正常人周围血初始和记忆T细胞亚群及细胞因子表达的探讨

目的：利用多色流式检测技术探讨初始和记忆T细胞亚群与细胞因子表达之间的关系。方法：自正常人静脉血中分离PBMC,经超抗原（SEB）刺激5h后,加入多种抗细胞表面标记和抗细胞因子抗体进行染色,利用流式细胞术检测,并利用Flow Jo软件分析结果。结果：根据CD45RO表达与否,将CD4^＋和CD8^＋T细胞分为初始和记忆T细胞,再根据归巢受体（CD62L）和趋化因子受体（CCR7）的表达与否,将初始和记忆T细胞进一步分为不同的亚群。当T细胞受到SEB激活后,CD45RO^＋和CD45RO^-的CD4^＋或CD8^＋T细胞均表达IL-2、IFN-γ和TNF-α。进一步分析结果表明,CD62L^hi和CD62L^hiCCR7^＋细胞不表达细胞因子,而CD62L^loCCR7^lo和CCR7^＋T细胞均表达细胞因子,其中CD62L^loCCR7^lo细胞表达细胞因子的阳性率明显高于CCR7^＋细胞亚群。结论：只利用CD45RO表达与否区分初始和记忆T细胞是不够准确的,同时检测CD62L的表达,可明显地提高其准确性。     

Differential production of macrophage inflammatory protein 1gamma (MIP-1gamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro and in vivo

Due to differential expression of chemokine receptors, the Th1 and Th2 subsets of CD4(+) T cells differ in their migratory responses to chemokines. These differences in the migration patterns are likely to play a role in the initiation and regulation of Th1 and Th2 immune responses, inflammatory processes, and T-cell-mediated pathology. In the present study we evaluated the role of activated Th cells as producers of chemokines. Three different sources of murine Th cells were used, i.e., long-term-cultured Th1 and Th2 cell clones, Th1 and Th2 cells differentiated from na?ve CD4(+) spleen and lymph node cells in vitro, and Th1 and Th2 subsets polarized in vivo using a murine experimental Leishmania major infection model. Following stimulation with anti-CD3, macrophage inflammatory protein 1gamma (MIP-1gamma) and lymphotactin were produced selectively by Th1 cells but not by Th2 cells. In contrast, only Th2 cells produced MIP-2. The possible biological relevance of these data was substantiated by the finding that in vivo-polarized Th1 cells, but not Th2 cells, produced MIP-1gamma and lymphotactin while in vivo-polarized Th2 cells secreted MIP-2. The above data demonstrate that Th1 and Th2 cells differ in their ability to produce chemokines, suggesting that Th1 and Th2 subsets differentially contribute to recruitment of cells into inflammatory foci.     

Expression of L-selectin (CD62L) discriminates Th1- and Th2-like cytokine-producing memory CD4+ T cells.

Human memory (CD45RO+) CD4+ T cells can be distinguished into two subpopulations on the basis of expression of the lymph node homing receptor, L-selectin (CD62L). In a prior study we showed that human L-selectin-positive memory T-helper (Th) cells promote the maturation of IgG- and IgA-producing cells by naive B cells. To further elucidate the contribution of memory CD4+ T cells to B-cell differentiation, human memory CD4+ T cells with or without L-selectin expression were evaluated for production of cytokines that participate in regulation of immunoglobulin production. It was found that L-selectin-positive human memory CD4+ T cells produce mainly interleukin (IL)-4 and IL-5, whereas L-selectin-negative CD4+ T cells produce mainly interferon-gamma (IFN-gamma). This profile of cytokine expression coincides with the profile that distinguishes Th1 and Th2 subsets. In contrast to the murine system, IL-10 production was similarly contributed by human L-selectin-positive and -negative memory CD4+ T-cell subpopulations. These results suggest that the human L-selectin-negative and -positive subpopulations of human memory CD4+ T cells contain Th1-like and Th2-like cytokine-producing cells, respectively.     

Changes in the expression of CD31 and CXCR3 in CD4+ naïve T cells in elderly persons

So far, very few studies exist on the na?ve T cell population of elderly persons. Only recently an increase in the percentage of long lived CD4(+)CD31(-) na?ve T cells has been claimed to occur with aging. We, therefore, characterised CD31(+) and CD31(-) CD45RA(+) CD4(+) T cells in young and healthy elderly persons. The production of IL-2 and IFN-gamma by the different subpopulations was studied following stimulation with PMA and Ionomycin. The expression of CD28, CD11a, CD62L, CXCR3 and CCR7 was also analysed. The results of this study demonstrate a pronounced increase in the percentage of CD31(-) CD45RA(+) T cells within the CD4 subpopulation of elderly persons. Both, CD31(-) and CD31(+) CD45RA(+) cells expressed CD28, CD62L, were CD11a (dim) and produced IL-2 but no IFN-gamma. This phenotype confirms that they were na?ve T cells. IL-2 production by na?ve T cells was not impaired in elderly persons. Interestingly, CD31(+) as well as CD31(-) na?ve T cells contained a subpopulation of CXCR3(+) cells in elderly individuals, but not in young ones. In spite of expressing this chemokine receptor that enables the cells to migrate into inflammatory tissues, they were still CCR7(+) and CD62L(+). We speculate that due to previous contact with local environmental factors, this subset of na?ve T cells acquires a different chemokine receptor phenotype, resulting in an altered migratory capacity in old age. Aberrant contact with antigen and effector cell differentiation in unorthodox locations may be the consequence. This could also affect Th1/Th2 polarisation, which is known to be impaired in elderly persons.     

Beta interferon restricts the inflammatory potential of CD4+ cells through the boost of the Th2 phenotype, the inhibition of Th17 response and the prevalence of naturally occurring T regulatory cells

Beta-interferon (IFN-beta) is a valuable therapy for multiple sclerosis (MS) which is also effective in the animal model of experimental autoimmune encephalomyelitis (EAE). However, the accurate mechanisms to explain its anti-inflammatory activity in the disease are not fully revealed. Available data support that T lymphocytes are among the main cell targets of IFN-beta. We have found that in vitro anti-CD3 stimulation of uncommitted murine na?ve T cells under IFN-beta treatment results in skewing the T cell differentiation process towards the T2 phenotype, in a prevention from apoptosis of naturally occurring CD4+ T regulatory cells (nTreg) in correlation with an increase in Bcl-XL expression, and in a decrease of IL-17 expression. Elimination of nTreg from the primary culture of na?ve CD4+ cells abolished the down-regulation of IL-17 driven by IFN-beta, what suggests the interaction between Th17 and nTreg subsets. Experiments in EAE induced in SJL mice, showed in vivo evidence for the accumulation of spleen CD4+CD25+GITR+Foxp3+ cells after IFN-beta treatment. On the other hand, treated animals showed a striking decrease of IL-17 expression by peripheral CD4+ cells (Th17) and MBP-specific spinal cord cells. Both the in vivo and in vitro results point out new targets through which IFN-beta could exert its therapeutic action.     

瘦素系统对Th17细胞分化和功能的影响

Th17细胞在介导炎性反应、自身免疫性疾病、移植排斥和肿瘤等的发生发展中发挥着重要的作用.瘦素是一类脂肪源性细胞因子,具有多种免疫调控作用.瘦素可以通过上调RORγt的表达和转录,促进初始CD4＋T细胞向Th17细胞分化,但不影响该细胞的增殖.雷帕霉素哺乳动物靶位（mTOR）可以调控初始CD4＋T细胞的分化方向,mTORC1和mTORC2分别调节不同Th亚群的生成.研究瘦素系统与Th17细胞的关系和调控机制,将有助于通过靶向瘦素系统和AKT-mTOR信号通路来调控病理环境中的Th17细胞的免疫应答,可以为疾病的治疗提供新思路和新策略.     

U87MG培养上清致Na觙ve CD4分化为Th2细胞的能力降低

目的肿瘤微环境或者肿瘤细胞本身对免疫细胞的分化具有定向诱导作用,本研究目的在于了解胶质瘤细胞株U87MG外泌分子对Na觙ve CD4细胞分化可能的影响。方法外周血PBMC中分离Na觙ve CD4细胞,U87MG培养液上清以1/5量加至Na觙veCD4细胞培养体系中,培养7 d,检测Na觙ve CD4细胞分化增殖情况以及分化为Th1、Th2、Th17、Treg四个亚群量的变化。结果加有1/5量U87MG培养上清的Na觙ve CD4细胞分化成Th2减低,较对照组有显著性差异8.17%±2.08%vs 9.63%±2.48%(P<0.05),Th1较对照组均值低,但无显著性差异,Th17、Treg细胞在2组之间无明显差异。结论观察到U87MG外泌分子Na觙ve CD4分化为Th2细胞能力减弱,同时初步观察了Na觙ve CD4细胞分化为其他亚群的变化规律,为理解肿瘤细胞对Na觙ve CD4细胞分化影响提供了实验基础。     

Organ-specific distribution of CD4+ T1/ST2+ Th2 cells in Leishmania major infection

Activated CD4(+) T helper cells (Th) comprise at least two functionally distinct subsets, Th1 and Th2, which mediate different immunological effector functions. Experimental leishmaniasis is widely used to study the effector function of Th cell subsets in vivo. Healing and nonhealing Leishmania major infections have been correlated with polarized Th1 and Th2 responses, respectively. In the study presented here, a stable cell surface marker expressed on Th2 cells, T1/ST2, has been used to assess the distribution of CD4(+) T1/ST2(+) T cells in different organs of healer and nonhealer strains of mice during the course of L. major infection. The frequency of CD4(+) T cells expressing the T1/ST2 cell surface marker and Th2 cytokines in the lymphoid organs was low in both strains of infected mice; however, CD4(+) T1/ST2(+) T cells could be enriched from the lymphoid organs of infected nonhealer but not from healer strains of mice. The highest frequency of CD4(+) T1/ST2(+) T cells was detected in the footpads of mice with nonhealing disease, showing that CD4(+) T1/ST2(+) T cells home to the footpads. Since the majority of parasites persist at the local site of infection in nonhealing BALB/c mice, these results show that CD4(+) T1/ST2(+) T cells are localized at the site of active infection and inflammation in this model.     

Heterogeneity of CD4(+) and CD8(+) T cells

There is extensive plasticity in the T-cell response to antigen. Helper CD4(+) T cells, cytotoxic CD8(+) T cells, the progression from na?ve to effector and memory T cells, and differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. More recently it has become apparent that T-cell populations display additional diversity in terms of phenotype, anatomical distribution and effector function.     

CD8alpha(-) and CD8alpha(+) subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of na?ve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8alpha expression. We have studied the phenotype and maturation state of mouse splenic CD8alpha(-) and CD8alpha(+) DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8alpha(-) and CD8alpha(+) DCs into potent and equally efficient activators of na?ve CD4(+) and CD8(+) T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.     

Cross-regulation of CD86 by CD80 differentially regulates T helper responses from Mycobacterium tuberculosis secretory antigen-activated dendritic cell subsets

We report that stimulation of Mycobacterium tuberculosis secretory antigen- and tumor necrosis factor alpha-matured BALB/c mouse bone marrow dendritic cells (BMDCs) with anti-CD80 monoclonal antibody up-regulated CD86 levels on the cell surface. Coculture of these BMDCs with na?ve, allogeneic T cells now down-regulated T helper cell type 1 (Th1) responses and up-regulated suppressor responses. Similar results were obtained with splenic CD11c(+)/CD8a(-) DCs but not to the same extent with CD11c(+)/CD8a(+) DCs. Following coculture with T cells, only BMDCs and CD11c(+)/CD8a(-) DCs and not CD11c(+)/CD8a(+) DCs displayed increased levels of surface CD86, and further, coculturing these DCs with a fresh set of T cells attenuated Th1 responses and increased suppressor responses. Not only na?ve but even antigen-specific recall responses of the Th1-committed cells were modulated by DCs expressing up-regulated surface CD86. Further analyses showed that stimulation with anti-CD80 increased interleukin (IL)-10 and transforming growth factor-beta-1 levels with a concomitant reduction in IL-12p40 and interferon-gamma levels from BMDCs and CD11c(+)/CD8a(-) DCs and to a lesser extent, from CD11c(+)/CD8a(+) DCs. These results suggest that cross-talk between costimulatory molecules differentially regulates their relative surface densities leading to modulation of Th responses initiated from some DC subsets, and Th1-committed DCs such as CD11c(+)/CD8a(+) DCs may not allow for such modulation. Cognate antigen-presenting cell (APC):T cell interactions then impart a level of polarization on APCs mediated via cross-regulation of costimulatory molecules, which govern the nature of subsequent Th responses.     

Cd4(+) T cell response to Leishmania spp. in non-infected individuals

T cell mediated immunity is known to play a central role in the host response to control intra-cellular pathogens. This work demonstrates the presence of specific CD4(+) T cells to Leishmania spp. antigens in peripheral mononuclear cells of na?ve individuals (normal volunteers from non-endemic regions). The responder population was expanded by generation of antigen-specific T cell lines, which were produced by repeated stimulation with fixed promastigotes and autologous irradiated PBMC as antigen presenting cells. The leishmania-T cell lines were shown to proliferate in response to different species of the parasite (L. amazonensis, L. braziliensis, and L. donovani), but not to other recall antigens such as Candida albicans or tetanus toxoid. A preferential expansion of IFNgamma and IL-2 producing Th1-like T cells was observed. The leishmania-reactive cells were distributed between CD4(+) CD45RA(+) ("na?ve") and CD4(+) CD45R0(+) ("memory") populations. Although limiting dilution analysis showed a precursor frequency 3 times lower within the na?ve compartment, similar numbers of T cell lines were derived from both purified subpopulations. This study using leishmania-specific CD4(+) T cell lines produced from normal individuals should provide information on cellular immune responses that are triggered by the parasite and how infection impacts the na?ve T cell repertoire.     

T cell co-stimulatory molecules other than CD28.

CD28 is the primary co-stimulatory receptor for inducing high-level IL-2 production and survival of na?ve CD4(+) T cells. While no other cell surface receptor can be considered fully redundant with CD28, recent developments suggest that additional co-stimulatory pathways have preferential effects at different stages of T cell activation, on different subsets of T cells or contribute to the development of different effector functions.     

Expansion and activation of CD4(+)CD25(+) regulatory T cells in Heligmosomoides polygyrus infection

Regulatory T cell responses to infectious organisms influence not only immunity and immunopathology, but also responses to bystander antigens. Mice infected with the gastrointestinal nematode parasite Heligmosomoides polygyrus show an early Th2-dominated immune response (days 7-14), but by day 28 a strongly regulatory profile is evident with antigen-specific IL-10 release and elevated frequency of CD4(+) T cells bearing surface TGF-beta. CD4(+)CD25(+) T cells from infected mice show enhanced capacity to block in vitro effector T cell proliferation. CD4(+)CD25(+) cell numbers expand dramatically during infection, with parallel growth of both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets. CTLA-4 and glucocorticoid-induced tolerance-associated receptor, also associated with regulatory T cell function, become more prominent, due to both expanded CD25(+) cell numbers and increased expression among the CD25(-) population. Both intensity and frequency of CD103 expression by CD4(+) T cells rise significantly, with greatest expansion among CD25(+)Foxp3(+) cells. While TGF-beta expression is observed among both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets, it is the latter population which shows higher TGF-beta staining following infection. These data demonstrate in a chronic helminth infection that Foxp3(+) regulatory T cells are stimulated, increasing CD103 expression in particular, but that significant changes occur to other populations including expansion of CD25(+)TGF-beta(+)Foxp3(-) cells, and induction of CTLA-4 on CD25(-) non-regulatory lymphocytes.     

Mineralocorticoid receptor signaling reduces numbers of circulating human naïve T cells and increases their CD62L,CCR7, and CXCR4 expression

The role of mineralocorticoid receptors (MRs) in human T‐cell migration is not yet understood. We have recently shown that the MR antagonist spironolactone selectively increases the numbers of circulating naïve and central memory T cells during early sleep, which is the time period in the 24 h cycle hallmarked by predominant MR activation. To investigate whether this effect is specific to spironolactone's blockade of MRs and to study the underlying molecular mechanisms, healthy humans were given the selective MR‐agonist fludrocortisone or placebo and numbers of eight T‐cell subsets and their CD62L and CXCR4 expression were analyzed. Fludrocortisone selectively reduced counts of naïve CD4⁺, central memory CD4⁺, and naïve CD8⁺ T cells and increased CXCR4 expression on the naïve subsets. In complementing in vitro studies, fludrocortisone enhanced CXCR4 and CD62L expression, which was counteracted by spironolactone. Incubation of naïve T cells with spironolactone alone reduced CD62L and CCR7 expression. Our results indicate a regulatory influence of MR signaling on human T‐cell migration and suggest a role for endogenous aldosterone in the redistribution of T‐cell subsets to lymph nodes, involving CD62L, CCR7, and CXCR4. Facilitation of T‐cell homing following sleep‐dependent aldosterone release might thus essentially contribute to sleep's well‐known role in supporting adaptive immunity.     

CD154(CD40L)的表达与初始和记忆CD4+T细胞相关性的探讨

目的:探讨CD154 细胞的表型特征与初始和记忆CD4 T细胞以及与细胞因子表达之间的关系。方法:正常人外周血单个核细胞(PBMC),经抗CD3 抗CD28单克隆抗体(mAb)刺激培养后,利用多种抗细胞表面分子和抗细胞因子以及抗CD154抗体进行标记,用流式细胞术检测。结果:体外刺激PBMC6h后,CD154表达于CD4 T细胞,而不表达于CD8 T细胞。对比分析CD4 CD154 T和CD4 CD154- T细胞上CD45RA(初始)和CD45RO(记忆)表面分子的结果表明,大多数CD4 CD154 T细胞为记忆细胞。当利用抗CD3或抗CD3 抗CD28mAb刺激后,分泌IFN-γ、IL-2和TNF-α的CD4 T细胞均为CD4 CD154 T细胞,而且单个细胞可以同时分泌两种以上细胞因子。进一步研究表明,CD4 CD154 T细胞低表达或不表达CD25,不表达CD62L,50%左右的细胞表达CCR7。结论:体外短时间刺激PBMC,经过对细胞表型和细胞因子表达的研究,表明CD154分子结合其他表面标记或细胞因子的表达可以用于鉴别初始和记忆CD4 T细胞。     

Novel phenotypes and migratory properties distinguish memory CD4 T cell subsets in lymphoid and lung tissue

Memory T cells are heterogeneous in expression of lymph node homing receptors, delineating "central-memory" (TCM, CD62Lhi/CCR7+) and "effector-memory" (TEM, CD62Llo/CCR7-) subsets that migrate to lymphoid and non-lymphoid tissues, respectively. It is not known how these subsets arise or how homing receptor expression and tissue origin determine their functional and migratory properties. Here, we investigated the role of CD62L expression in the generation, function, distribution and migration of heterogeneous memory CD4 T cells specific for influenza hemagglutinin (HA). We found that CD62Lhi and CD62Llo memory subsets are generated independent of CD62L expression by the activated precursor, and both subsets distribute into spleen and lung. Functionally, spleen- and lung-derived CD62L memory subsets produce effector cytokines at similar kinetics but differ strikingly in cell surface phenotype and migration: the CD62Llo memory subset expresses a classic memory phenotype (CD45RBlo/CD44hi/CD11a(hi)), while the CD62Lhi subset expresses an unconventional phenotype (CD45RBhi/CD44int/CD11a(int)), defining a new polyclonal memory subset. The CD62Lhi subset also trafficked more efficiently than CD62Llo cells into lymph nodes; however, only lung but not spleen CD62Llo memory T cells homed to lung. Our results reveal novel phenotypic heterogeneity of memory CD4 T cells co-segregating with CD62L expression and tissue-specific tropism of non-lymphoid memory CD4 T cells.