Transplantation tolerance: gene expression profiles comparing allotolerance vs. allorejection

An understanding of the molecular basis of immune regulation will allow development of therapies for diseases caused by immune dysregulation and for therapeutic exploitation of the immune response in transplantation of organ grafts or stem cells. To identify critical regulatory factors in immunity, we have used a mouse model wherein infectious regulatory tolerance is inducible by CD4/CD8 blockade in recipients of vascularised heart grafts. Once established, this transplantation tolerance is robust and isolated "tolerant" spleen cells show powerful immune regulatory properties, being able to impose donor-specific allotolerance upon fully immune competent naive recipients. Here, we present a compound comparison of four gene arrays (tolerance vs. rejection, at 48 h, and at 123 h) where a relatively small number of differentially expressed genes occurred. In rejection, there was a strong progressive amplification of IFNgamma and granzyme B mRNAs. In tolerance, both ELKL motif kinase and axotrophin occurred in the group of upregulated genes. Mice lacking ELKL motif kinase develop autoimmune disease, whilst axotrophin is a newly discovered stem cell gene that has only been explored in the context of neural development. This gene expression data is the first to demonstrate a link between axotrophin and regulatory tolerance and, since axotrophin, LIF, STAT3 and c-kit each function in stem cells, we propose that common mechanisms play a central role both in developmental regulation of stem cells, and in immune regulation.     

CD8 blockade promotes the expansion of antigen-specific CD4+ FOXP3+ regulatory T cells in vivo

Treatment with a cocktail of CD4 and CD8-specific monoclonal antibodies (mAb) induces long-term transplantation tolerance and regulatory CD4(+) T cells that induce tolerance in non-tolerant T cells. In contrast, treatment with a CD4-specific mAb alone fails to induce long-term tolerance. The current study was designed to test the hypothesis that CD8 blockade plays a role in promoting the development of CD4(+) regulatory T cells. Using the DO11.10 CD4(+) TCR transgenic mouse model we show that treatment with a CD4/CD8-specific mAb cocktail induces antigen-specific tolerance to OVA, measured by a significant decrease in OVA-specific IgG, on challenge with antigen. Although treatment with OVA and the CD4-specific mAb alone also induces a significant decrease in OVA-specific antibody, the number of DO11.10 cells is significantly greater in mice treated with the CD4/CD8-specific mAb cocktail, and this is associated with a significant increase in proliferation of DO11.10 cells in response to specific antigen. DO11.10 cells sorted from mice made tolerant to OVA with the CD4/CD8-specific mAb cocktail promote an OVA-specific IgG1 (Th2-type) response but not an OVA-specific IgG3 (Th1-type) response on transfer into new syngeneic recipients, suggesting their ability to regulate the type of antigen-specific immune response that ensues after priming with antigen. In addition, DO11.10 cells from tolerant mice express markers that are characteristic of CD4(+) regulatory cells, including FOXP3, GITR and CTLA4, but not CD25. Taken as a whole, these data suggest that CD8 blockade promotes CD4(+) FOXP3(+) regulatory CD4(+) T cells by promoting their proliferation in tolerant mice.     

Prevention of graft-versus-host disease by a novel immunosuppressant, (5R)-5-hydroxytriptolide (LLDT-8), through expansion of regulatory T cells

(5R)-5-hydroxytriptolide (LLDT-8) is a new compound derived from triptolide, which is the major immunosuppressive fraction of Tripterygium wilfordii Hook. F (TWHF). In this study, we demonstrated that administration of LLDT-8 (1 mg/kg/day, p.o.) effectively prevented weight loss and death induced by allo-BMT (BLAB/c, H-2d to C57BL/6, H-2b), and extended survival in allo-BMT model of aGVHD. Following days 7 to 28 after allo-BMT, the allogeneic graft survived by increasing the number of engrafted cells (H-2d) in spleens of recipient mice with LLDT-8 treatment. To construe the immunosuppressive effects of LLDT-8, the splenocytes (H-2d) of LLDT-8 treated recipients (H-2b) were tested for the proliferative responses to donor antigen (H-2d), host antigen (H-2b) and mitogen (ConA) stimulations, respectively, the results indicated that LLDT-8 induced the T cells' unresponsiveness to donor and host antigens, while still maintaining response to ConA; Compared with the vehicle group of GVHD mice, administration of LLDT-8 significantly inhibited T cells to produce IFN-gamma with or without host antigen or ConA stimulation. Further studies indicated LLDT-8 had a normalizing effect on the ratio of CD4+/CD8+ T cells, and increased CD4+CD25+ T regulatory cells with the Foxp3 expression in splenocytes from LLDT-8 treated mice. The results outline the great potential of LLDT-8 as a therapeutic tool to induce suppression in GVHD.     

大鼠肝移植自然耐受模型移植术后Foxp3基因表达的变化

目的 探讨大鼠肝移植自然耐受模型移植术后Foxp3基因表达的变化及意义.方法 分别建立大鼠急性排斥(DA→LEW)、自然耐受(LEW→DA)、同基因组(DA→DA)肝移植模型,收集术后7、14、28d外周血、脾脏、肝脏标本,FCM检测标本淋巴细胞中CD4 CD25 Tr (regulatory T cell)比例变化,RT-PCR, Western blot分别检测肝脏内Foxp3基因及其scrufin蛋白的表达.结果 术后7d,各组外周血、脾脏、肝脏中CD4 CD25 Tr细胞比例无明显差异.术后14d自然耐受组脾脏、肝脏内CD4 CD25 Tr细胞比例明显高于同基因组(P<0.05),外周血中差异无统计学意义(P>0.05).术后28d自然耐受组与同基因组间无差异(P>0.05).术后7d,自然耐受组移植物Foxp3 mRNA水平明显高于排斥组和同基因组(P<0.05);术后14d,自然耐受组移植物Foxp3 mRNA水平明显高于同基因组(P<0.05);术后28d,自然耐受组与同基因组移植物Foxp3 mRNA表达无差异(P>0.05).自然耐受组与同基因术后14、28d scrufin蛋白条带明显强于7d,且自然耐受组表达强于同基因组.结论 大鼠肝移植术后1～2周内,移植肝内Foxp3基因表达可诱导机体对移植物产生免疫耐受.     

系统性红斑狼疮患者外周血CD4~+调节性T细胞CD25~+和FOXP3~+的表达及意义

目的探讨系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)中CD4+T细胞CD25和FOXP3的表达及其临床意义。方法流式细胞术检测20例SLE患者(9例为狼疮性肾炎)及10例健康对照组PBMC中CD4+T细胞中CD25及Foxp3的表达。结果 SLE患者CD4+CD25highT细胞中FOXP3+T细胞的表达明显低于健康对照组(P<0.05),与SLE疾病活动指数SLEDAI积分明显负相关(r=-0.514,P<0.01),与补体C4正相关(r=0.362,P<0.05),与ESR及CRP负相关(r分别=-0.23、-0.216,P<0.05),与外周血免疫球蛋白IgG、IgA、IgM及补体C3水平无明显相关性。将SLE患者分为狼疮性肾炎(LN)组及非肾损(SLE-NR)组,两组CD4+CD25highT细胞及CD4+CD25lowT细胞中FOXP3+T细胞的表达均明显低于健康对照组(P<0.05);LN组CD4+T细胞中CD25high及CD25low的表达均较SLE-NR组增高(P<0.05)。结论 SLE患者外周血中CD4+CD25high T细胞中FOXP3+的表达水平明显降低,且与疾病活动性相关,而LN患者代偿增高的无FOXP3表达的CD4+CD25+T细胞可能与其发病机制有关。     

CD4+CD25+Foxp3+调节性T细胞与慢性乙型肝炎病毒感染的关系

目的 探讨调节性T细胞（Treg）在慢性乙型肝炎病毒感染中的免疫调节作用。方法 采用流式细胞术检测慢性活动性肝炎患者、乙型肝炎病毒携带者、乙型肝炎恢复者及健康对照者外周血CIN^＋T细胞中的CIN^＋CD25^＋Foxp3^＋调节性T细胞的比例。采用免疫组织化学法检测肝组织中Foxp3的表达。统计分析外周血中CIN^＋CD25^＋Foxp3^＋调节性T细胞的比例数与血清HBeAg和HBVDNA含量的关系。结果 外周血CIN^＋T细胞中CIN^＋CD25^＋Foxp3^＋调节性T细胞的比例数在慢性活动型肝炎组和乙型肝炎病毒携带组均高于健康对照组（P〈0．05），乙型肝炎病毒携带者组高于乙型肝炎恢复组（P〈0．05）。Foxp3在慢性活动型肝炎组肝组织中的阳性率和阳性细胞数均高于乙型肝炎病毒携带者组（P〈0．05）。外周血HBeAg（＋）患者Treg的比例数高于血清HBeAg（-）患者Treg的比例数（t=1．67．P〈0．05）。患者外周血CIN^＋CD25^＋Foxp3^＋Treg的比例数与血清HBV DNA含量呈正相关（r=0．56，P〈0．01）。结论 CIN^＋CD25^＋Foxp3^＋调节性T细胞可能与乙型肝炎病毒感染慢性化及乙型肝炎病毒的清除有关。     

外源性LTB4对CIA小鼠Treg/Th17脾细胞分化的作用

目的探讨外源性白三烯B4(LTB4)对胶原诱导型关节炎(collagen-induced arthritis,CIA)小鼠脾细胞调节性T细胞(Treg)和Th17细胞分化的调节,进一步阐明LTB4在类风湿关节炎(RA)发病中的作用机制。方法建立CIA小鼠模型,取造模d28的脾细胞,体外实验分析外源性LTB4对Treg和Th17细胞分化的影响。应用流式细胞术检测CD4+CD25+Foxp3+细胞的数量,荧光定量PCR技术检测调控Treg和Th17细胞分化的特异性转录因子Foxp3和RORγt的mRNA的表达,酶联免疫吸附(ELISA)方法检测培养细胞上清IL-17的含量。结果成功建立CIA小鼠模型;造模d28分离小鼠脾细胞,体外培养加入鸡Ⅱ型胶原(CⅡ)共同孵育,随着LTB4浓度增加(0.01、0.1、1μmol·L-1),CD4+CD25+Foxp3+细胞数量相应减少,Foxp3 mRNA的表达相应降低;相反,IL-17的含量相应增加,RORγt mRNA的表达相应升高。结论LTB4抑制CIA模型脾细胞Treg细胞的分化,促进Th17细胞的分化,提示LTB4在CIA发病过程中具有一定的促炎活性。     

Unexpected modulation of recall B and T cell responses after immunization with rotavirus-like particles in the presence of LT-R192G

LT-R192G, a mutant of the thermolabile enterotoxin of E. coli, is a potent adjuvant of immunization. Immune responses are generally analyzed at the end of protocols including at least 2 administrations, but rarely after a prime. To investigate this point, we compared B and T cell responses in mice after one and two intrarectal immunizations with 2/6 rotavirus-like particles (2/6-VLP) and LT-R192G. After a boost, we found, an unexpected lower B cell expansion measured by flow cytometry, despite a secondary antibody response. We then analyzed CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) and CD4(+)CD25(+)Foxp3(-) helper T cells after in vitro (re)stimulation of mesenteric lymph node cells with the antigen (2/6-VLP), the adjuvant (LT-R192G) or both. 2/6-VLP did not activate CD4(+)CD25(+)Foxp3(-) nor Foxp3(+) T cells from non-immunized and 2/6-VLP immunized mice, whereas they did activate both subsets from mice immunized with 2/6-VLP in the presence of adjuvant. LT-R192G dramatically decreased CD4(+)CD25(+)Foxp3(+) T cells from non-immunized and 2/6-VLP immunized mice but not from mice immunized with 2/6-VLP and adjuvant. Moreover, in this case, LT-R192G increased Foxp3 expression on CD4(+)CD25(+)Foxp3(+) cells, suggesting specific Treg activation during the recall. Finally, when both 2/6-VLP and LT-R192G were used for restimulation, LT-R192G clearly suppressed both 2/6-VLP-specific CD4(+)CD25(+)Foxp3(-) and Foxp3(+) T cells. All together, these results suggest that LT-R192G exerts different effects on CD4(+)CD25(+)Foxp3(+) T cells, depending on a first or a second contact. The unexpected immunomodulation observed during the recall should be considered in designing vaccination protocols.     

Dynamic Frequency of Blood CD4+CD25+ Regulatory T Cells in Rats with Collagen-induced Arthritis

CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the frequency of CD4+CD25+ Tregs in peripheral blood (PB) of collagen-induced arthritis (CIA) rats during the development of arthritis, to determine whether their frequency is involved in the immunoregulation of this disease. The results showed that normal rats had similar frequencies of CD4+CD25+ Tregs in PB during the experiment time, expressed as a percentage of CD4+CD25+Foxp3+ T cells among the CD4+ T lymphocyte population. In contrast, the frequency of CD4+CD25+Foxp3+ T cells in CIA rats was found to change during the development of arthritis. In CIA rats, there is a significant negative correlation between the frequency of CD4+CD25+Foxp3+ T cells and paw swelling (r=-0.786, p< 0.01). The relationship between the frequency of CD4+CD25+Foxp3+ T and immune activation was not found in normal rats. During the time course, the frequency of CD4+CD25+Foxp3+ T was lower in CIA rats than in normal ones. The data suggest that the frequency of PB CD4+CD25+ Tregs may be a promising marker for arthritis activity.     

CD8+CD103+ regulatory T cells in spontaneous tolerance of liver allografts

It has been shown that rat liver allografts between certain inbred major histocompatibility complex (MHC) disparate strains are accepted spontaneously, and regulatory T cells (Tregs) have been suggested to play a role in the spontaneous liver tolerance. CD8⁺CD103⁺ T cells bear the phenotypes of effector cells, and they are implicated in allograft destruction. Here we provide evidence that CD8⁺CD103⁺ T cells possess regulatory function and may contribute to prevent liver allograft rejection. We show that the expression of CD103 in the CD8⁺ T cells was increased in spontaneous liver grafts tolerant recipients. We further show that CD8⁺CD103⁻ T cells can also upregulate the expression of CD103 and Foxp3 after stimulation with alloantigen or TGF-β in vitro, and the CD8⁺CD103⁺ T cells acquired regulatory properties. The suppressive function of the alloantigen or TGF-β conditioned CD8⁺CD103⁺ T cells was cell–cell contact dependent. These results imply that liver-specific factor(s) would be involved in the generation of CD8⁺CD103⁺ Tregs that contribute to spontaneous liver allografts tolerance.     

The effects of Foxp3-expressing regulatory T cells expanded with CD28 superagonist antibody in DSS-induced mice colitis

Regulatory T (Treg) cells play an important role in the pathogenesis of inflammatory bowel disease (IBD). In the present study, we found that a superagonistic CD28-specific monoclonal antibody (supCD28mAb, D665) could preferentially stimulate expansion of CD4+Foxp3+ Treg cells. Foxp3(EGFP) mice were orally administrated with 3.5% DSS for 5days, and intraperitoneally injected supCD28mAb 1mg/mice in treated group. All of the mice were sacrificed on day 8, and both clinical and histological parameters showed that the severity of colitis was significantly reduced in treated group compared to controls. In treated group, the proportion of CD103, CD152 and CD62L expression on Foxp3+Treg cells in the spleen and mesenteric lymph node were higher than controls. Furthermore, qRT-PCR analysis showed that expression of anti-inflammatory cytokines such as IL-10, TGF-β was significantly increased in treated group. Taken together, our data demonstrated that supCD28mAb targets CD4+Foxp3+Treg cells expansion in vivo, maintains and enhances their regulatory functions, to reduce the damage of colon in dextran sulfate sodium (DSS)-induced mouse colitis by secreting a large amount of IL-10. It represents a major advance towards the therapeutic use of polyclonally activated Treg cells as cellular therapy for treatment of IBD.     

Expression of costimulatory molecules (CD80, CD86, CD28, CD152), accessory molecules (TCR alphabeta, TCR gammadelta) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy patients using Mycobacterium leprae antigen (MLCWA) with murabutide and T cell peptide of Trat protein

In leprosy, cell-mediated immunity (CMI) is more significant than humoral response to eliminate intracellular pathogen. T cell defect is a common feature in lepromatous leprosy (LL) patients as compared to tuberculoid type (TT) patients. For efficient initiation of CD4+, T cell response requires T cell receptor (TCR) activation and costimulation provided by molecules on antigen-presenting cells (APC) and their counter receptors on T cells. In our previous study, the defective T cell function in LL patients was restored to a proliferating state with the release of TH1 type cytokines using mycobacterial antigen(s) with two immunomodulators (Murabutide (MDP-BE) and T cell epitope of Trat protein of Escherichia coli) by presenting the antigen in particulate form in vitro to PBMC derived from leprosy patients. This observation prompted us to study the expression of the costimulatory molecules (CD80, CD86, CD28, CD152), other accessory molecules (TCR alphabeta/gammadelta) and T cell lineage molecules (CD4+ and CD8+) during constitutive and activated state of peripheral blood mononuclear cells (PBMC) derived from normal and leprosy individuals using different formulations of Mycobacterium leprae total cell wall antigen (MLCWA), Trat and MDP-BE using flow cytometric analysis. An increased surface expression of CD80, CD86 and CD28 but decreased CD152 expression was observed when PBMC of normal, BT/TT (tuberculoid) and BL/LL (lepromatous) patients were stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite results were obtained with the antigen alone. Antibody inhibition study using antihuman CD80 or CD86 completely abolished the T cell lymphoproliferation, thereby reconfirming the importance of these costimulatory molecules during T cell activation/differentiation. Though the liposome-entrapped antigen formulation has no effect on expression of alphabeta/gammadelta T cell receptor, the constitutive levels of TCR gammadelta were high in lepromatous patients. Thus, TCR bearing gammadelta appears to have a negligible regulatory role in peripheral blood of leprosy patients. The percentage of cells positive for CD4+ are increased in inducible state in all the three groups, while CD8+-positive cells were decreased in LL patients, thereby reconfirming the fact that priming of CD4+ cells are necessary for producing final effector functions. Lastly, intracellular cytokine staining experiment indicated that CD4+ cells are the major producers of IFN-gamma but not NK cells. The study highlights the reversal of T cell anergy especially in lepromatous patients through the modulation of costimulatory molecule expression under the influence of Th1 cytokines, i.e., IL-2 and IFNgamma.     

miR-31协同5-Fu发挥抗肿瘤作用的体内研究

目的：探讨miR-31在胃癌细胞对化疗药物5-氟尿嘧啶（5-Fu）敏感性方面的影响,以及在小鼠体内miR-31联合5-Fu对胃癌生长的影响和机制。方法实时定量PCR的方法检测临床耐药胃癌患者癌组织和非胃癌患者胃组织中以及PBMC中的miR-31的表达,野生型MFC细胞和耐5-Fu 的MFC-R细胞中miR-31的表达,同时,流式细胞术检测耐药胃癌患者癌组织和非胃癌患者外周血中Treg细胞的比例,分析miR-31和FOXP3的相关性。 MTS检测miR-31对MFC细胞增殖的影响。 miR-31和5-Fu联合用药后对小鼠体内肿瘤生长的影响, LDH释放实验检测CD8＋T细胞对MFC的杀伤影响。结果临床耐药胃癌患者癌组织和PBMC中的miR-31水平显著低于非胃癌患者（P＜0唵．05）,而外周CD3＋CD4＋CD25＋FOXP3＋T细胞比例显著高于非癌症患者（ P ＜0．05）,且二者呈负相关（ P ＜0．05）。而在耐5-Fu的MFC细胞中,miR-31的水平降低。当转染miR-31到MFC细胞中后,miR-31的高表达增加了细胞对5-Fu的敏感性（ P ＜0．05）。小鼠体内实验证实,miR-31协同5-Fu可以显著抑制MFC细胞在小鼠体内生长,并且miR-31可以降低荷瘤小鼠肿瘤浸润以及外周的Treg的比例,增强CD8＋T细胞的CTL功能（ P ＜0．05）。结论miR-31能够抑制胃癌MFC细胞的生长可能是通过直接增加癌细胞对化疗药物的敏感性,并且通过抑制Treg细胞的功能来实现的。     

Type of lymphocytes affected by the islet-activating protein (IAP)

By flow cytometric analysis, we identified the subclass of lymphocytes that proliferates in response to islet-activating protein (IAP), both in vitro (human peripheral blood mononuclear cells, MNC, cultured with IAP) and in vivo (peripheral blood MNC derived from A/J mice treated with IAP). IAP caused a preferential proliferation of CD8+ T cells. These cells expressed the IL-2 receptors on their surface. CD4+ CD8+ T cells could also be detected in these cultures, IAP caused human MNC to produce IL-1 and to induce expression of HLA-DR antigen. These effects may play an important role in the T-cell proliferation induced by IAP, although IAP by itself suppressed the proliferative action of IL-1 in mouse thymocytes. IAP induced proliferation of the purified CD4+ cells but had a smaller effect on the purified CD8+ cells. This suggests that the proliferation of CD8+ cells in IAP-treated MNC depends on the function of other types of cell, e.g. CD4+ cell and macrophage.     

Adoptive transfer of CD4+CD25+ regulatory cells combined with low-dose sirolimus and anti-thymocyte globulin delays acute rejection of renal allografts in Cynomolgus monkeys

Although donor alloantigen specific Treg cells play an important role in transplant tolerance, therapeutic applications are limited by their low frequency. In this study, isolated Tregs from Cynomolgus monkeys were efficiently expanded by a co-culture system, and maintained suppressive function on the proliferation of CD4(+) effector cells in vitro. Adoptive transfer of expanded donor alloantigen specific Tregs without any immunosuppressants could prolong survival of MHC-mismatched allografts in Cynomolgus monkeys. To reach the feasibility of clinical transplantation, our objectives focused on whether exposure of monkey Tregs to immunosuppressants could preserve suppressive function in vitro and in vivo. The results showed that low-dose sirolimus selectively expanded Tregs, increased the expression of CD25(bright) and Foxp3 markers, and suppressed TCR- or allo-antigens induced CD4(+) T cell proliferation in vitro. In vivo, after pre-treated with anti-thymocyte globulin (ATG) for consecutive 3days, a 14-day therapy of adoptive infusion of donor alloantigen-specific Tregs combined with low-dose sirolimus delayed acute rejection of renal allografts in Cynomolgus monkeys, showing an MST of 48.5days as compared with those of untreated and sirolimus-treated monkeys (7days and 22days). The frequencies of CD4(+)CD25(bright) T cells were significantly elevated in mesenteric lymph nodes vs. those in inguino lymph nodes and peripheral blood. In summary, expanded donor alloantigen specific Tregs exposed to sirolimus can preserve inhibition in vitro and in vivo. Tregs are more resistant to sirolimus than other T cells. Expanded donor alloantigen specific Tregs combined with sirolimus and ATG prolongs renal allograft survival in monkeys, suggesting that sirolimus might be one of the best synergists for Tregs therapy.