中药泰山盘石饮对局部移植物抗宿主反应(GVHR)的影响及其机制的初步研究

先前的工作表明,泰山盘石饮灌入小鼠,可显著延长同种游离心脏组织移植物的存活时间。鉴于移植物抗宿主反应(GVHR)是移植物中免疫活性细胞对组织型不相配的、免疫缺陷的宿主进行攻击而引起的病理过程,不仅其机制与移植排斥反应相似,而且是某些同种移植如骨髓移植、胸腺移植及肝脏移植时的重要并发症,我们就泰山盘石饮对异种局部GVHR的影响进行了实验观察,并对其机制进行了初步研究,结果表明,泰山盘石饮有抑制异种局部GVHR的作用,经泰山盘石饮处理的小鼠脾脏中组胺受体_2阳性细胞明显增加。     

新生小鼠移植耐受的诱导及其机理探讨

本文用去T细胞的同种细胞及杂交一代(F_1)细胞成功地建立了新生小鼠移植耐受模型。通过耐受小鼠脾淋巴结细胞体内过继转移试验及移植物抗宿主反应试验(GVHR),对耐受的机理进行了探讨。实验结果表明,新生期诱导的移植耐受是特异性的,耐受的维持与T抑制细胞(Ts)参与的主动抑制机理有关。     

抗γc单克隆抗体联合供者特异性输注延长移植物存活

目的 探讨抗γ公共链单克隆抗体(抗γc单抗)联合供者脾细胞特异性输注(DST)诱导移植物长期存活的可行性及相关机制.方法 建立小鼠颈部异位心脏移植模型,组Ⅰ为单纯实施心脏移植组,组Ⅱ于移植前给予DST,组Ⅲ术前予DST联合抗γc单克隆抗体处理,术后观察移植物的存活时间及相关指标.结果 组Ⅲ心脏移植物平均存活时间明显长于组Ⅰ及组Ⅱ (P＜0.05).FACS分析显示组Ⅲ受者脾细胞中Tr比例较组Ⅰ及组Ⅱ显著增高(P＜0.05),同时脾细胞增殖活性明显减低(P＜0.05).结论 抗γ公共链单克隆抗体联合DST通过增加供者体内Tr的比例,减低对同种抗原的应答能力,显著延长同种心脏移植模型中移植物存活时间.     

抗激活小鼠T细胞抗原单抗抑制同种移植物排斥反应的作用及其机理

为阐明活化 T 细胞主动免疫同系小鼠诱导同种移植物存活期延长的机理,利用本室制备的抗活化小鼠 T 细胞抗原单克隆抗体(2H_3)观察它对同种免疫反应的影响;结果表明给接受同种移植物的受者转输2H_3能显著地延长同种心肌移植物的存活时间;在 ConA 诱导的淋巴细胞增生反应、混合淋巴细胞反应和诱导同种 T 杀伤性细胞的体外实验中加入2H_3,观察到2H_3对上述反应的明显抑制作用。提示活化 T细胞主动免疫同系小鼠诱导同种移植物存活期延长的机理,可能与免疫动物体内产生了相应的抗体,抑制了同种免疫反应的进行有关。     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅱ.抗独特型T细胞免疫反应

同种特异T细胞疫苗(TCV)免疫诱导出同种免疫反应低下,同种移植物存活时间显著延长,推测其作用机制可能是同种特异TCV免疫诱导机体抗同种特异TCV(独特型)T细胞的上调.实验证实了“抗TCV-T细胞”的存在.以同种特异TCV免疫动物可诱导出对TCV特异的细胞增殖反应.TCV免疫小鼠淋巴细胞能特异杀伤TCV细胞.将TCV免疫小鼠脾细胞作为调节细胞,观察到它能显著地抑制同种MLR,表明它是一“抑制性T细胞”.这些结果提示,同种特异TCV免疫可诱导机体免疫网络中独特型-抗独特型的上调,产生了同种反应T细胞的独特型T细胞,从而保护同种移植物免受同种反应性T细胞的攻击使同种移植物存活时间显著延长.     

α-黑素细胞刺激素基因修饰的树突状细胞延长小鼠移植心脏的存活时间

为观察α-黑素细胞刺激素(α-MSH)基因修饰的树突状细胞(DC)抑制同种异型基因小鼠心脏移植排斥反应的效果,以腺相关病毒(AAV)为载体将α-MSH基因导入BALB/c小鼠骨髓来源的未成熟DC,制备了α-MSH基因修饰的DC(α-MSH-DC)。在小鼠心脏移植前7 d,将α-MSH-DC输至受者C57BL/6小鼠体内,通过免疫组化方法观察供者α-MSH-DC在受者脾脏内存在的情况,用混合淋巴细胞反应(MLR)测定受者脾脏T细胞对供者同种抗原的反应性。通过颈部Cuff法小鼠异位心脏移植模型,观察心脏移植物存活时间,并用ELISA方法测定受者血清细胞因子水平的变化。结果显示,α-MSH-DC在受者脾脏内存在时间延长,能诱导受者脾脏T细胞的抗原特异性低反应性,使移植心脏存活天数延长至(19.3±2.35)d,较PBS对照组的(7.0±0.33)d明显延长(P<0.01)。使受者小鼠血清IL-2和IFNγ-水平显著降低(P<0.01),明显升高IL-4、IL-10水平(P<0.01)。表明移植前输入供者α-MSH基因修饰的DC能延长同种异型基因心脏移植的存活时间。     

抑制NF-κB活性诱导同种异体小鼠心脏移植耐受的早期作用

目的 研究调节性T细胞(Tr)和Th17细胞在特异性NF-κB抑制诱导同种异体小鼠心脏移植耐受的早期作用机制.方法 以BALB/c小鼠为供体,C57BL/6小鼠(对照组)和IκBα△N-Tg小鼠(实验组)分别作为受体建立同种异体腹部异位心脏移植模型;流式细胞术分别检测两组受鼠脾脏内Tr在移植前以及移植术后7 d、30 d和100 d的变化规律,以及Th17细胞在移植术后5 d时的变化;Western blot检测两组心脏移植物内IL-17在移植后3 d和5 d的表达.结果 实验组心脏移植物存活时间均大于100 d,HE染色未见心脏移植物内有明显淋巴细胞浸润;实验组Tr比例在移植后7 d和30 d时明显升高(21.23±3.95,23.17±4.11 vs 11.64±1.96,P0.05),而对照组Tr在移植前后则无明显变化;与对照组相比,实验组Th17细胞及移植物内IL-17表达在移植术后5 d时均明显下降.结论 特异性受体T细胞NF-κB功能缺陷可以打破受体内Tr/Th17细胞平衡,在移植术后早期促进T细胞向Tr分化而抑制向Th17细胞分化,从而阻止急性排斥反应发生,诱导耐受.     

建立预测同种异体间组织相容性细胞模型

目的：建立了预测受、供者间组织相容性信息的体外实验模型，探讨了借助此模型预测的组织相容性与BMT术后GVHR发生时间的相关性及其与小鼠移植皮肤存活时间的相关性。方法：借助所建立的实验模型和细胞模型预测供、受者间组织相容性差异。结果：①显示其与BMT术后GVHR发生时间呈一定相关性，即实验模型检测的受、供者间杀伤活性比值愈大，GVHR发生时间愈晚；②显示其与同种异体小鼠皮肤移植物存活时间呈正相关，即实验模型检测的供、受者间杀伤活性比值愈大，小鼠皮肤移植物存活时间愈长。结论：该模型对选择组织兼容性的移植物供者和判断移植术的预后，提供了实验依据和研究探索。     

甲基转移酶抑制剂延长小鼠心脏移植物存活期的作用研究

探讨甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷调控体内幼稚T细胞转化对同种心脏移植物存活期的影响及其机制。将C57BL/6小鼠分为实验与对照2组,实验组每日腹腔注射5-氮杂-2′-脱氧胞苷(0.25mg/kg体质量),对照组接受等体积的二甲亚砜腹腔注射。然后,提取小鼠脾细胞,抗CD3与抗CD28抗体协同共刺激后,检测CD4+Foxp3+T细胞占CD4+T细胞比例。以BALB/c小鼠为供鼠,两组C57BL/6小鼠为受鼠,建立腹部异位心脏移植模型,监测两组移植心存活时间,检测受鼠脾细胞中CD4+T细胞凋亡率,观察移植心脏组织病理学变化。结果显示,实验组移植前脾细胞中CD4+Foxp3+T细胞占CD4+T细胞比例为(22.7±2.6)%,明显高于对照组的(12.6±1.1)%,差异有统计学意义(P0.05);实验组移植物存活(22.8±2.8)d,明显长于对照组(8±1.8)d,P0.05);实验组移植后脾细胞中CD4+T细胞凋亡率(11.5±1.22)%,高于对照组的(6.6±0.91)%,差异有统计学意义(P0.05)。实验组移植物淋巴细胞浸润数量少于对照组。以上结果提示甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷受体腹腔注射可延长同种移植心脏存活期,其机制可能与调控幼稚T细胞转化为CD4+Foxp3+T细胞,诱导CD4+T细胞凋亡有关。     

茶碱引起哮喘者细胞免疫的改变

20例口服一年以上茶碱的哮喘患者为A组,B组为10例用其它药物治疗的哮喘患者,C组为不用药的健康对照组。A组与B、C两组进行比较研究。发现A组哮喘患者T抑制细胞(Ts)数目增多,A组患者Ts占T细胞总数的37±7％,B组Ts占T细胞总数的19±4％(P<0.01),C组Ts占T细胞总数的26±6％(P<0.01),而T细胞总数无差异。另发现移植物抗宿主反应(GVHR)减弱,A组20例患者中17例GVHR阴性,阴性率达85％,而B、C两组,除一例为阴     

Suppressor, helper and immunoregulatory T cells in normal human blood as defined by theophylline sensitivity

Two functionally distinct subpopulations of human T cells, one T suppressor and the other T-helper lymphocytes, were separated from normal donor human peripheral blood and tested for immunoregulatory properties. The separation of these two populations was performed by the aid of theophylline sensitivity as described by Shore et al. [1]. In order to assess the activity of the suppressor and helper T lymphocytes, a local xenogeneic graft-versus-host reaction (GVHR) according to the method of Shohat et al. [13] was used. These studied demonstrated that the theophylline sensitive (TS) T-suppressor cells have a suppressor effect on normal human T cells. They were further found to consist of two cell subsets, one suppressive and radiosensitive and the other radioresistant and having the ability to induce feedback help, a finding which may explain the inverse relationship found between the quantity of TS cells added and the degree of suppression obtained in the GVHR as well as the enhancement of the GVHR obtained after addition of irradiated TS cells to autologous T cells. The theophylline resistant (TR) T cells were found to have a helper action when added to autologous T cells and were radioresistant. Soluble cell-free factors from both TS and TR cells were found to mimic the function of the cells from which they were extracted.     

Specific immunosuppressive therapy by monoclonal anti-IL 2 receptor antibody and its synergistic action with cyclosporin

The effects of anti-IL 2R mab treatment on local GVHR and on heterotopic cardiac allograft survival in rats were investigated. Anti IL 2 mab inhibited specifically both GVHR as well as allograft rejection. IL 2R-targeted therapy was superior to that of anti-T helper/inducer cell treatment, as it did not affect T subset distribution and did not reduce the number of circulating T lymphocytes. This therapy when combined with a low subtherapeutic dose of CsA exerted a strongly synergistic effect. The results support the concept of IL 2R-targeted therapy in suppressing undesired immune reactions with limited side effects.     

Suppressor cells induced in the human autologous mixed lymphocyte reaction

Theophylline, a phosphodiesterase inhibitor, also reversibly inhibits the SRBC receptor of a subpopulation of human T lymphocytes. We have previously reported that the precursor for the Concanavalin A (Con A) inducible suppressor cell (SC) is found predominantly in the theophylline-sensitive (Ts) population; whereas, the precursor of the mitomycin resistant suppressor cell induced in the allogeneic mixed lymphocyte reaction (MLR) is predominantly in the theophylline-resistant (TR) subset. This study examines the theophylline sensitivity and functional characteristics of suppressor cells induced in autologous MLR (AMLR). Both TS and TR proliferate moderately in AMLR but differ in their ability to form suppressors. AMLR activated Ts and TR cells demonstrate early blastogenesis and weak suppression in the Con A assay system, but suppression in an MLR assay system appeared to be confined largely to the TR subset. AMLR suppressors were mitomycin sensitive. These data illustrate that suppressors generated in allogeneic and autologous MLR have significant functional and phenotypic similarities, and strengthen the association between these two immunologic phenomena.     

Liver nonparenchymal cells involved in hyporesponsiveness induced by portal vein injection of alloantigen

INTRODUCTION: Intrahepatic injection of alloantigen prolongs allograft survival and inhibits T-lymphocyte release of both IL-2 and IFN-gamma but not IL-4. This suggests that intrahepatic processing of antigen lead to a predominance of Th2 cell population with inhibition of Th1 cell type. This study examines the effects of hepatic nonparenchymal cells (NPCs) on T cell function and cytokine mRNA expression profiles. MATERIALS AND METHODS: Following portal vein (p.v.) injection of allogeneic splenic mononuclear cells (SMNC) in mice, heterotopic cardiac allograft survival and donor-specific immune responses were assessed. The cytokine profiles were evaluated in heart grafts and spleens from transplanted mice, or in recipient lymphocytes stimulated in vitro with alloantigen. The immunoregulatory role of NPCs from p.v. injected mice was evaluated. RESULTS: Transplanted mice with prolonged graft survival demonstrated increased IL-4, TGF-beta and IL-10 and/or decreased IFN-gamma and IL-2 mRNA expression within the spleen and the transplanted graft. This correlated with increased antigen-specific IL-4, IL-10 and TGF-beta expression in lymphocytes isolated from the p.v. injected mice. In mixed lymphocyte cultures using NPC from p.v. injected mice as regulatory cells, there was decreased proliferation of lymphocytes from the p.v. injected mice in response to allogeneic stimulation, associated with increased IL-4, TGF-beta and IL-10 production and decreased IFN-gamma and IL-2 production. The regulatory effects of the NPC was reversed by prostaglandin E inhibitor. CONCLUSIONS: Interactions between allogeneic lymphocytes and NPCs results in an impaired Th1 response and preferential shift towards a Th2 cytokine response which may regulate allograft rejection.     

The lack of specificity of the sheep erythrocyte-T lymphocyte rosetting phenomenon.

One subpopulation of lymphocytes, thymus-derived (T) cells, is identified by its characteristic ability to form spontaneous rosettes with sheep erythrocytes (SRBC). However, the mechanism by which the rosettes form remains unknown. To gain more insight into the specificity of this phenomenon, the ability of SRBC to rosette with cells other thah lymphocytes was studied. All of the cell types utilized in this study (L cells, monkey liver cells, HeLa cells, and human liver, bladder, lung, parathyroid, and dermal fibroblasts), except neoplastic B lymphocytes, rosetted with SRBC. Viability was not a factor in the rosette formation. These findings suggest that the process of T lymphocyte-SRBC rosette formation is not due to a T cell-specific membrane receptor or antigen but may be due to a widely distributed basic substructure of the cell membrane.     

Autologous mixed lymphocyte reaction in man. III. Regulation of autologous MLR by theophylline-resistant and -sensitive human T-lymphocyte subpopulations

Human peripheral blood T lymphocytes were separated into theophylline-resistant (TR) and -sensitive (TS) subpopulations. Proliferative responses TR, TS and unfractionated T cells were studied, using irradiated autologous or allogeneic non-T cells as stimulators. TR cells proliferated vigorously in both autologous and allogeneic mixed lymphocyte cultures (MLC). TS cells. which constitute about 20% of unfractionated T cells, exhibited poor proliferative responses to autologous and allogeneic stimulation. The magnitude of proliferation in autologous and in allogeneic MLC was found to be directly dependent on the number of TR cells in the culture. Mitomycin-C (MMC)-treated TR cells augmented and MMC-treated TS cells suppressed (P less than 0.05) the autologous and allogeneic MLC responses of unfractionated T cells. However, the response of TS cells did not increase in autologous or allogeneic MLC when co-cultured with MMC-treated TR cells. MMC-treated TS cells, when co-cultured with TR cells, suppressed the responses of TR cells (P less than 0.05). The enhancing effect of TR cells was radiation-resistant. Suppressor influence of TS cells, in contrast, was abolished by irradiation (P less than 0.05). These findings demonstrate that TR cells are the responding cell in autologous MLR and augment the MLC responses of unfractionated T cells. TS cells, on the other hand, respond poorly in autologous or allogeneic MLC and suppress the response of TR cells.     

A combination of anergic cells' adoptive transfer and rapamycin therapy prolongs cardiac allograft survival in mice

The in vivo immunoregulatory effect of anergic cells induced by blocking the costimulatory pathway was investigated in this study. Anergic cells were generated in vitro by mixed culture of murine splenic cells from BALB/c and C3H/HeJ under the blockade of anti-CD154 and anti-CD80 monoclonal antibodies, and the in vitro activity of anergic cells were observed. The 3.0 Gy gamma-irradiated BALB/c mice received cardic allografts from C3H/HeJ, and anergic cells were intravenously injected immediately after transplantation. Recipient mice injected with anergic cells also received rapamycin therapy (1 mg/kg/day) for 14 days. On day 7 after transplantation, the subsets of peripheral blood T lymphocytes, the pathology of grafts and the infiltration of lymphocytes in grafts were analysed. Untreated gamma-irradiated animals showed a graft median survival time (MST) of 9 days. Animals injected with anergic cells only or receiving rapamycin therapy alone showed MST of 11 and 17 days, respectively. MST of allograft in mice treated with control cells plus rapamycin therapy was 9 days. Animals injected with anergic cells plus rapamycin therapy, but receiving third-party allografts (C57BL/6J), showed an MST of 15 days. However, anergic cell injection plus rapamycin therapy prolonged allograft survival significantly (MST 28 days, P < 0.01). The rejection was mild and tissue architecture was preserved in recipient mice receiving anergic cell injection plus rapamycin therapy. Furthermore, anergic cells and rapamycin therapy decreased the percentage of peripheral blood CD4+ and CD8+ T cells (including CD25+, CD152+, CD154+ and CD28+ subsets) and greatly reduced the infiltrating lymphocytes in allografts (including CD3+, CD4+, CD8+ and CD25+ T cells). In conclusion, the treatment based on anergic cells' adoptive transfer plus rapamycin therapy demonstrated a significant prolongation of murine cardiac allograft survival in a donor antigen-specific manner. This therapeutic protocol alleviated allograft rejection to solid allograft in vivo.     

Spontaneous and alloantigen-induced cytotoxicity by human T-lymphocyte subpopulations

Freshly isolated human T lymphocytes were separated into two subpopulations on the basis of their ability to form E rosettes after treatment with the phosphodiesterase inhibitor, theophylline. T cells that retained the ability to form E rosettes (T-res cells) and those that failed to form E rosettes (T-sens cells) were assayed for natural killer (NK) cell activity against 51Cr-labeled K562 tumor cells and for the ability to proliferate and kill allogeneic cells in mixed-lymphocyte culture (MLC). T-sens cells were highly enriched for NK activity. In contrast, T-res cells exhibited much less activity than either T-sens or unseparated T cells (T-sens greater than unseparated T cells approximately equal to unseparated PBL approximately equal to non-T cells greater than T-res cells). T-sens cells were poorly responsive to allogeneic cells in proliferation assays and demonstrated greater levels of cytotoxicity against allogeneic cells than T-res cells. T cells stimulated with allogeneic lymphocytes for 7 days were cytotoxic for K562 targets while comparably stimulated non-T cells and T cells cultured with medium were not cytotoxic. Cold target inhibition experiments suggested that within the T-sens subset there are overlapping populations which mediate cytotoxicity against K562 and allogeneic cells. These studies demonstrate that freshly isolated human T cells are composed of heterogeneous populations which differ in their ability to mediate NK and to generate cytotoxic T lymphocytes in culture.     

Lack of suppressor T cells in renal transplant recipients and activation by aminophylline

Immunoregulatory T-cell subsets were determined in 16 patients after renal transplantation. Suppressor and helper T lymphocytes were isolated with the aid of theophylline according to the method described by Shore et al. [1]. The system model used for assessing the function of these subsets was the local xenogeneic graft-versus-host reaction (GVHR). Lack of suppressor T cells was found in 6 out of the 16 patients, all 6 in acute steroid nonresponsive rejection crisis. Four of these patients received aminophylline per os at a dose of 1000 mg/kg per day and the T-lymphocyte subsets were retested several days afterwards. Active suppressor T cells (TS) appeared in all 4 treated patients, paralleled by disappearance of rejection crisis and a dramatic decrease of serum creatinine levels. These findings suggest that the inactivation of the immunoregulatory suppressor subset, probably by the immunosuppressive treatment, may play an important role in acute rejection and that activation of this subset of T suppressors could be of beneficial effect and prevent renal rejection.     

Effects of in-vivo administration of a monoclonal antibody specific for the interleukin-2 receptor on the acute graft-versus-host reaction in mice.

Parental strain T lymphocyte injected into F1 mice respond to allogeneic MHC antigens and so induce the symptoms of a graft-versus-host reaction (GVHR). We have measured the local GVHR by the popliteal lymph node assay, and showed the suppression of the local GVHR in mice by treatment with the monoclonal antibody (MoAb) AMT-13 which is specific against the interleukin 2 (IL-2) receptor on activated mouse lymphocytes. The inhibitory effect of the AMT-13 administration was comparable with the suppression of the local GVHR by treatment with L3T4, an MoAb directed against the T helper subset. The L3T4 administration caused a dramatic decrease in the proportion of the cells with the L3T4 phenotype in the circulation and a marginal reduction of these cells in the lymph nodes. In contrast, the AMT-13 treated mice showed no changes in the distribution of the T lymphocyte subsets besides those in the GVHR-stimulated lymph nodes. Obviously, only the small subset of antigen-activated IL-2 receptor-bearing lymphocytes was influenced by treatment with AMT-13. MoAb directed against antigens whose expression is restricted to activated lymphocytes, such as the IL-2 receptor, might become useful for a short term immunosuppression with limited side effects.