HBV特异性T细胞表位肽的研究和应用

HBV特异性T细胞表位肽能够刺激机体产生特异的T细胞免疫应答反应.寻找能快速有力激发T细胞应答的抗原肽,提高患者自身的免疫水平,达到清除病毒的目的.随着免疫学和生物信息学的高速发展,研究T细胞表位肽的方法不断进步,越来越多的T细胞表位肽被发现,T细胞表位肽在HBV持续感染治疗中也有非常重要的应用.     

免疫应答类型的重新分类

过渡性免疫应答由原属固有免疫应答的原始B1细胞、γδT细胞和NKT细胞所组成.其中原始B1细胞承担过渡性免疫应答的体液免疫,γδT细胞和NKT细胞承担过渡性免疫应答的细胞免疫,主要识别和排除TI抗原.固有免疫应答的细胞包括吞噬细胞、树突状细胞、NK细胞等,体液免疫组包括补体、急性期蛋白、溶菌酶等,其主要作用是非特异性地清除或递呈抗原.适应性免疫应答的细胞由B2细胞和αβT细胞组成,B2细胞承担适应性体液免疫应答,αβT细胞承担适应性细胞免疫应答,主要识别和排除胸腺依赖性抗原(TD).免疫应答分为3种类型是对免疫系统进化的客观阐述,符合物种进化的基本规律,准确反映了免疫功能的发生和发展,对完善免疫学基础理论以及指导临床免疫学研究具有重要的意义.     

T细胞受体基因转导的研究和应用

T细胞受体（TCR）作为T细胞识别抗原的分子,在免疫应答和免疫调节中发挥重要的作用。利用转基因技术,将识别特定抗原的TCR基因转导到正常T细胞中,使其强制性表达了识别特异抗原TCR,从而达到发挥特异性免疫效应的目的,为产生有效的特异性免疫治疗开辟了一条新途径。     

小鼠γδT细胞抗原呈递功能的实验研究

目的:证实小鼠γδT细胞抗原也具有呈递功能.方法:观察了高度纯化的小鼠γδT细胞体外激活后MHC Ⅱ分子及其他共刺激分子的表达与其表面特征性TCR的关系.用自身反应抗原IRBP/MOG特异性T细胞株作为反应细胞,以增殖反应及细胞因子表达作为效应指标,观察γδT细胞的抗原呈递功能.结果:小鼠γδT细胞在激活后可表面表达MHC Ⅱ分子,并能有效呈递抗原给T细胞,使之发生抗原特异性免疫应答.结论:小鼠γδT细胞具有抗原呈递功能,在启动特异性免疫应答的过程中起重要作用.另一方面,不象人γδT细胞,表达MHC Ⅱ分子限于新激活的小鼠γδT细胞,后者失去了表面γδTCR.当γδT细胞重新获得表面γδTCR时,其MHC Ⅱ抗原表达则逐渐减少.     

人黑色素瘤抗原-3冲击的树突状细胞诱导抗肿瘤免疫应答

目的:观察经MAGE-3蛋白抗原冲击的树突状细胞在体外诱导抗肿瘤免疫应答的能力。方法:用粒-巨噬细胞集落刺激因子和白介素-4从人外周血分化、诱导树突状细胞,经MAGE-3蛋白冲击后,与T淋巴细胞共培养7d,收集T淋巴细胞进行免疫应答及肿瘤细胞杀伤试验。结果:与MAGE-3冲击后的树突状细胞共培养的T淋巴细胞在体外能有效地杀伤MAGE-3阳性的靶细胞。结论:经MAGE-3蛋白抗原冲击的树突状细胞,在体外能有效提呈抗原,激活抗原特异性CTL,诱导特异性抗肿瘤免疫应答。     

卵清白蛋白特异性T细胞克隆的建立及T细胞受体使用情况分析

目的：体外建立可长期培养的抗原特异性T细胞系及克隆，并分析其T细胞受体的使用情况。方法：用卵清白蛋白（OVA）免疫BALB／c小鼠，取淋巴结细胞在体外用抗原原复刺激建立抗原特异性T细胞系，通过能限稀释法进行克隆，应用锚定PCR方法分析其特异的T细胞受体的应用。结果：建立了H－2^d限制的OVA特异性T细胞系，获得了3株特异性较高的T细胞克隆，T细胞系中TCR AV11S4和BV4S1应用频率最高，3株T细胞克隆的TCR均为AV5S2和BV2S1，这些T细胞所使用的TCRα链和β链在CDR3区有明显的共性，结论：获得了卵清白蛋白特异的T细胞系和克隆，且其T细胞受体的应用有一定的限制性。     

记忆CD8+T细胞亚群在感染和肿瘤免疫应答中的作用

在初次免疫应答的过程中,当抗原被清除以后,大多数效应T细胞凋亡,只有少数细胞存活并分化成稳定、长寿的记忆细胞。与初始T细胞相比,当再次接触同一抗原时,记忆T细胞能够介导快速、强烈、有效的免疫应答[1-3]。记忆细胞高表达抗凋亡分子并以非抗原依赖的方式增殖来维持记忆细胞     

乙肝病毒感染不同阶段抗原肽特异性细胞毒性T 淋巴细胞的初步研究

目的:为了阐明HBV 感染不同阶段机体免疫功能的不同。方法:分别构建了我国最高频等位基因HLA鄄A*0201 三种常见抗原肽表位(HBVcore18-27、pol575-583、env335-343)四聚体复合物,并用这些四聚体检测HBV 感染外周血单个核细胞(PBMCs)中HLA-A*0201 限制性抗原特异性CD8+ T 细胞的频率、功能及CD127 的表达。结果:在大多数自限性HBV感染者的PBMCs 中抗原特异性CD8+ T 细胞的频率和增殖能力都高于慢性HBV 感染者。在低病毒载量的免疫低复制期抗原特异性CD8+ T 的数量和在高病毒载量、肝脏损害免疫清除期及免疫耐受期抗原特异性CD8+ T 细胞频率是相似的,同病毒定量及ALT 水平无显著相关性。慢性HBV 感染者抗原特异性CD8+ T 细胞增殖能力和病毒滴度呈反比。在自限性HBV 感染患者抗原特异性CD8+ T 细胞分泌IFN 的功能明显高于慢性HBV 患者,在免疫耐受期基本丧失产生细胞因子的能力;HBV 感染后不同免疫状态的HBsAg 水平不同,免疫耐受期最高,其次为免疫清除期、再活动期,低复制期的最低。而且随着HBsAg 水平下降,同HBV DNA 的相关性也逐渐下降。慢性HBV 感染者中CD8+ CD127+ T 细胞要低于对照组及自限性感染组,特别是在HBeAg 阳性的免疫耐受期及免疫清除期组患者中CD8+ CD127+ T 细胞比例更低。结论:慢性HBV 感染中抗原特异性CD8+ T细胞的频率不是决定免疫应答的唯一因素,慢性乙肝患者体内记忆性的抗原特异性CD8+ T 细胞并未被完全清除或缺失,这为治疗性疫苗和免疫恢复治疗提供了可行性。     

087 树突状细胞诱导T细胞免疫耐受的机理

摘要DC是一类专职的抗原呈递细胞,不仅具有强大的免疫应答诱导能力,而且能够通过多种机制诱导T细胞产生抗原特异性免疫耐受,这些机制包括诱导T细胞无能、专职地释放免疫抑制性细胞因子、介导T细胞的克隆清除、诱导和募集调节性T细胞和诱导免疫偏离.DC诱导免疫耐受机制研究的深入必将为DC的临床应用提供更坚实的基础.     

可溶性MHC-肽四聚复合物法及其进展

抗原特异性细胞毒性T淋巴细胞(CTL)主要是CD8^ T细胞，少数为CD4^ T细胞，CTL对抗原的识别及自身的活化受MHC分子的限制。抗原特异性CTL在抗感染免疫、移植物免疫和肿瘤免疫中发挥重要作用，定量分析抗原特异性CTL可为阐明免疫应答的自然发生过程提供重要信息，但是传统的检测分析方法均为间接的活性测定，费时费力。1996年Ahman等首创的可溶性MHC-肽四聚复合物法则能够直接定量检测抗原特异性CTL的比率，     

T-cell reconstitution without T-cell immunopathology in two models of T-cell-mediated tissue destruction

Antigen-specific T cells play a pivotal role in adaptive immune responses. However, they also contribute to the progression of a variety of diseases including autoimmune disorders, graft rejection and graft-versus-host disease (GVHD). Non-specific immune-ablation treatments compromise the ability of the host to respond to infection, whereas the selective removal of epitope-specific T cells could theoretically ameliorate T-cell-mediated pathology while preserving the rest of the host immune function. In this study we investigated whether it is possible to destroy specific unwanted antigen-specific T cells by incubating polyclonal T-cell populations with major histocompatibility complex (MHC) tetramers that are conjugated to the ribosomal-inactivating toxin, saporin. This strategy resulted in a dramatic reduction in the number of targeted antigen (Ag)-specific CD8 T cells with no observable bystander toxicity in vitro. Moreover, in a model of transferable T-cell-dependent neurological disease induced by intracerebral (i.c.) lymphocytic choriomeningitis virus (LCMV) infection, the targeted killing of LCMV-specific CD8 T cells extended the survival of mice or fully prevented their death, depending on the dose of cells transferred. In addition, the tetramer– saporin conjugate also reduced liver damage in a model of donor T-cell-mediated hepatic destruction. These data provide a proof of principle that MHC tetramers could be exploited for the elimination or clinical manipulation of T-cell responses by linking effector molecules (a toxin in this case) to MHC tetramers. Also, the results suggest that it may be feasible to remodel T-cell responses, especially in immunocompromised hosts who receive adoptive cell transfers with many potential alloreactive cells.     

Antigen-Specific Killer Polylactic-Co-Glycolic Acid (PLGA) Microspheres Can Prolong Alloskin Graft Survival in a Murine Model

Background: The strategy of specifically depleting antigen-specific T cells can potentially be used for the treatment of allograft rejection and autoimmunity because it does not suppress the overall immune systems.

Methods: In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling major histocompatibility complex (MHC) class I antigens and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto PLGA microspheres. A modified double-emulsion method was used for the preparation of cell-sized PLGA microspheres. H-2K^b/peptide monomers were generated in-house and analyzed through flow cytometry. The killer PLGA microspheres were administered intravenously into BALB/c mice (H-2K^d) that had previously been grafted with skin squares from C57BL/6 mice (H-2K^b). Tumor cell challenge and third-party mixed lymphocyte culture were used to assess the general immune functions of host.

Results: The alloskin graft survival was prolonged by 4?days. The killer PLGA microspheres could specifically deplete the H-2K^b alloantigen-reactive CD8⁺ T cells that infiltrated into the alloskin graft but not CD4⁺ T cells, without impairment of host overall immune function.

Conclusions: Here, we initially report that PLGA microspheres, which have been widely used as medicine-delivering carriers, were used to prepare antigen-specific killer complexes and treat allograft rejection. Our data highlight the therapeutic potential of this biocompatible and biodegradable antigen-specific killer effector for the treatment of allograft rejection and autoimmune disease.     

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 _103–111/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 _103–111 peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.     

Immune checkpoints and the regulation of tolerogenicity in dendritic cells: Implications for autoimmunity and immunotherapy

The immune system is responsible for defending the host from a large variety of potential pathogens, while simultaneously avoiding immune reactivity towards self-components. Self-tolerance has to be tightly maintained throughout several central and peripheral processes; immune checkpoints are imperative for regulating the immunity/tolerance balance. Dendritic cells (DCs) are specialized cells that capture antigens, and either activate or inhibit antigen-specific T cells. Therefore, they play a key role at inducing and maintaining immune tolerance. DCs that suppress the immune response have been called tolerogenic dendritic cells (tolDCs). Given their potential as a therapy to prevent transplant rejection and autoimmune damage, several strategies are under development to generate tolDCs, in order to avoid activation and expansion of self-reactive T cells. In this article, we summarize the current knowledge relative to the main features of tolDCs, their mechanisms of action and their therapeutic use for autoimmune diseases. Based on the literature reviewed, autologous antigen-specific tolDCs might constitute a promising strategy to suppress autoreactive T cells and reduce detrimental inflammatory processes.     

Human tumor antigens: implications for cancer vaccine development

The adoptive transfer of tumor-infiltrating lymphocytes along with interleukin 2 into autologous patients resulted in the objective regression of tumor in about 30% of patients with melanoma, indicating that these T cells play a role in tumor rejection. To understand the molecular basis of the T cell-cancer cell interaction we and others started to search for tumor antigens expressed on cancer cells recognized by T cells. This led to the identification of several major histocompatibility complex (MHC) class I restricted tumor antigens. These tumor antigens have been classified into several categories: tissue-specific differentiation antigens, tumor-specific shared antigens, and tumor-specific unique antigens. Because CD4+ T cells play a central role in orchestrating the host immune response against cancer, infectious diseases, and autoimmune diseases, a novel genetic approach has recently been developed to identify these MHC class II restricted tumor antigens. The identification of both MHC class I and II restricted tumor antigens provides new opportunities for the development of therapeutic strategies against cancer. This review summarizes the current status of tumor antigens and their potential applications to cancer treatment.     

Restimulation-induced cell death: new medical and research perspectives

In the periphery, homeostasis of the immune system depends on the equilibrium of expanding and contracting T lymphocytes during immune response. An important mechanism of lymphocyte contraction is clonal depletion of activated T cells by cytokine withdrawal induced death (CWID) and TCR restimulation induced cell death (RICD). Deficiencies in signaling components for CWID and RICD leads to autoimmunune lymphoproliferative disorders in mouse and human. The most important feature of CWID and RICD is clonal specificity, which lends great appeal as a strategy for targeted tolerance induction and treatment of autoimmune diseases, allergic disorders, and graft rejection by depleting undesired disease-causing T cells while keeping the overall host immunity intact.     

Peptide-activated double-negative T cells can prevent autoimmune type-1 diabetes development

Autoimmune diseases may develop because of defective maturation, activation, differentiation and function of regulatory T cells. Previous studies have shown that exposure to donor antigen activates peripheral TCRalphabeta+CD3+CD4-CD8-NK1.1-, double-negative (DN) T cells, which specifically suppress anti-donor T cells and enhance survival of skin and heart grafts from allogeneic and xenogeneic donors. However, the role of DN T cells in preventing T cell-mediated autoimmune disease is unknown. Here, we analyzed the ability of DN T cells to recognize peptides expressed on self MHC and to suppress peptide-reactive CD8+ T cells, using the P14 mouse model that expresses a transgenic TCR specific for gp33 peptide presented on self MHC class I-Db. We found that injection of gp33 peptide resulted in increased DN and decreased CD8+ T cell numbers in the lymph nodes when compared to untreated mice. Injection of gp33, but not TCR-non-specific AV peptide, increased expression of T cell activation markers on DN T cells. Moreover, gp33-activated DN T cells suppressed proliferation of syngeneic CD8+ T cells via killing activated CD8+ T cells in an antigen-specific fashion in vitro. Furthermore, transferring gp33-activated DN T cells inhibited the development of autoimmune diabetes, suggesting that DN T cells may provide a novel therapy for T cell-mediated autoimmune diseases.     

MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete

Selective expansion and activation of a very small number of antigen-specific CD4(+) T cells is a remarkable and essential property of the adaptive immune response. Antigen-specific T cells were until recently identified only indirectly by functional assays, such as antigen-induced cytokine secretion and proliferation. The advent of MHC Class II tetramers has added a pivotal tool to our research armamentarium, allowing the definition of allo- and autoimmune responses in deeper detail. Rare antigen-specific CD4(+) cells can now be selectively identified, isolated and characterized. The same tetramer reagents also provide a new mean of stimulating T cells, more closely reproducing the MHC-peptide/TCR interaction. This property allows the use of tetramers to direct T cells toward the more desirable outcome, that is, activation (in malignancies and infectious diseases) or Th2/T regulatory cell deviation, anergy and deletion (in autoimmune diseases). These experimental approaches hold promise for diagnostic, prognostic and therapeutic applications.     

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of α-chain in the case of MHC I, or β-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal I to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.     

MHC tetramers: tracking specific immunity

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells. T cells play important role in the control of infection and in the development of protective immunity. These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease. The specificity of T cells is determined by T cell receptors (TCR). Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes. Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria. MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C. CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells. A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome. Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses. The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding. Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye. Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e. approximately 1 in 50.000 lymphocytes). The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation. This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies. There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections. Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.