cognate interactions leading to the differentiation of B7+ B cells. Anti-CD28 mAb 9.3 specifically inhibited proliferative responses of CD4+ T cells to both allogeneic B cells and soluble Ag-presenting autologous non-T cells. In addition, anti-CD28 mAb 9.3 inhibited Th-induced differentiation of alloantigen-presenting B cells into ISC. Similar inhibition of both Ag-induced Th activation and B cell differentiation into ISC was observed using mAb BB1 which recognizes a B cell-associated molecule B7. In contrast, non-cognate Th-independent exogenous interleukin 6-induced differentiation of B7+ B cells into ISC was not inhibited by mAb to either molecule. These results clearly demonstrate the involvement of CD28 on Th and its ligand B7 on B cells during cognate Th-B interactions leading to the differentiation of B cells. Furthermore, these results also suggest the development of new mAb-based therapeutic approaches for exaggerated B cell activation associated with certain autoimmune diseases such as systemic lupus erythematosus.     

Involvement of GANP in B cell activation in T cell-dependent antigen response

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.     

Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells

T-cell recognition of peptide-MHC complexes on APCs requires cell-cell interactions. The molecular events leading to T-cell activation have been extensively investigated, but the underlying physical binding forces between T-cells and APCs are largely unknown. We used single cell force spectroscopy for quantitation of interaction forces between T-cells and APCs presenting a tolerogenic peptide derived from myelin basic protein. When T-cells were brought into contact with peptide-loaded APCs, interaction forces increased with time from about 0.5nN after 10s interaction to about 15nN after 30min. In the absence of antigen, or when ICAM-1-negative APC was used, no increase in binding forces was observed. The temporal development of interaction forces correlated with the kinetics of immune synapse formation, as determined by LFA-1 and TCR enrichment at the interface of T-cell/APC conjugates using high throughput multispectral imaging flow cytometry. Together, these results suggest that ICAM-1/LFA-1 redistribution to the contact area is mainly responsible for development of strong interaction forces. High forces will keep T-cells and APCs in tight contact, thereby providing a platform for optimal interaction between TCRs and peptide-MHC complexes.     

T and B lymphocytes in multiple sclerosis.

The percentage and total number of E and EAC rosettes, as indicators of T and B lymphocytes respectively, were studied in the blood of subjects with multiple sclerosis (MS) and normals. MS patients in acute exacerbation were found to have a decrease in E rosettes and an increase in EAC rosettes. The relationship of these findings to the pathogenesis of MS is unclear; several possible pathogenetic implications are considered.     

siRNA沉默抗原递呈细胞表面CD86的表达对T淋巴细胞激活的影响

目的:通过小干扰RNA(small interfering RNA,siRNA)沉默抗原递呈细胞(Antigen presenting cells,APC)表面共刺激分子CD86的表达,分析其对T细胞增殖和白介素10及IFNγ-分泌的影响,为移植排异和自身免疫性疾病的治疗提供新的思路和方法。方法:设计并通过化学方法合成三对针对人CD86 mRNA的siRNA片段(siRNA-1、2、3),脂质体法转染人B淋巴瘤Raji细胞;转染24、48、72小时后,流式细胞术(Flow cytometry,FCM)检测Raji细胞表面CD86蛋白水平的表达;荧光定量PCR方法检测CD86mRNA水平的表达;分别采用siRNA抑制Raji细胞表面CD86分子的表达、抗人CD86单克隆抗体封闭CD86分子以及二者的联合来阻断CD86-CD28信号通路、MTT和ELISA方法分别检测CD86信号通路被抑制后对T细胞增殖和细胞因子IL-10及IFNγ-分泌的影响。结果:FCM结果显示,Raji细胞表面天然表达CD86的阳性率约为98.0%;转染siRNA后24小时时,仅siRNA-2组表现较为明显的抑制效应,此时细胞表面CD86的阳性表达率约为61.7%,抑制率为37.0%;转染后48小时,control和siRNA-1组Raji细胞表面CD86的表达仍没有变化,而siRNA-2组CD86的表达下降至39.6%,抑制率为59.6%,siRNA-3组CD86的表达下降至72.6%,抑制率为25.9%;siRNA转染后72小时,各组Raji细胞表面CD86的表达均有不同程度的变化,分别为:control组,90.4%;siRNA-1组,83.1%;siRNA-2组,15.2%和siRNA-3组,46.2%;各组的抑制率依次为7.6%,15.2%,84.5%及52.8%。荧光定量PCR结果显示,转染后72小时,仅siRNA-2和siRNA-3组CD86 mRNA水平的表达与Raji细胞相比有显著差异,抑制率分别为78.7%和45.9%。CD86的表达被siRNA-2抑制后,可抑制Raji细胞对T细胞的活化、增殖和IL-10及IFNγ-的分泌;抗人CD86单克隆抗体同样可以抑制Raji细胞对T细胞的激活;并且siRNA-2和抗人CD86单克隆抗体对T细胞的活化抑制具有协同效应。结论:通过siRNA沉默抗原递呈细胞表面共刺激分子CD86的表达,从而阻断CD86/CD28共刺激信号通路,可有效抑制T淋巴细胞的活化、增殖及细胞因子IL-10、IFNγ-的分泌,由此削弱了T细胞应答来诱导免疫耐受。     

HLA-DR antigen expression on T cells from cerebrospinal fluid in multiple sclerosis and aseptic meningo-encephalitis.

HLA-DR expression on T lymphocytes in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and acute aseptic meningo-encephalitis (AM), and from blood only from healthy controls was examined by a new double-immunofluorescence labelling assay using species-specific second layers on prefixed cell samples. Thirteen of 16 patients with AM (81%) had an elevated percentage of DR positive T cells in CSF against only two of 20 patients with MS (10%). Our data indicate that AM, an acute infection of the central nervous system (CNS), is accompanied by accumulation in CSF of activated, DR positive T cells as a reflection of actively involved cellular immunity within the CNS, while this accumulation of DR positive T cells is not seen in MS, a chronic inflammatory CNS disease, despite some of the patients being examined during clinical exacerbations.     

Regulation of T follicular helper cell formation and function by antigen presenting cells

CD4+ T cells can differentiate into numerous subsets characterized by expression of a suite of cytokines and effector molecules that endow them with specialized functions. By mediating the differentiation of B cells into memory and plasma cells following exposure to T-dependent antigens (Ag), T follicular helper (TFH) cells have emerged as the predominant subset of CD4+ T cells responsible for regulating humoral immunity. The generation of TFH cells from na?ve precursors typically involves sequential cognate interactions with distinct populations of Ag-presenting cells (APCs): dendritic cells within the T-cell zone of lymphoid tissues, and activated B cells at the border of the T-zone and follicle, and then within a germinal center. Recent studies have illuminated the key roles of APCs in TFH development, and have also re-defined the role of B cells in this process.     

Differences in IgG subclass do not effect immune complex-enhanced T cell activation despite differential binding to antigen presenting cells.

Ag presentation to CD4 T cells is a critical event in the generation of protective immunity. IgG, in the form of IgG-pathogen (Ag) complexes, is capable of mediating FcgammaR-dependent Ag presentation, and thereby enhanced T cell activation. Therefore, it is important to understand the ability of the individual human IgG subclasses to function in enhanced T cell activation. We hypothesized that increased delivery of Ag to monocyte FcgammaR by high affinity human IgG subclasses, IgG1 and IgG3, would lead to increased Ag presentation, as compared to low affinity IgG subclasses, IgG2 and IgG4. To create immune complexes, we linked biotinylated IgG subclasses to biotinylated Ag via an avidin bridge, and examined T cell responses to them. Although IgG2- and IgG4-Ag complexes bound to monocytes at significantly lower levels than those made with IgG1 and IgG3, we observed no significant difference in the ability of the four human IgG subclasses to mediate enhanced T cell activation. Studies suggest the explanation for this dichotomy lies within the first 24 h of Ag processing, and that processing efficiency may vary with IgG subclass. They also suggest the existence of a highly efficient, and selective processing pathway, which is dependent on IgG subclass, and can compensate for low level production and FcgammaR binding of IgG2- and IgG4-Ag complexes.     

Subsets of CD4 T cells and B cell activation

All helper T cells recognise foreign protein antigens presented by class II molecules of the major histocompatibility complex (MHC) and express the cell surface molecule CD4. However, not all CD4 T cells behave as helper T cells when assayed for their ability to activate B cells to produce antigen specific antibody. In this review, after discussing the background information about CD4 T cell subsets, a series of questions will be asked: whether selective activation of distinct functional subsets of CD4 T cell accounts for the difference between humoral and cell-mediated immunity; whether the same subsets exist in all mammalian species studied to date, or whether there are major species differences; whether cells belonging to distinct functional subsets can be distinguished on the basis of a subset-specific cell surface molecule; what models of subset development can account for existing data; and whether or not the subsets defined to date are all comparable in their ability to activate B cells?     

MHC-restricted autoantigen-reactive T cell clones in multiple sclerosis

Multiple sclerosis is a demyelinating disease of the central nervous system with genetic, viral and autoimmune characteristics. Myelin basic protein (MBP) is a suspected target autoantigen since it induces experimental autoimmune encephalomyelitis, an animal model closely resembling multiple sclerosis. The disease is mediated by Class II restricted, MBP-reactive T cells possessing the T helper/inducer phenotype. In the present study, we have isolated MBP-reactive T cell clones from the peripheral blood of a chronic progressive multiple sclerosis patient. The clones displayed blastogenic memory responses when rechallenged with the autoantigen and irradiated autologous lymphocytes. MBP recognition by the autoantigen-reactive T lymphocytes was restricted by major histocompatibility complex Class II antigens. Both CD4+8- and CD4-8+ MBP-reactive T cell clones were obtained.     

Stimulation of adult oligodendrogenesis by myelin-specific T cells

In multiple sclerosis (MS), myelin-specific T cells are normally associated with destruction of myelin and axonal damage. However, in acute MS plaque, remyelination occurs concurrent with T-cell infiltration, which raises the question of whether T cells might stimulate myelin repair. We investigated the effect of myelin-specific T cells on oligodendrocyte formation at sites of axonal damage in the mouse hippocampal dentate gyrus. Infiltrating T cells specific for myelin proteolipid protein stimulated proliferation of chondroitin sulfate NG2-expressing oligodendrocyte precursor cells early after induction via axonal transection, resulting in a 25% increase in the numbers of oligodendrocytes. In contrast, T cells specific for ovalbumin did not stimulate the formation of new oligodendrocytes. In addition, infiltration of myelin-specific T cells enhanced the sprouting response of calretinergic associational/commissural fibers within the dentate gyrus. These results have implications for the perception of MS pathogenesis because they show that infiltrating myelin-specific T cells can stimulate oligodendrogenesis in the adult central nervous system.     

Thymic epithelial cells: antigen presenting cells that regulate T cell repertoire and tolerance development

During thymocyte development bone marrow-derived precursors in the thymus undergo a series of differentiation steps to produce self-tolerant, mature T lymphocytes. The thymus contains two functionally distinct anatomical compartments, consisting of a centrally located medulla surrounded by the thymic cortex. These compartments in turn are comprised of two major cellular components: (1) the T lymphoid compartment of developing thymocytes and (2) the thymic stroma consisting mainly of thymic epithelial cells (TECs). These epithelial cells are further separated into cortical and medullary TECs (cTECs and mTECs) based on their localization within the thymic cortex or medulla respectively. Reciprocal interactions between thymocytes and epithelial cells are required for the development of both cellular components into a functional thymic organ. Thymocytes provide trophic factors for the development of a complex three-dimensional epithelial cell network, while epithelial cells regulate T cell development through expression and presentation of self-antigens on major histocompatibility molecules. Our work focuses on how thymic epithelial cells regulate T cell development and function and on elucidating the mechanisms of thymic epithelial cell differentiation. Here we review current knowledge and provide our own insight into the development, differentiation and antigen presenting properties of TECs. We focus specifically on how mTECs regulate T cell repertoire selection and central tolerance.     

T cells from multiple sclerosis patients recognize multiple epitopes on Self-IgG

The highly diversified variable regions of immunoglobulin (Ig) molecules contain immunogenic determinants denoted idiotopes. We have previously reported that T cells from multiple sclerosis (MS) patients recognize IgG from autologous cerebrospinal fluid (CSF), and mapped a T-cell epitope to an IgG idiotope. To test the ability of CSF IgG molecules to elicit a broad polyclonal T-cell response in MS, we have analysed T-cell responses in the blood and CSF against idiotope peptides spanning complementarity determining region (CDR) 3 and somatic mutations within the variable regions of monoclonal CSF IgG. Consistent with a diversified idiotope-specific T-cell repertoire, CD4(+) T cells from both patients recognized several idiotope peptides presented by HLA-DR molecules. Mutations were critical for T-cell recognition, as T cells specific for a mutated CDR1 peptide did not recognize corresponding germline-encoded peptides. One T-cell clone recognized both an idiotope peptide and the B-cell clone expressing this idiotope, compatible with endogenous processing and presentation of this idiotope by B cells. These results suggest that mutated CSF IgG from MS patients carry several T-cell epitopes, which could mediate intrathecal IgG production and inflammation in MS through idiotope-driven T-B-cell collaboration.