Lymphocyte Dynamics

淋巴细胞动力学

• 批准号: 8745403
• 负责人: William Paul
• 金额: $ 91.86万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8745403
• 关键词: Academy; Accounting; Adjuvant; Antigens; Biological Models; Blastomyces; C-Peptide; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Count; Cell Death; Cell Fraction; Cell Proliferation; Cells; Cessation of life; Collaborations; Complex; Computational Biology; Data; Dendritic Cells; Frequencies; Genes; Goals; HIV; HIV Infections; Heating; Homeostasis; IL2RA gene; Immune response; Immunization; Individual; Infection; Interferon Type II; Interleukin-1; Interleukin-10; Interleukin-12; Interleukin-15; Interleukin-17; Interleukin-2; Interleukin-7; Knock-out; Lead; Listeria monocytogenes; Lung; Lymphocyte; Lymphocyte Count; Lymphocytic choriomeningitis virus; Measurement; Measures; Mediating; Memory; Molecular; Mus; NR4A1 gene; Pattern; Peptide/MHC Complex; Peptides; Phenotype; Physiological; Population; Process; Production; Proliferating; Proteins; RNA; Recording of previous events; Regimen; Regulation; Regulatory T-Lymphocyte; Relative (related person); Role; SIV; Science; Scientist; Self Stimulation; Series; Specificity; Supplementation; T cell response; T-Cell Depletion; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; TLR3 gene; TNF gene; Time; Tissues; Transgenic Organisms; Vaccines; Vaccinia; Vaccinium; antigen challenge; base; cytokine; granzyme B; in vivo; insight; interdisciplinary approach; killings; lymphocyte proliferation; mathematical model; memory CD4 T lymphocyte; pathogen; precursor cell; professor; receptor; research study; response; tool development

Lymphocyte numbers are regulated both by responses to conventional exogenous antigens and endogenous microflora, by stimulation by self-peptide/MHC complexes and by the action of a series of cytokines. This multifaceted regulation permits individuals to maintain a broad repertoire of lymphocytes of distinctive specificities, allowing responses against a vast array of foreign substances and, at the same time, providing a pattern of memory based on the immunization history of the individual. The study of the process of lymphocyte dynamics that underlies this regulation requires a multidisciplinary approach, aimed both at the molecular underpinnings of the processes through which lymphocytes survive and proliferate and a systemics/ computational biology approach to appreciate the overall mechanisms governing total numbers of lymphocytes of distinct phenotype and distinct specificity. Emphasis has been placed on four aspects of this problem: the priming and expansion of naive CD4 T cells in response to antigen challenge, the dynamics of lymphocyte memory and of memory phenotype cells, the mechanisms underlying CD4 T cell depletion in HIV infection, and the process of homeostatic proliferation and death. Unit scientists have shown that primary responses are highly dependent upon the number of precursor cells that can respond to antigenic challenge. Using both real time PCR and flow cytometric analysis to measure the response when TCR transgenic cells are transferred to intact recipients, it has been shown that the factor of expansion (FE) of the antigen-stimulated CD4 T cells is highly dependent upon the number of specific precursors. In a model system based on responses of T cell receptor transgenic cells specific for a cyctochrome C peptide, when the frequency of precursors in the recipient is 3 or less, FE is 1500, at 300 cells it is 200 and at 30,000 it is 20. Limitation in expansion does not result from a smaller fraction of cells responding but rather, at least in part, from diminished proliferative rates of responding cells. Diminution in FE as number of precursors increase cannot be accounted for by Fas, TNF or IFNg-mediated cell death nor can it be due to limitation in numbers of dendritic cells or in amounts of antigen as increasing either DC number or amount of antigen does not alter the relationship of FE and precursor number. Furthermore, the effect is not altered by supplementation with IL-1, IL-2, IL-7 or IL-15. The relative frequency of regulatory T cells, either derived from the responding cells or from the host, is not altered by precursor frequency and the difference in FE occurs even when responding cells are unable to develop into regulatory T cells. The effect is highly antigen specific in that large numbers of cells of one specificity do not effect the rate of expansion of small numbers of cells of another specificity. In collaboration with Professor Gennady Bocharov of the Russian Academy of Sciences, a mathematical model of the proliferation of these cells has been developed conforms very well to the observed data. In the course of analyzing the control of FE on the part of both nave and memory cells, it was observed that the most potent stimulant of FE was the cytokine IL-1. When expansion of CD4 TCR transgenic T cells in a syngeneic host in response to antigen was measured, it was found that administering IL-1 over a 3 to 5 day period caused a ten fold enhancement in FE when compared to that seen using conventional adjuvants such as LPS. This was equally true for naive and memory cells and was not mediated by other cytokines. The effect could only be partially explained by enhanced proliferation so that greater survival was also implicated. The use of recipients that were IL-1 receptor knockouts and IL-1 receptor-sufficient donors of TCR transgenic T cells showed that IL-1 could act directly on the responding CD4 or CD8 T cells to mediate expansion. The IL-1 receptor antagonist diminished the adjuvant effect of LPS indicating that a substantial portion of the effect of this conventional adjuvant was due to endogenous production of IL-1. Initial analysis of genes activated and suppressed in cells responding to antigen in the presence of LPS suggest avenues for further analysis that may lead to a mechanistic understanding of the IL-1 effect. The very robust effect of IL-1 suggests it may have a role in certain immunization strategies. IL-1 acts directly on CD4 T cells to enhance their differentiation into IL-17 producing cells. However, although IL-1 acts directly on CD8 cells to mediate their expansion, differentiation of CD8 cells requires the action of IL-1 on non T cells. Strikingly, the effects of IL-1 during priming are retained at the time of secondary challnege even though IL-1 is not administered again. Thus, the secondary response in mice primed in the presence of IL-1 includes CD8 cells increased in number, found in the tissues, and siaplying a high degree of granzyme B expression and IFN gamma production. Experiments involving the use weak vaccines, such as heat killed Listeria monocytogenes, the gD2 protein of H. simplex, heat killed Blastomyces or peptides associated with vaccinia, result in strikingly induced protection if IL-1 is included in the priming regimen. Memory CD4 T cell proliferation was shown to be quite slow. Specific T cells from mice infected with LCMV divide at a rate of 2% per day. By contrast CD44bright CD25- CD4 T cells divide much more rapidly, at 8 to 10% per day. Furthermore this rapid steady state proliferation of "memory phenotype" CD4 T cells is similar in conventional and germfree mice. Analysis of the repertoire of memory phenotype undergoing proliferation revealed no difference in receptor complexity from that of non-dividing memory phenotype cells. This implies that division is largely stochastic and probably dominantly driven by cytokines rather than by peptide/ MHC complexes, whether of exogenous or endogenous origin. Memory phenbotype and autehntic memory cells differ from one another not only in their proliferative rates but, based onan RNA-SEQ analysis, also on the expression of NUR77, the latter being highly expressed on authentic memory cells even 30 days after priming, when they are already quiescent. Efforts to understand the role of distinct priming regimens to induce particular phenotypic CD4 T cells responses to lung immunization reveal a profound effect of different "adjuvants". Priming in the presence of LPS leads to a TH17 response whereas priming in the presence of polyI:C to an exclusive IFNgamma response. Based on studies with KO mice, the results have been interpreted as indicated that LPS, through its activation of Myd88, results in robust IL-10 production, blocking IL-12 production and Th1 priming. In parallel, though its activation of TRIF/TRAM, it activates IL-1 production, markedly enhancing Th17 priming. By contrast, polyI:C, acting through TLR3 and TRIF activates type I intewrferon and IL-12, enhancing IFNgamma priming and blocking Th17 induction. These results give important insight into the precise resultation of CD4 T cells responses to distinct pathogens in the lung.

淋巴细胞数量通过对常规外源抗原和内源微生物群的反应、自肽/MHC复合物的刺激以及一系列细胞因子的作用来调节。这种多方面的调节使个体能够维持具有独特特异性的广泛淋巴细胞库，从而能够对大量外来物质做出反应，同时提供基于个体免疫史的记忆模式。对作为这一调节基础的淋巴细胞动力学过程的研究需要采用多学科方法，既针对淋巴细胞生存和增殖过程的分子基础，又针对系统学/计算生物学方法，以了解控制不同表型和不同特异性的淋巴细胞总数的总体机制。重点关注该问题的四个方面：初始 CD4 T 细胞响应抗原挑战的启动和扩增、淋巴细胞记忆和记忆表型细胞的动态、HIV 感染中 CD4 T 细胞耗竭的机制以及稳态增殖和死亡的过程。 单位科学家表明，初级反应高度依赖于能够对抗原攻击做出反应的前体细胞的数量。使用实时 PCR 和流式细胞术分析测量 TCR 转基因细胞转移至完整受体时的反应，结果表明，抗原刺激的 CD4 T 细胞的扩增因子 (FE) 高度依赖于特定前体细胞的数量。在基于对细胞色素C肽特异的T细胞受体转基因细胞的反应的模型系统中，当受体中前体细胞的频率为3或更少时，FE为1500，在300个细胞时为200，在30,000个细胞时为20。扩增的限制不是由于响应细胞的比例较小，而是至少部分由于响应细胞的增殖率降低而导致。 FE 随着前体细胞数量增加而减少，不能用 Fas、TNF 或 IFNg 介导的细胞死亡来解释，也不能归因于树突细胞数量或抗原量的限制，因为增加 DC 数量或抗原量不会改变 FE 和前体细胞数量的关系。此外，补充 IL-1、IL-2、IL-7 或 IL-15 不会改变其效果。调节性 T 细胞（无论是来自应答细胞还是来自宿主）的相对频率不会因前体频率而改变，即使当应答细胞无法发育为调节性 T 细胞时，FE 也会出现差异。该效应是高度抗原特异性的，因为一种特异性的大量细胞不会影响另一种特异性的少量细胞的扩增速率。与俄罗斯科学院的根纳季·博恰洛夫教授合作，开发了这些细胞增殖的数学模型，该模型与观察到的数据非常吻合。 在分析 FE 对幼稚细胞和记忆细胞的控制过程中，观察到 FE 最有效的刺激物是细胞因子 IL-1。当测量同系宿主中 CD4 TCR 转基因 T 细胞响应抗原的扩增时，发现与使用 LPS 等传统佐剂相比，在 3 至 5 天的时间内施用 IL-1 导致 FE 增强十倍。对于幼稚细胞和记忆细胞来说同样如此，并且不受其他细胞因子介导。这种效应只能通过增殖增强来部分解释，因此生存率也更高。使用IL-1受体敲除的受体和IL-1受体充足的TCR转基因T细胞供体表明，IL-1可以直接作用于响应的CD4或CD8 T细胞以介导扩增。 IL-1受体拮抗剂减弱了LPS的佐剂作用，表明这种常规佐剂的作用的很大一部分是由于IL-1的内源性产生。对 LPS 存在下响应抗原的细胞中激活和抑制的基因的初步分析表明了进一步分析的途径，这可能导致对 IL-1 效应的机制理解。 IL-1 的强大作用表明它可能在某些免疫策略中发挥作用。 IL-1 直接作用于 CD4 T 细胞，增强其分化为产生 IL-17 的细胞。 然而，尽管IL-1直接作用于CD8细胞以介导其扩增，但CD8细胞的分化需要IL-1对非T细胞的作用。引人注目的是，即使不再给予IL-1，在引发期间IL-1的作用在二次攻击时仍保留。 因此，在IL-1存在下引发的小鼠的二次反应包括组织中发现的CD8细胞数量增加，以及颗粒酶B表达和IFNγ的高度产生。 涉及使用弱疫苗的实验，例如热灭活单核细胞增生李斯特氏菌、单纯螺杆菌的 gD2 蛋白、热灭活芽生菌或与牛痘相关的肽，如果在引发方案中包含 IL-1，则会产生显着诱导的保护作用。 记忆 CD4 T 细胞增殖速度相当缓慢。 感染 LCMV 的小鼠的特定 T 细胞每天以 2% 的速度分裂。 相比之下，CD44bright CD25-CD4 T 细胞的分裂速度要快得多，每天为 8% 至 10%。 此外，“记忆表型”CD4 T 细胞的这种快速稳态增殖在传统小鼠和无菌小鼠中是相似的。对正在增殖的记忆表型的全部成分的分析表明，受体复杂性与非分裂记忆表型细胞的受体复杂性没有差异。 这意味着分裂在很大程度上是随机的，并且可能主要由细胞因子驱动，而不是由肽/MHC复合物驱动，无论是外源性还是内源性的。 记忆表型和真实记忆细胞不仅在增殖率方面存在差异，而且基于 RNA-SEQ 分析，NUR77 的表达也存在差异，后者甚至在启动后 30 天（此时它们已经处于静止状态）在真实记忆细胞上仍高度表达。 努力了解不同的启动方案在诱导特定表型 CD4 T 细胞对肺部免疫反应的作用，揭示了不同“佐剂”的深远影响。 在 LPS 存在的情况下引发会导致 TH17 反应，而在 PolyI:C 存在的情况下引发会产生独有的 IFNγ 反应。 基于对 KO 小鼠的研究，结果表明 LPS 通过激活 Myd88，导致大量 IL-10 产生，阻断 IL-12 产生和 Th1 启动。 同时，它通过激活 TRIF/TRAM，激活 IL-1 的产生，显着增强 Th17 启动。 相比之下，polyI:C 通过 TLR3 和 TRIF 发挥作用，激活 I 型干扰素和 IL-12，增强 IFNgamma 启动并阻断 Th17 诱导。 这些结果为了解 CD4 T 细胞对肺部不同病原体反应的精确结果提供了重要的见解。