脐带间充质干细胞体外对脐带血CD4+ T 淋巴细胞免疫调节作用的研究

目的:研究脐带间充质干细胞(UCMSCs)在体外对异源脐血CD4+ T 淋巴细胞增殖、凋亡及向CD4+ CD25+ 调节性T 细胞(Treg)分化的免疫调控作用。方法:建立不同比例UCMSCs 与植物血凝素(PHA)活化的脐血CD4+ T 淋巴细胞共培养,或与Transwell 共培养体系,检测CD4+ T 淋巴细胞增殖情况、CD4+ CD25+ / CD4+ 比率的变化及调节性T 淋巴细胞标志基因Foxp3 相对表达量的变化。同时,建立UCMSCs 与地塞米松(DXM)刺激的脐血CD4+ T 淋巴细胞共培养,或与Transwell 共培养体系,检测CD4+ T 淋巴细胞凋亡情况。结果:PHA 活化的脐血CD4+ T 淋巴细胞在与UCMSCs 共培养3 d 后,较无UCMSCs对照组相比,细胞数量明显减少(P<0. 05)且CD4+ CD25+ / CD4+ T 淋巴细胞的比率及Foxp3 的相对表达量显著增加(P<0. 01),并随UCMSCs 数量增加,作用增强(P<0. 05)。DXM 诱导的脐血CD4+ T 淋巴细胞与UCMSCs 共培养7 d 后,细胞凋亡比率较无UCMSCs 对照组显著降低(P<0. 01)。上述效应在Transwell 共培养中部分减弱但无法消除。结论:UCMSCs 对脐血CD4+ T细胞介导的免疫反应具有负调节作用,其作用主要表现在对细胞增殖能力和分化的调控而不是促进凋亡。     

RIA法测定白细胞介素-2在肺部疾病中的临床应用

白细胞介素-2(IL-2)是活化的辅助性T淋巴细胞(TH)产生的一种可溶性介质(或称淋巴因子).它通过作用于能表达膜IL-2受体(MIL-2R)的免疫细胞发挥免疫调节作用,促进T细胞的增殖[1],并能维持T细胞在体外的生长.     

JM细胞培养上清对人B淋巴细胞和小鼠脾细胞增殖的免疫调节作用

JM 是来自人胸腺不成熟 T 细胞的急性淋巴细胞白血病细胞系.本文以 SAC 刺激的人外周血纯化 B 淋巴细胞和小鼠脾细胞增殖为模型,观察了 JM 细胞培养上清(SPN_(JM))对人 B 淋巴细胞和小鼠脾细胞增殖的免疫调节作用.发现具有 T 细胞抑制活性的 SPN_(JM)对人和小鼠 B 淋巴细胞的增殖具有促进作用。在无 SAC 诱导时,SPN_(JM)与 HrIL—2协同对 B 细胞仍有促增殖作用.本实验还发现,高浓度时对 T 细胞具有抑制作用的 SPN_(JM),在低浓度时(1:640)对 T 细胞增殖亦具有促进作用.     

大肠癌浸润淋巴细胞的免疫活性及表型特征

为明确大肠癌患者的细胞免疫活性状态及大肠癌肿瘤浸润淋巴细胞(TIL)活性,分析比较了 TIL 区域淋巴结淋巴细胞(RNL)、外周血淋巴细胞(PBL)对 rIL—2刺激的增殖反应、表型特征、细胞毒活性及 IL—2产生能力.TIL 在 rIL—2培养早期呈现延迟的增殖反应和较高的总扩增能力.流式细胞仪分析表明肿瘤局部 T 细胞为主,各组淋巴细胞 T 亚群中 CD4~ 细胞减少,CD8~ 增加,Tac 抗原表达增加.乳酸脱氢酶(LDH)释放法检测结果显示大肠癌患者淋巴细胞 NK 活性、ADCC 普遍降低,同位素掺入法显示 IL—2产生能力亦降低.IL—2培养后淋巴细胞的 NK 活性、ADCC 提高,并获得得 LAK 活性.     

HBsAg抗体增强人T淋巴细胞对HBsAg的免疫应答

T淋巴细胞对多数抗原的免疫应答的调节起着十分重要的作用.抗原提呈细胞表面存在着主要组织相容性基因复合体编码的分子(Ia或DR),T细胞只识别与这些分子相关连的抗原.T细胞受抗原刺激后,增殖并分泌一些因子,这些因子增强或抑制免疫效应分子和细胞(如抗体和细胞毒性T淋巴细胞)的产生.和大多数感染性疾病一样.对乙型肝炎病毒(HBV)的抵抗主要是靠保护性抗体和免疫效应细胞.急性乙肝病毒感染后恢复的机体,由于产生了抗病毒外膜分子即HBsAg的抗体,便获得了对HBV的免疫性.事实上,使用灭活并纯化的HBsAg制剂,已     

红景天苷对小鼠原代T淋巴细胞体外行为的影响

目的:分析红景天苷(Sal)对小鼠原代T淋巴细胞体外行为的影响。方法:无菌分离并培养BALB/c近交系小鼠淋巴结细胞,MTT比色法检测并分析Sal对小鼠T淋巴细胞活力的影响;荧光染料标记的单克隆抗体染色结合流式细胞术(FCM)检测刀豆蛋白A(Con A)诱导作用下Sal对T淋巴细胞活化抗原CD69表达水平的影响。羧基二乙酸荧光素琥珀酰亚胺酯(CFDA-SE)染色分析T淋巴细胞体外增殖情况。2',7'-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光染色检测小鼠T淋巴细胞胞内活性氧簇(ROS)生成。Di OC6(3)染色检测Sal对T淋巴细胞胞内线粒体活性的影响以及地塞米松(DEX)诱导作用下Sal对T淋巴细胞线粒体膜电位的影响。制备小鼠胸腺T淋巴细胞悬液,FCM检测DEX诱导作用下胸腺T淋巴细胞的凋亡率。结果:终浓度为80、160和320μmol/L的Sal均能提高T淋巴细胞活化抗原CD69的表达水平(P0.05)。Sal能促进Con A诱导72 h条件下T淋巴细胞体外增殖(P0.01),且能减少T淋巴细胞胞内ROS生成量(P0.05),对T淋巴细胞线粒体膜电位有保护作用(P0.01)。Sal能明显降低DEX诱导条件下的胸腺T淋巴细胞凋亡率(P0.01)。结论:Sal能够促进T淋巴细胞的体外活化和增殖,减少T淋巴细胞内ROS的产生,维持细胞内线粒体膜电位稳定性,抑制DEX诱导条件下胸腺T淋巴细胞的凋亡。     

SEB活化的人外周血T细胞CD25,CD69的表达

本文用超抗原葡萄球菌肠毒素B(SEB)诱导外周血淋巴细胞增殖。结果显示出微量超抗原能诱导外周血淋巴细胞的增殖,大量的增殖反应发生在SEB刺激后的第5天,增殖的细胞是CD4~+T细胞,它们由刺激前的27％增加到42％。外周血活化的T细胞不依赖外源性IL-2:大量内源性IL-2的存在抑制T细胞的增殖反应。伴随CD4~+T细胞的增殖,CD25和CD69分子表达明显增加。提示SEB能改变外周血T细胞表面的分子表达。     

间充质干细胞对混合淋巴细胞反应体系中T淋巴细胞的免疫调节作用

目的:探讨间充质干细胞(MSC)在体外对T淋巴细胞免疫调节作用的特点。方法:通过Ficoll梯度密度离心法分离出正常人骨髓单个核细胞,体外培养扩增MSC,获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养(MLC)体系中,在第3、5天,采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况,再用流式细胞术分析其与MSC共孵育前后细胞表面标记的变化情况。结果:加入了MSC的MLC体系中,MSC对T淋巴细胞的增殖抑制具有剂量依赖性,且随着时间延长,抑制程度增强;其中,CD4 T细胞亚群受抑不如CD8 T细胞亚群显著;另外,T淋巴细胞表面CD25的表达虽然较对照组有所下降,但是CD4、CD25共表达的细胞却较对照组有明显的上升趋势。T淋巴细胞表面活化抗原HLA-DR的表达较对照组有轻度减低。结论:MSC在体外能够明显抑制T淋巴细胞的增殖,其主要是针对CD8 T细胞(CTL)。另外,它还能下调活化T淋巴细胞上一些较特异的表面标记CD25和HLA-DR的表达。     

重组白介素—4促进外周血淋巴细胞增殖和抗辐射的作用

目的 研究重组人白介素－4(rhIL-4)对正常人外周血淋巴细胞亚群的促增殖和对离体照射淋巴细胞的保护作用。方法 用MTT、碱性磷酸酶免疫组化和TUNEL方法,检测淋巴细胞的增殖,T、B淋巴细胞亚群和淋巴细胞凋亡。结果（1）在适当浓度PWM存在下,一定浓度的rhIL-4能明显促进正常人外周血淋巴细胞增殖,其中以无血清体系和培养后24h的作用较强;（2）rhIL－4的促增殖作用主要表现在,促进TH、Ts细胞亚群和B细胞的增殖;（3）一定浓度的rhIL-4能明显抑制6Gy γ-线照射引起的淋巴细胞总数、TH,Ts细胞亚群和B细胞数量的减少;（4）rhIL-4能明显抑制急性照射引起的大量淋巴细胞凋亡,可能是减轻淋巴细胞辐射损伤和改善机体免疫功能的重要原因。结论rhIL-4能使正常人外周血T、B淋巴细胞数量增加,对致死剂量照射引起的淋巴细胞减少也具有明显的抑制作用。     

慢性HBV感染者调节性T细胞、黏附细胞对细胞毒T淋巴细胞体外增殖的影响

乙型肝炎患者体内细胞毒T淋巴细胞(cytotoxic T lymphocytes,CTL)的功能和数量直接影响着病毒的清除和肝细胞的损伤.本实验主要探讨慢性HBV感染者调节性T细胞(Tr)、黏附细胞(AC)对HLA-A2限制的HBV特异性CTL体外增殖的影响.     

Homeostatic proliferation of naïve CD8+ T cells depends on CD62L/L‐selectin‐mediated homing to peripheral LN

Adoptive transfer of naïve CD8⁺ T cells into lymphopenic recipients results both in spontaneous proliferation and in partial activation of T cells, a phenomenon termed homeostatic proliferation (HP). HP of CD8⁺ T cells is dependent on host IL‐7, IL‐15, and MHC‐class I and has been shown to prevent T‐cell tolerance, reverse T‐cell anergy and support T‐cell‐mediated tumor control in vivo. However, the initial anatomic site of HP is still under debate. Since we observed that the earliest detectable HP occurs within LN and that T cells undergoing HP retain a CD62L^bright phenotype, we investigated the functional role of CD62L for this process. We found that CD62L‐expression on T cells is required for optimal HP and HP was impaired in lymphotoxin‐αβ^?/? mice, indicating the necessity for intact host secondary lymphoid organ structures. Use of the LN egression inhibitor FTY720 indicated that LN structures were pivotal to yield homeostatically proliferated T cells detected in other compartments. Consistent with these results, HP‐supported control of MC57‐SIY tumors depended on CD62L. Our data indicate a critical role for CD62L and LN homing for the process of HP, which has implications for adoptive immunotherapy approaches of cancer.     

IL-7 Is the Limiting Homeostatic Factor that Constrains Homeostatic Proliferation of CD8+ T Cells after Allogeneic Stem Cell Transplantation and Graft-versus-Host Disease

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4⁺ T cell HP during GVHD. However, it is not clear whether these alterations to the peripheral CD4⁺ T cell niche also contribute to impair CD8⁺ T cell regeneration during GVHD. We found that IL-7 therapy was sufficient for restoring CD8⁺ T cell HP in GVHD hosts while forcing DC regeneration with Flt3-L had only a modest effect on CD8⁺ T cell HP in IL-7 treated mice. Using bone marrow chimeras, we showed that HP of naïve CD8⁺ T cells is primarily regulated by MHC class I on radio-resistant stromal cells, yet optimal recovery of CD8⁺ T cell counts still requires expression of MHC class I on both radio-resistant and radio-sensitive hematopoietic cells. Thus, IL-7 level is the primary limiting factor that constrains naïve CD8⁺ T cell HP during GVHD, and accessibility of MHC class I on stromal cells explains how IL-7 therapy, as a single agent, can induce robust CD8 ⁺ T cell HP in the absence of DCs.     

T cell immune reconstitution following lymphodepletion

T cell reconstitution following lymphopenia from chemotherapy or stem cell transplant is often slow and incompetent, contributing to the development of infectious diseases, relapse, and graft-versus-host disease. This is due to the fact that de novo T cell production is impaired following cytoreductive regimens. T cells can be generated from two pathways: (1) thymus derived through active thymopoiesis and (2) peripherally expanded clones through homeostatic proliferation. During recovery from lymphopenia, the thymic pathway is commonly compromised in adults and T cells rely upon peripheral expansion to restore T cell numbers. This homeostatic proliferation exploits the high cytokine levels following lymphopenia to rapidly generate T cells in the periphery. Moreover, this early peripheral expansion of T cells can also be driven by exogenous antigen. This results in loss of T cell repertoire diversity and may predispose to auto- or allo-immunity. Alternatively, the high homeostatic proliferation following lymphopenia may facilitate expansion of anti-tumor immunity. Murine and human studies have provided insight into the cytokine and cellular regulators of these two pathways of T cell generation and the disparate portraits of T cell immunity created through robust thymopoiesis or peripheral expansion following lymphopenia. This insight has permitted the manipulation of the immune system to maximize anti-tumor immunity through lymphopenia and led to an appreciation of mechanisms that underlie graft versus host disease.     

Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8+ T cells induced by TCR triggering or homeostatic cytokines

Production of indoleamine 2,3-dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L-kynurenine (L-kyn) and 3-hydroxyanthranilic acid (3-HAA) on survival and antigen-dependent and independent proliferation of CD8(+) cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8(+) T cells. Non toxic concentrations of L-kyn or 3-HAA did not significantly inhibit antigen-specific CTL responses. However, 3-HAA, but not L-kyn markedly inhibited antigen-independent proliferation of CD8(+) T cells induced by common receptor gamma-chain cytokines IL-2, -7 and -15. Our data suggest that CD8(+) T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3-HAA profoundly affects homeostatic proliferation of CD8(+) T cells.     

CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8+ T cells in a manner dependent on IL-4

CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide (alpha-GalCer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that alpha-GalCer enhanced homeostatic proliferation of CD8+ but not CD4+ T cells and this effect of alpha-GalCer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8+ T cell homeostatic proliferation. Importantly, the expression of IL-4Ralpha and STAT6 in CD8+ T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8+ T cells. Consistent with this, the level of IL-4Ralpha expression on memory phenotype CD8(+) T cells was higher than that on naive phenotype one and CD4+ T cells. Thus, these results showed the 'involvement' of IL-4 that is produced from activated NKT cells for CD8+ T cell homeostatic proliferation in vivo.     

IL-2-targeted therapy ameliorates the severity of graft-versus-host disease: ex vivo selective depletion of host-reactive T cells and in vivo therapy

T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.     

T cell homeostasis in tolerance and immunity

The size of the peripheral T cell pool is remarkably stable throughout life, reflecting precise regulation of cellular survival, proliferation, and apoptosis. Homeostatic proliferation refers to the process by which T cells spontaneously proliferate in a lymphopenic host. The critical signals driving this expansion are "space," contact with self-major histocompatibility complex (MHC)/peptide complexes, and cytokine stimulation. A number of studies have delineated an association between T cell lymphopenia, compensatory homeostatic expansion, and the development of diverse autoimmune syndromes. In the nonobese diabetic mouse model of type 1 diabetes, lymphopenia-induced homeostatic expansion fuels the generation of islet-specific T cells. Excess interleukin-21 facilitates T cell cycling but limited survival, resulting in recurrent stimulation of T cells specific for self-peptide/MHC complexes. Indeed, data from several experimental models of autoimmunity indicate that a full T cell compartment restrains homeostatic expansion of self-reactive cells that could otherwise dominate the repertoire. This review describes the mechanisms that govern T cell homeostatic expansion and outlines the evidence that lymphopenia presents a risk for development of autoimmune disease.     

The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells

Antigen-specific memory T cells are a critical component of protective immunity because of their increased frequency and enhanced reactivity after restimulation. However, it is unclear whether 'memory-like' T cells generated during lymphopenia-induced homeostatic proliferation can also offer protection against pathogens. Here we show that homeostatic proliferation-induced memory (HP-memory) CD8(+) T cells controlled bacterial infection as effectively as 'true' memory CD8(+) T cells, but their protective capacity required the presence of CD4(+) T cells during homeostatic proliferation. The necessity for CD4 help was overcome, however, if the HP-memory CD8(+) T cells lacked expression of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand; also called Apo-2L). Thus, like conventional CD8(+) memory T cells, the protective function of HP-memory CD8(+) T cells shows dependence on CD4(+) T cell help.     

Altered naive CD4 and CD8 T cell homeostasis in patients with relapsing-remitting multiple sclerosis: thymic versus peripheral (non-thymic) mechanisms

We have reported previously that naive T cells from relapsing-remitting multiple sclerosis (RRMS) patients have T cell receptor (TCR) repertoire shifts, but the basis of these TCR repertoire shifts was uncertain. Here, we questioned whether RRMS patients have altered naive CD4 and CD8 T cell homeostasis by studying homeostatic proliferation and thymic production in RRMS patients and healthy controls. We measured thymic production by quantifying signal joint T cell receptor excision circles (sjTRECs). Both naive T subsets from controls showed an age-associated decrease in sjTRECs, i.e. evidence of progressive thymic involution, but we detected no age-associated decrease in sjTRECs in RRMS patients. Instead, naive CD8 T cells from patients had lower sjTRECs (P = 0.012) and higher Ki-67 proliferation levels (P = 0.04) than controls. Naive CD4 T cell sjTRECs did not differ between patients and controls. However, in RRMS these sjTRECs correlated strongly with CD31, a marker expressed by newly generated CD4 T cells but not by naive CD4 T cells that have undergone homeostatic proliferation. HLA-DR2 positivity correlated negatively with naive CD4 T cell CD31 expression in RRMS (P = 0.002). We conclude in RRMS that naive T subsets have homeostatic abnormalities due probably to peripheral (non-thymic) mechanisms. These abnormalities could have relevance for MS pathogenesis, as naive T cell changes may precede MS onset.