TIM-3-TIM-3L通路在天然调节性T细胞和诱导调节性T细胞免疫抑制效应中的研究

发布时间：2018-06-10 18:59

本文选题：TGF-β_1 + Foxp3　；参考：《华中科技大学》2007年博士论文

【摘要】： 第一部分TGF-β1对同种反应性T细胞增殖能力及CD25表达的影响 [目的]研究TGF-β1对同种反应性T细胞增殖能力及CD25表达水平的影响。 [方法]以丝裂霉素处理的Balb/C(H-2d)裸鼠脾细胞为刺激细胞,CFDA-SE标记的C57BL/6(H-2b)小鼠的T细胞为反应细胞进行混合淋巴细胞培养(mixed lymphocytes culture, MLC)。分别设立对照组(TGF-β1 0ng/ml)、低浓度组(TGF-β1 1.0ng/ml)、中等浓度组(TGF-β1 5.0ng/ml)和高浓度组(TGF-β1 10.0ng/ml)。MLC结束后利用Alamar Blue法检测T细胞增殖活性,流式细胞仪(FACS)检测CD3、CD25表达水平。同时设立IL-2组(TGF-β1 5.0ng/ml +外源性IL-2 100u/ml),以观测其对TGF-β1生物学效应的影响。 [结果] TGF-β1可明显降低同种抗原活化T细胞的增殖反应强度(对照组vs低浓度组,p0.05),而且随着TGF-β1剂量加大,免疫抑制能力增强(低浓度组vs中浓度组,p0.05)。TGF-β1在抑制T细胞增殖的同时,也使得CD25+T细胞比例上调(对照组vs低浓度组,p0.05),但并无明显的剂量依耐性。加入外源性IL-2后,T细胞增殖活性增强(IL-2组vs中浓度组,p0.05),同时CD25+T细胞比例降低(IL-2组vs中浓度组,p0.05)。 [结论] TGF-β1可明显抑制同种抗原活化T细胞的增殖,并使CD25+T细胞表达增加,而外源性IL-2则可逆转TGF-β1的免疫抑制功能,下调CD25+T细胞比例,说明TGF-β1的生物学效应要受到外源性IL-2的影响。第二部分TGF-β1诱导CD4+CD25-T细胞分化为CD4+CD25+调节性T细胞 [目的]研究TGF-β1诱导CD4+CD25+调节性T细胞分化的能力及其相关机理。 [方法] 1、利用丝裂霉素处理的Balb/C裸鼠脾细胞刺激C57BL/6小鼠T细胞,同时给予TGF-β1予以干预(按TGF-β1剂量不同设立4组:对照组,低浓度组,中浓度组和高浓度组),共同培养5天后,用流式细胞仪检测CD4+CD25+T细胞比例;同时用RT-PCR检测培养细胞中Foxp3的表达水平。2、在第一部分实验基础上,通过MACS分离获取C57BL/6小鼠CD4+CD25-T细胞,用同种抗原刺激后,给予TGF-β1干预,培养5天后分离CD4+CD25+T细胞,利用混合淋巴细胞培养系统检测其抑制淋巴细胞增殖的能力。[结果] T细胞经同种抗原刺激后,在中、高浓度TGF-β1诱导下CD4+CD25+T细胞比例均明显升高,Foxp3的表达水平也相应增加,与对照组相比有显著性差异(p0.05)。TGF-β1可直接诱导CD4+CD25-T细胞转化为CD4+CD25+T细胞,转化后的CD4+CD25+T细胞能有效抑制淋巴细胞增殖。 [结论] TGF-β1可诱导CD4+CD25-T细胞转化为CD4+CD25+Treg,促进其表达Foxp3,并能抑制淋巴细胞增殖。第三部分TGF-β1诱导的调节性T细胞体内输注延长小鼠皮肤移植物存活 [目的]利用TGF-β1体外诱导na?ve T细胞分化为调节性T细胞,通过体内输注延长小鼠皮肤移植物存活时间,并研究其相关机理。 [方法]根据诱导条件不同分为三组:对照组(加入IL-2培养的C57BL/6小鼠T细胞)、MLR组(经同种抗原刺激活化的C57BL/6小鼠T细胞)和TGF-β组(经同种抗原刺激活化的C57BL/6小鼠T细胞,同时加入5.0ng/ml TGF-β1诱导)。利用FACS检测CD4+CD25+T细胞比例,并用RT-PCR检测Foxp3的表达水平。建立小鼠皮肤移植模型,并于0、1、2和3d输注上述细胞,观察皮肤移植物存活时间。术后第9天,取部分受鼠行移植物病理检测,用FACS检测脾脏中Th1、Th2和CD4+CD25+Treg比例,并用Alamar Blue法检测淋巴细胞增殖能力。 [结果] TGF-β组中CD4+CD25+T细胞比例高于对照组和MLR组(p0.05),且其高表达Foxp3。将培养的细胞输注给受鼠后发现,输注MLR组细胞的受鼠其移植物平均存活时间(mean survival time, MST)为(9.4±1.3)d,低于对照组(p0.05);而输注TGF-β组细胞小鼠MST为(22.8±1.9)d,较对照组和MLR组明显延长(p0.05)。病理检测亦显示TGF-β组受鼠移植物结构完整,无明显淋巴细胞浸润。FACS结果显示TGF-β组小鼠体内Th1细胞(CD4+TIM-3+)比例低于对照组和MLR组(p0.05),而Th2细胞(CD4+TIM-1+)比例则与MLR组类似(p0.05),但低于对照组(p0.05);而且TGF-β组中CD4+CD25+Treg比例明显高于对照组和MLR组(p0.05)。用Alamar Blue法检测受鼠外周淋巴细胞增殖活性显示,TGF-β组受鼠淋巴细胞增殖能力被明显抑制,低于对照组(p0.05)。 [结论] TGF-β1可诱导T细胞分化为具有抑制能力的调节性T细胞,将诱导后的细胞进行过继输注可使小鼠体内CD4+CD25+Treg比例升高,同时抑制Th1和Th2细胞分化扩增,并使得外周淋巴细胞增殖能力减弱,从而延长小鼠皮肤移植物存活时间。第四部分调节性T细胞通过TIM-3-TIM-3L通路抑制淋巴细胞增殖 [目的]研究TIM-3-TIM-3L通路在调节性T细胞免疫抑制效应中的作用。 [方法]用MACS从成年C57BL/6分离获取天然调节性T细胞(natural Treg, nTreg),并利用TGF-β1诱导na?ve T细胞分化为诱导性调节性T细胞(induced Treg, iTreg),随后通过RT-PCR检测其表达Foxp3和TIM-3L的水平,及其对淋巴细胞增殖的抑制能力。使用TIM-3融合蛋白(TIM-3.Ig)和anti-TIM-3处理nTreg和iTreg,对TIM-3-TIM-3L通路进行阻断,随后利用MLC体系检测其免疫抑制能力的变化情况。 [结果]分离获取的nTreg和iTreg均可表达Foxp3。TIM-3L(galectin-9)在脾细胞高表达,同时在nTreg和iTreg也检测到有galectin-9表达,但表达水平低于脾细胞(p0.05)。用TIM-3.Ig对nTreg和iTreg进行干预后发现,nTreg和iTreg抑制淋巴细胞增殖的能力部分减弱,与未干预组差异显著(p0.05),但nTreg和iTreg之间无差异(p0.05);而用anti-TIM-3处理nTreg和iTreg后并未明显减弱其抑制淋巴细胞增殖的能力(p0.05)。 [结论] TGF-β1可诱导iTreg产生。nTreg和iTreg均表达TIM-3L,并可通过TIM-3-TIM-3L通路抑制淋巴细胞增殖;但阻断TIM-3-TIM-3L通路后Treg仍可部分抑制淋巴细胞增殖,表明TIM-3-TIM-3L通路不是Treg发挥免疫抑制效应的唯一通路。
[Abstract]:Part one the effect of TGF- beta 1 on the proliferation and CD25 expression of allogeneic T cells
[Objective] to study the effect of TGF- beta 1 on the proliferation and CD25 expression of allogeneic T cells.
[Methods] Balb/C (H-2d) nude mice were treated with mitomycin as the stimulating cells, and the T cells of the CFDA-SE labeled C57BL/6 (H-2b) mice were used for mixed lymphocyte culture (mixed lymphocytes culture, MLC). The control group (TGF- beta 1 0ng/ml), the low concentration group (beta 1), and the medium concentration group (C57BL/6 beta 1) were set up respectively. After the end of the high concentration group (TGF- beta 1 10.0ng/ml).MLC, the proliferation activity of T cells was detected by Alamar Blue, and the level of CD3 and CD25 expression was detected by flow cytometry (FACS), and the IL-2 group (TGF- beta 1 5.0ng/ml + exogenous hormones) was set up to observe the effect of its biological effect on the beta 1.
[results] TGF- beta 1 could significantly reduce the proliferation reaction intensity of the alloantigen activated T cells (the control group, vs low concentration group, P0.05), and with the increase of TGF- beta 1, the immunosuppressive ability enhanced (the concentration group of vs in the low concentration group, P0.05).TGF- beta 1, while inhibiting the T cell proliferation, also increased the ratio of CD25+T cells (vs low concentration group of control group, P0). .05), but there was no obvious dose dependent. After adding exogenous IL-2, the proliferation activity of T cells was enhanced (the concentration group of vs in group IL-2, P0.05), and the proportion of CD25+T cells decreased (the concentration group in IL-2 group vs, P0.05).
[Conclusion] TGF- beta 1 can inhibit the proliferation of T cells and increase the expression of CD25+T cells, while exogenous IL-2 can reverse the immunosuppressive function of TGF- beta 1 and reduce the proportion of CD25+T cells, indicating that the biological effect of TGF- beta 1 is affected by exogenous IL-2.
The second part, TGF- beta 1, induces CD4+CD25-T cells to differentiate into CD4+CD25+ regulatory T cells.
[Objective] to investigate the ability of TGF- beta 1 to induce differentiation of CD4+CD25+ regulatory T cells and its related mechanisms.
[method] 1, Balb/C nude mouse splenocytes treated with mitomycin were used to stimulate T cells in C57BL/6 mice, and TGF- beta 1 was given at the same time. 4 groups were set up according to the dosage of TGF- beta 1: the control group, the low concentration group, the medium concentration group and the high concentration group. The proportion of CD4+CD25+T cells was detected by the flow cytometry for 5 days, and the culture was detected by RT-PCR. The expression level of Foxp3 in the cells was.2. On the basis of the first part of the experiment, the CD4+CD25-T cells of C57BL/6 mice were obtained by MACS separation. After the stimulation of the same antigen, TGF- beta 1 was given, and CD4+CD25+T cells were isolated after 5 days, and the ability to inhibit the proliferation of lymphocytes was detected by mixed lymphocyte culture system. [results] T cells were the same. After the stimulation of the antigen, the proportion of CD4+CD25+T cells induced by high concentration of TGF- beta 1 increased obviously, and the expression level of Foxp3 increased correspondingly. Compared with the control group, there was a significant difference (P0.05).TGF- beta 1 could directly induce the transformation of CD4+CD25-T cells into CD4+CD25+T cells, and the transformed CD4+CD25+T cells could effectively inhibit the proliferation of lymphocytes.
[Conclusion] TGF- beta 1 can induce CD4+CD25-T cells to transform into CD4+CD25+Treg, promote its expression of Foxp3, and inhibit lymphocyte proliferation.
The third part is the prolongation of mouse skin graft survival by in vivo infusion of TGF- beta 1 regulatory T cells.
[Objective] to induce na? Ve T cells to differentiate into regulatory T cells by using TGF- beta 1 in vitro, and to prolong the survival time of mouse skin graft by infusion in vivo, and to study its mechanism.
[Methods] according to the different induction conditions, the control group was divided into three groups: the control group (C57BL/6 mouse T cells cultured with IL-2), the MLR group (C57BL/6 mice T cells activated by the same antigen stimulation) and the TGF- beta group (T cells activated by the alloantigen stimulated C57BL/6 mice, and 5.0ng/ml TGF- beta 1 induced by 5.0ng/ml). The expression level of Foxp3 was detected by RT-PCR. The mice skin transplantation model was established and the cells were transfused with 0,1,2 and 3D to observe the survival time of the skin graft. Ninth days after the operation, the pathological examination of the graft was taken, the proportion of Th1, Th2 and CD4+CD25+Treg in the spleen was detected by FACS, and the lymphocyte proliferation was detected by Alamar Blue.
[results] the proportion of CD4+CD25+T cells in the TGF- beta group was higher than that of the control group and the MLR group (P0.05), and the high expression of Foxp3. showed that the cells of the cultured cells were transfused to the rats. The average survival time (mean survival time, MST) of the transfused MLR group was (9.4 + 1.3) d, lower than the control group (P0.05). (22. 8 + 1.9) d, compared with the control group and MLR group (P0.05), the pathological examination also showed that the structure of the TGF- beta group was complete, and no obvious lymphocyte infiltration.FACS results showed that the proportion of Th1 cells (CD4+TIM-3+) in the TGF- beta group was lower than that of the control group and the MLR group (P0.05), but the proportion of Th2 cells (CD4+TIM-1+) was similar to that of the group, but lower than that of the group. In the control group (P0.05), the proportion of CD4+CD25+Treg in the TGF- beta group was significantly higher than that in the control group and the MLR group (P0.05). The proliferation activity of the peripheral lymphocyte in the rats was detected by Alamar Blue method, and the proliferation ability of the lymphocytes in the TGF- beta group was significantly inhibited, lower than that of the control group (P0.05).
[Conclusion] TGF- beta 1 can induce T cells to differentiate into regulatory T cells with inhibitory ability. The adoptive infusion of induced cells can increase the proportion of CD4+CD25+Treg in mice, inhibit the differentiation and amplification of Th1 and Th2 cells, and reduce the proliferation of peripheral lymphocytes, thus prolonging the survival time of the skin graft in mice.
The fourth part of regulatory T cells inhibit lymphocyte proliferation through TIM-3-TIM-3L pathway.
[Objective] to study the role of TIM-3-TIM-3L pathway in regulating the immunosuppressive effect of regulatory T cells.
[Methods] natural regulatory T cells (NATURAL Treg, nTreg) were isolated from adult C57BL/6, and na? Ve T cells were induced by TGF- beta 1 to differentiate into inducible T cells (induced). TIM-3.Ig (anti-TIM-3) and nTreg (nTreg) and iTreg were used to block the TIM-3-TIM-3L pathway, and then the MLC system was used to detect the changes of the immunosuppressive ability.
[results] the isolated nTreg and iTreg could express the high expression of Foxp3.TIM-3L (galectin-9) in the spleen cells, while the expression of galectin-9 was also detected in nTreg and iTreg, but the expression level was lower than that of the spleen cells (P0.05). There was significant difference (P0.05) in the untreated group, but there was no difference between nTreg and iTreg (P0.05), while nTreg and iTreg treated with anti-TIM-3 did not significantly weaken the ability to inhibit lymphocyte proliferation (P0.05).
[Conclusion] TGF- beta 1 can induce iTreg to produce.NTreg and iTreg to express TIM-3L, and can inhibit lymphocyte proliferation through TIM-3-TIM-3L pathway, but Treg still partially inhibits lymphocyte proliferation after blocking TIM-3-TIM-3L pathway, indicating that TIM-3-TIM-3L pathway is not the only pathway for Treg to play an immunosuppressive response.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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