免疫抑制状态下小鼠MD-1表达的影响因素及其在皮肤移植中的效应

目的 探讨免疫抑制状态下小鼠MD-1表达的影响因素及在皮肤移植中的效应。方法采用小鼠同种皮肤移植模型，以环孢霉素A（CsA）、他克莫司（FK506）、霉酚酸酯（MMF）或雷帕霉素（SRL）为免疫抑制手段。结果CsA和MMF能抑制脾细胞的MD-1表达和增殖反应强度，下调血清IL-2水平和上调IL-10水平，并显著延长皮肤移植物平均存活时间（MST）；FK506对IL-10水平无明显影响；而SRL对MD-1表达及IL-2与IL-10水平均无明显影响。MD-1表达水平与脾细胞增殖反应强度和血清IL-2水平呈极显著正相关，和IL-10水平呈极显著负相关。结论CsA、FK506和MMF能抑制小鼠MD-1的表达，其作用与血清IL-2水平下调有关。在免疫抑制状态下，MD-1表达下调与同种皮肤移植物存活时间延长有关，而其表达上调对排斥反应的发生具有一定的指示作用。     

Lymphotactin: a key regulator of lymphocyte trafficking during acute graft rejection.

The attraction of leucocytes to allografts is essential for rejection. The process is controlled by chemokines. In order to clarify the role of lymphotactin (a cytokine that represents a novel branch of the chemokine superfamily) in regulating leucocyte trafficking during graft rejection, we used rat renal transplantation models to examine its gene expression and the distribution of lymphotactin-expressing cells in renal grafts. Lymphotactin mRNA was upregulated strongly in acutely rejecting renal allografts. The mRNA was undetectable in isografts, chronically rejecting renal allografts or normal kidney. Once lymphotactin was expressed, large numbers of infiltrating lymphocytes were seen. Moreover extended studies demonstrated that in cultured rat spleen cells the expression of lymphotactin mRNA was markedly induced by phytohaemagglutinin (PHA) or phorbol myristate acetate (PMA), and such induction was inhibited by the immunosuppressive drugs FK506 and cyclosporin. Collectively, these observations provide new evidence demonstrating that lymphotactin is a key regulator of lymphocyte motility and adhesiveness during acute allograft rejection. FK506 and cyclosporin inhibition of lymphotactin expression is likely to represent an important molecular mechanism of the action of the drugs.     

Prolonged skin allograft survival by IL-10 gene-introduced CD4 T cell administration

Both CD4 and CD8 T cells play crucial roles in immune responses in transplantation. Immunosuppressive drugs, such as FK506 and cyclosporin A, block the priming of alloreactive CD4 T(h) cells and the subsequent induction of allospecific CD8 cytotoxic effector T cells and inhibit allograft rejection. However, the desire to minimize chronic complications that may arise from the use of immunosuppressive agents drives the search for additional strategies for immunosuppression of allograft rejection. In this study, CD4 or CD8 T cells into which the IL-10 gene is introduced using an adenovirus vector containing human IL-10 (hIL-10) cDNA (Ad-hIL-10) and into mouse T cells transgenic for the Coxsackie virus and adenovirus receptor form a model system to study the effect of administration of IL-10-secreting T cells on the survival of the allogenic skin grafts. Ad-hIL-10-infected CD4 and CD8 T cells secreted a large amount of hIL-10 for 3-4 days in culture in vitro. Ad-hIL-10-infected CD4 T cells administered in vivo could be detected in the spleen for 7 days post-transfer. Significantly prolonged survival of grafts was observed in animals that received either Ad-hIL-10-infected activated CD4 T cells or T(h)2-skewed CD4 T cells as compared with controls. Furthermore, substantial enhancement of the effect was observed in B6.C-H2(bm1)/ByJ transplants. Thus, a direct manipulation of T cells through the introduction of the immunosuppressive cytokine gene IL-10 may be a novel strategy for the control of allograft rejection.     

The effect of cyclosporin A,FK506 and rapamycin on the murine contact sensitivity reaction

We have evaluated the effects of three potent immunosuppressive agents, cyclosporin A (CsA), FK506 and rapamycin, on the murine contact sensitivity (CS) reaction to the hapten trinitrochlorobenzene. Development of CS reaction requires participation of three distinct T cell subsets: αβ⁺, CD4⁺ T lymphocytes, which are the classical effector cell of the CS reaction, γδ⁺ T lymphocytes, and αβ⁺, double-negative (CD4^? CD8^?) T lymphocytes that express the B220 molecule and produce IL-4. We found that all three drugs inhibit the development of the CS reaction, but they affect different target cells. In fact, rapamycin and FK-506 block both αβ⁺, CD4⁺ and γδ⁺ T lymphocytes, while CsA inhibits only the αβ⁺, CD4⁺ T lymphocyte. None of the three drugs exerted any inhibitory activity on the αβ⁺, double-negative (CD4^? CD8^?) T lymphocytes. Hapten-immune lymph node cells from mice treated in vivo with CsA or FK506 failed to proliferate and to produce IL-2 when re-exposed to the specific antigen in vitro. In contrast, immune lymph node cells from mice that had been treated in vivo with rapamycin gave optimal antigen-specific proliferation and IL-2 production in vitro. The implications of these observations are discussed in relation to the use of these immunosuppressive agents for prevention of allograft rejection.     

Inhibition of endogenous MHC class II-restricted antigen presentation by tacrolimus (FK506) via FKBP51

The effect of tacrolimus (FK506) on down-regulation of IL-2 production by T cells is considered to be mainly responsible for its strong suppression of immunological events. In this study, we show that FK506 also has an affect on antigen presentation by antigen-presenting cells in vitro. FK506 was able to inhibit the presentation of endogenous MHC class II-restricted minor histocompatibility antigens in primary dendritic cells (DC) in vitro, but cyclosporine A (CsA) and rapamycin (RAP) were not. RNA interference (RNAi)-mediated reduction of endogenous FK506-binding protein (FKBP)51 expression resulted in a marked decrease in antigen presentation, suggesting that FKBP51 plays a role in endogenous MHC class II-restricted antigen presentation. Since our model used naturally expressed cytosolic antigens in primary DC, these effects might have been due to novel properties of the immunosuppressive drugs and may allow us to elucidate a new paradigm for the immunosuppressive mechanism of FK506.     

Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts

Immune dysfunction is well documented during tumor progression and likely contributes to tumor immune evasion. CD8⁺ cytotoxic T lymphocytes (CTLs) are involved in antigen-specific tumor destruction and CD4⁺ T cells are essential for helping this CD8⁺ T cell-dependent tumor eradication. Tumors often target and inhibit T-cell function to escape from immune surveillance. This dysfunction includes loss of effector and memory T cells, bias towards type 2 cytokines and expansion of T regulatory (Treg) cells. Curcumin has previously been shown to have antitumor activity and some research has addressed the immunoprotective potential of this plant-derived polyphenol in tumor-bearing hosts. Here we examined the role of curcumin in the prevention of tumor-induced dysfunction of T cell-based immune responses. We observed severe loss of both effector and memory T-cell populations, downregulation of type 1 and upregulation of type 2 immune responses and decreased proliferation of effector T cells in the presence of tumors. Curcumin, in turn, prevented this loss of T cells, expanded central memory T cell (T_CM)/effector memory T cell (T_EM) populations, reversed the type 2 immune bias and attenuated the tumor-induced inhibition of T-cell proliferation in tumor-bearing hosts. Further investigation revealed that tumor burden upregulated Treg cell populations and stimulated the production of the immunosuppressive cytokines transforming growth factor (TGF)-β and IL-10 in these cells. Curcumin, however, inhibited the suppressive activity of Treg cells by downregulating the production of TGF-β and IL-10 in these cells. More importantly, curcumin treatment enhanced the ability of effector T cells to kill cancer cells. Overall, our observations suggest that the unique properties of curcumin may be exploited for successful attenuation of tumor-induced suppression of cell-mediated immune responses.     

Effect of FK506 on the interleukin 15-driven proliferation and apoptosis of anti-CD3-activated umbilical cord blood T cells.

BACKGROUND: Increased susceptibility of umbilical cord blood (CB) T cells to FK506 immunosuppression may contribute to the lessened severity of graft-vs-host disease in CB transplantation. OBJECTIVE: To investigate the FK506 sensitivity of interleukin 15 (IL-15)- and IL-2-driven proliferation and apoptosis of anti-CD3-stimulated CB T cells compared with adult peripheral blood (APB) T cells. METHODS: Surface flow cytometric analysis (CD25 and CD95), carboxyfluorescein diacetae succinimidyl ester staining to track CD3+ T-cell division, and flow cytometric analysis of apoptotic cell death using Annexin V were performed to determine the effect of FK506 on CD3+ T-cell activation and apoptosis after anti-CD3 stimulation in the presence of IL-15 or IL-2. RESULTS: IL-15 is superior to IL-2 in promoting CD25 expression and proliferation of anti-CD3-stimulated CB and APB T cells. Although IL-15-driven proliferation evaluated by carboxyfluorescein diacetae succinimidyl ester staining revealed comparable sensitivity to FK506 in anti-CD3-stimulated CB and APB T cells, IL-15-driven CD25 up-regulation in CB T cells was more sensitive to FK506 inhibition than APB T cells. FK506 down-regulated anti-CD3-induced apoptosis in CB and APB T cells (P < .01). However, the FK506 sensitivity of anti-CD3-induced T-cell apoptosis was lost in IL-15-supplemented CB cultures (P = .51) but not in corresponding APB cultures (P = .002). The IL-15-enhanced Fas expression on CB T cells (CD95) was decreased by FK506, similar to that observed with adults. CONCLUSIONS: We observed differential FK506 sensitivity of IL-15-driven CD25 up-regulation and apoptotic response comparing CB and APB T cells. This finding suggests the potential therapeutic benefit of FK506 in ameliorating graft-vs-host disease by decreasing IL-15-driven donor T-cell proliferation without inhibiting associated activation-induced apoptosis during CB transplantation.     

FR900506 (FK506) and 15-deoxyspergualin (15-DSG) modulate the kinetics of infiltrating cells in eyes with experimental autoimmune uveoretinitis.

In order to evaluate two new immunosuppressive agents, FR900506 (FK 506) and 15-Deoxyspergualine (15-DSG), the kinetics of infiltrating cells in the eyes of Lewis rats with experimental autoimmune uveoretinitis (EAU) were studied. Rats were immunized with retinal S-antigen and treated with different doses of either FK 506 or 15-DSG. The inflammatory ocular tissues obtained at various intervals during the process of EAU were examined using an immunoperoxidase technique. The results were compared with those eyes developing EAU without treatment or with suboptimal doses or a suboptimal dose of Cyclosporine (CsA). Both FK 506 and 15-DSG, like CsA, delayed the cellular kinetics during the course of EAU. However, FK 506 had the greatest effect on the kinetics of T lymphocyte subsets by causing the greatest increase in the recruitment time of the T suppressor/cytotoxic population. FK506 treatment resulted not only in the highest inhibition of expression of IL-2 receptors on T cells, but also in the prevention of the expression of MHC class II antigens on ocular resident cells. Treatment with 15-DSG resulted in general immunosuppression on various infiltrating inflammatory cells.     

Ssu72 is a T-cell receptor-responsive modifier that is indispensable for regulatory T cells

The homeostatic balance between effector T cells and regulatory T cells (Tregs) is crucial for adaptive immunity; however, epigenetic programs that inhibit phosphorylation to regulate Treg development, peripheral expression, and suppressive activity are elusive. Here, we found that the Ssu72 phosphatase is activated by various T-cell receptor signaling pathways, including the T-cell receptor and IL-2R pathways, and localizes at the cell membrane. Deletion of Ssu72 in T cells disrupts CD4⁺ T-cell differentiation into Tregs in the periphery via the production of high levels of the effector cytokines IL-2 and IFNγ, which induce CD4⁺ T-cell activation and differentiation into effector cell lineages. We also found a close correlation between downregulation of Ssu72 and severe defects in mucosal tolerance in patients. Interestingly, Ssu72 forms a complex with PLCγ1, which is an essential effector molecule for T-cell receptor signaling as well as Treg development and function. Ssu72 deficiency impairs PLCγ1 downstream signaling and results in failure of Foxp3 induction. Thus, our studies show that the Ssu72-mediated cytokine response coordinates the differentiation and function of Treg cells in the periphery.     

小鼠MD-1表达的影响因素及其在移植排斥反应中的作用

目的：探讨影响小鼠MD-1表达的因素以及MD-1在移植排斥反应中的作用。方法：实验采用C57BL／6-BALB／c小鼠同种皮肤移植模型，以非特异性免疫抑制剂CsA、FK506、MMF和SRL，以及特异性MD-1反义脱氧寡核苷酸(AS-ODNs)为干预手段，于术后第11天留取标本检测脾细胞MD-1表达水平和增殖反应强度、血清IL-2和IL-10水平以及皮肤移植物组织学改变，并记录移植物存活时间。结果：CsA、MMF和AS-ODNs处理能减少脾脏MD-1表达阳性细胞数、降低脾细胞增殖反应强度、下调血清IL-2水平和上调IL-10水平，并显著延长皮肤移植物平均存活时间(MST)；FK506与CsA和MMF处理结果的差异在于其对血清IL-10水平无明显影响；SRL处理仅能减低脾细胞增殖反应强度和延长皮肤移植物MST，对MD-1表达及IL-2与IL-10分泌无明显影响。MD-1表达水平与脾细胞增殖反应强度和血清IL-2水平呈极显著正相关，和IL-10水平呈极显著负相关。结论：CsA、FK506、MMF和AS-ODNs处理均能有效抑制小鼠MD-1的表达。免疫抑制剂干预MD-1表达的作用与血清IL-2水平下调有关。MD-1通过影响淋巴细胞增殖反应和血清IL-2、IL-10水平，在移植排斥反应中发挥重要作用。     

Inflammatory Cytokines and the Risk of Cardiovascular Complications in Type 2 Diabetes

This study evaluates peripheral blood T lymphocyte expression of inflammatory and proinflammatory cytokines as well as T regulatory (Treg) (FOXP3+CD25+CD4+) cells in type 2 diabetes (T2DM). Participants included 40 T2DM and 30 healthy control subjects. Twenty-four patients had no complications while 16 were afflicted with coronary heart disease (CHD). Relative to healthy subjects, all T2DM patients showed a significant increase in expression of CD4+IFN-ϒ+, CD4+TNF-α+, and CD4+IL-8+ T cells (P < 0.001) as well as CD4+IL-6+, CD4+IL-1β+, and IL-17+ T cells (P < 0.05) while the ratios of Treg/Th1(CD4+IFN-ϒ+) and Treg/Th-17(CD4+IL-17+) cells were significantly decreased (P < 0.05 and P < 0.01). T2DM patients with CHD showed a significant increase in CD4+IFN-ϒ+, CD4+TNF-α+, and CD4+IL-17+ T cells and a significant decrease in Treg/Th1 and Treg/IL-17 cells compared to T2DM patients without CHD (P < 0.05). In CHD-afflicted T2DM, HbA1c correlated positively with CD4+IFN-ϒ+ T cells (P < 0.01), HDL correlated negatively with each of CD4+IL-8+ T cells and CD4+IL-17+ T cells (P < 0.05), and LDL correlated positively with CD4+IL-1β+ T cells (P < 0.05). Conclusion. This study shows that hyperglycemia and dyslipidemia correlate with increased inflammatory cytokine expression and suggests the involvement of T cells in the development of diabetes and its complications.     

Inhibition of Pim2-prolonged skin allograft survival through the apoptosis regulation pathway

Recently, apoptosis has been considered to be an important regulator for allograft survival. The serine/threonine kinase Pim2 has been implicated in many apoptotic pathways. In a previous study, we found that pim2 was highly expressed in CD4⁺ T cells in an allograft model. Here, we further investigated the effects of Pim2 on allograft survival and the underlying mechanisms associated with apoptosis. The results showed that pim2 was overexpressed in grafts and spleens, particularly in spleen CD4⁺ T cells when acute allorejection occurred, and correlated positively with the extent of rejection. In T cells from the spleens of naive BALB/c mice treated with 5 µM 4a (a specific inhibitor of Pim2) for 24 h, the apoptosis rate increased and the phosphorylation of BAD was decreased. Furthermore, adoptive transfer of CD4⁺ T cells treated with 4a in vitro to allografted severe combined immunodeficiency (SCID) mice effectively prolonged allograft survival from 19.5±1.7 days to 31±2.3 days. Moreover, the results demonstrated that the CD4⁺CD25⁻ effector T-cell subset was the predominate expresser of the pim2 gene as compared with the CD4⁺CD25⁺ regulatory T (Treg) cell subset. Alloantigen-induced CD4⁺CD25⁺ T cells displayed less Foxp3 expression and a low suppression of apoptosis compared with effector CD4⁺CD25⁻ T cells treated with 4a. Collectively, these data revealed that Pim2 facilitated allograft rejection primarily by modulating the apoptosis of effector T cells and the function of Treg cells. These data suggested that Pim2 may be an important target for in vivo anti-rejection therapies and for the ex vivo expansion of CD4⁺CD25⁺ T cells.     

Efficient IL-2R signaling differentially affects the stability,function, and composition of the regulatory T-cell pool

Signaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25^Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25^Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.     

Suppression of adult T cell leukemia-derived factor/human thioredoxin induction by FK506 and cyclosporin A: a new mechanism of immune modulation via redox control

Adult T cell leukemia-derived factor (ADF), which is identicalto a disulfide reducing enzyme human thioredoxin (TRX), is producedand released by activated or virus-infected lymphocytes. Herewe report that, in peripheral blood mononuclear cells (PBMC)stimulated with phytohemagglutinin(PHA), ADF/TRX mRNA was inducedwithin 8 h after stimulation as detected by in situ hybridizationstudy. To analyze the mechanism of ADF/TRX induction duringT cell activation, the effects of immunosuppressants includingFK506, rapamycin (Rap) and cyclosporin A (CsA) on ADF/TRX expressionwere investigated by immunoblot analysis. ADF/TRX inductionin PBMC by PHA, Con A or OKT3 mAb was almost completely suppressedby FK506. Whereas CsA also inhibited ADF/TRX expression in OKT3mAb-stimulated PBMC, Rap falled to affect it in spite of exhibitinggrowth inhibition. In addition, exogenous IL-2 could not increaseADF/TRX production in FK506-treated PBMC or in PHA blasts. Theseresults indicate that ADF/TRX induction in T cell activationdepends on calclneurin-dependent events in the early phase andthat IL-2 production is not directly involved in ADF/TRX induction.Furthermore, when recombinant ADF (rADF) was added to a cultureof PBMC 1 h before the addition of PHA and FK506, the actionof FK506 was partially reversed as determined by [³H]thymidineincorporation and viable cell counts. These results suggestthat ADF/TRX produced and released from PBMC may be a crucialevent in lymphocyte activation, and that FK506 and CsA may exertthe immune suppression partly through inhibiting the inductionof the endogenous reducing factor ADF/TRX.     

Effects of a novel immunosuppressive agent, FK506, on human B cell activation.

We examined the effect of new immunosuppressive agent, FK506, on the human B cell function, in comparison with that of cyclosporin A (CyA) and tried to define the discrete activation step(s) which is selectively affected by FK506 and CyA. We used polyclonal B cell activators, Staphylococcus aureus Cowan I (SAC) and pokeweed mitogen (PWM). We found that (i) the initial B cell activation process by PWM, which is on the basis of T cell-dependent manner, is susceptible to the inhibitory effects of FK506 and CyA, while initial B cell activation on the basis of T cell-independent manner by SAC is resistant to these drugs; (ii) they also inhibit helper factor production by T cells; (iii) once they are activated, the B cells become resistant to inhibition by the drugs; and (iv) on an equimolar basis, FK506 exhibits 100-fold greater inhibitory activity than does CyA. Thus FK506 mainly interferes with interactions between T cells and other cells which are essential for B cell activation process, resulting in inhibition of B cell function.