阻断TIM-4/TIM-1共刺激信号诱导TIM-1+调节性B细胞介导移植耐受的机制与应用研究

It is now clear that B cells can play an important role in downregulating immune responses in an IL-10-dependent fashion. Other than a capacity to express IL-10 after ex vivo stimulation, these rare regulatory B cells (Bregs) lack specific markers and are found within various B cell subpopulations where their frequency can be as low as 1%. This has made study of Breg challenging. Surprisingly, a number of agents believed to induce allograft tolerance through their effects on T cells, were recently shown to be B cell dependent. This suggests that Bregs may be involved, and play a broad role in allograft tolerance. This is echoed by the "B cell profile" observed in tolerant human renal allograft recipients. T cell Ig and mucin domain-containing proteins family has 8 members (TIM-1~TIM-8) in mice, and 3 members(TIM-1,TIM-3,TIM-4)in humans. TIM-4 is preferentially expressed on antigen-presenting cells(DCs, M?s), and its counter-ligand, TIM-1, is thought to deliver co-stimulating signals to T cells, and helps regulate Th differentiation. Blockade of TIM-4/TIM-1signal with a low affinity anti-TIM-1 (RMT1-10) induces allograft tolerance thought to be Th2 dependent. Surprisingly, we found TIM-1 is expressed at much higher levels on B than T cells. Moreover, Th2 deviation and allograft tolerance induced by anti-TIM-1, were completely B cell dependent. In fact, in the absence of B cells, anti-TIM-1 actually accelerates rejection compared to untreated controls. TIM-1 is expressed by the large majority of IL-10+ B cells in all major subpopulations. TIM-1+, but not TIM-1-, B cells from allograft recipients can transfer tolerance to otherwise untreated allograft recipients. Armed with these novel insights, we can now examine whether blocking TIM-4/TIM-1 signal with anti-TIM-4 mAb has similar or better suppressiveness compared with anti-TIM-1, and establish the role of TIM-1+ Breg in allograft tolerance and determine the role of TIM-1 in Breg generation. Specifically, In Aim 1, we will examine the effect of blocking TIM-4/TIM-1 co-signaling with anti-TIM-4 mAb in allograft tolerance, and determine whether TIM-1+Breg cells are the specific B cell population required for the transplant tolerance induced by anti-TIM-4. In Aim 2, we will determine whether anti-TIM-4 acts synergistically with Rapamycin in promoting transplant survival. In Aim 3, we will examine the mechanisms of Breg induction through anti-TIM-4, and define the role of TIM-1+ Breg in allograft tolerance using MuMT(B-cell deficient) recipient mice, and examine the effect of TIM-1+Breg on Th1,Th2,Th17 and Tregs. In Aim 4, we will identify the specific transcription factors which are ciritical in Breg development. We believe these studies will greatly enhance our understanding of Breg immunobiology and provide therapeutic insight highly relevant to allograft tolerance.

调节性B细胞（Breg）通过分泌IL-10起免疫抑制作用。本人曾首次报导B细胞表达TIM-1，且TIM-1+B细胞富集IL-10，是Breg的最佳膜表面标志（TIM-1+Breg ）。anti-TIM-1单抗结合TIM-1而阻断TIM-4(配体)/TIM-1（受体）共信号诱导TIM-1+Breg并克服移植物排斥。本课题将研究：（1）以anti-TIM-4单抗封闭配体TIM-4从而阻断TIM-4/TIM-1信号，观察对小鼠Breg和胰岛移植物存活的影响；（2）探讨Breg介导免疫抑制的机制， 观察TIM-1+Breg对Treg、Th1、Th17等细胞的作用； (3) 探讨STAT6和Ca/Calcineurin/NFAT通路在Breg分化中的作用，并以高通量芯片筛选新的转录因子。本研究将加深对Breg发生和分化的机制理解，并提供诱导移植耐受的新型策略。

调节性B细胞(Breg)通过分泌IL-10起免疫抑制作用。本人曾首次报导B细胞表面表达TIM-1分子，且TIM-1+ B细胞富含IL-10，是Breg的最佳膜表面标志(TIM-1+Breg)。anti-TIM-1单抗阻断TIM-4(配体)/TIM-1（受体)信号诱导Breg并克服移植物排斥。内容: 1)以TIM-4单抗封闭配体TIM-4，观察对小鼠Breg和胰岛移植物存活的影响；（2）探讨Breg介导免疫抑制的机制， 观察TIM-1+ Breg对Treg、Th1、Th17等的作用； (3) 探讨STAT6和Ca/Calcineurin/NFAT通路以及microRNA-21在Breg分化中的作用。结果：1）anti-TIM-4单抗体内治疗封闭配体TIM-4从而阻断TIM-4/TIM-1信号后，同种异体移植物小鼠受体胰岛移植物平均存活39天（对照组14天，P<0.05），其中~35%受体移植物长期存活(>100天)。治疗后小鼠IL-10+ Breg细胞比例显著增加（治疗组~4% vs. 对照~1%, p<0.01);（2）输注TIM-1+Breg细胞能有效延长小鼠同种异体胰岛移植物存活(平均移植物存活26天 vs. 12天对照组)，并诱导炎症性Th1（IFN-g+下降52%）、Th17(IL-17+ 下降74%)细胞反应向保护性的Treg (Foxp3+， 上调33%)细胞反应转化。（3）通过基因芯片及定量PCR技术，发现anti-TIM-4治疗后，转录因子STAT6和Ca/Calcineurin/NFAT通路主要分子表达升高。并首次发现miR-21可特异性结合至IL-10分子3‘-UTR，负向调控IL-10+Breg的分化。miR-21抑制物体内转染后可减轻移植排斥及自身免疫性疾病EAE。Anti-TIM-4治体内转染后可减轻移植排斥及自身免疫性疾病EAE。Anti-TIM-4治疗通过降低体内miR-21水平，进而促进抑制性Breg产生，Breg通过抑制炎症性Th17细胞，从而诱导移植排斥减轻，促进移植物存活。传统的抗移植排斥治疗方案毒性大，对病人的损伤严重。本研究利用生物制剂单克隆抗体阻断免疫细胞表面的共信好分子TIM-4，对机体的毒性很小，且有诱导永久耐受的潜在作用，因而大大优于传统治疗。本研究发现miR-21是特异的免疫调控靶点，在未来药物研发中有潜在价值。

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