Breg通过GrB信号抑制效应性T细胞调节肝移植免疫的机理研究

Both clinical studies and animal experiments show that regulatory B cells play an important role in immune regulation following organ transplantation. B cells were first reported to be induced to secrete granzyme B in 2006. In our previous study we found that B lymphocytes purified from peripheral blood of healthy controls could produce granzyme B upon activation and exert regulatory function; In patients with kiddney transplantation this specific B cell subset could also be induced under certain condition and produce granzyme B upon activation. Furthermore, we detected that the granzyme B-producing B cells was associated with renal function in patients with renal transplantation when we analyzed various clinical risk factors. After coculture of the purified granzyme B-producing B cells and purified CD4+ T cells activated under certain conditions the production of IFN-γ secreted by CD4+ T cells decreased. Clinically, liver transplant patients are prone to induce immune tolerance while the exact mechanism remains unknown with respect to the function of regulatory B cells. As is proved that the TCR ζ chain is a substrate of granzyme B, we speculate that B cells can be activated to differentiate into granzyme B-producing B cells and thus to suppress T cell proliferation via granzyme B regulating immune tolerance. We aim to induce the granzyme B-producing B cells in patients with long-term normal liver function following liver transplantation, analyze the phenotype of the B cell subset and the molecular mechanism via cell coculture and signal blocking by serine proteinase inhibitor 9. Moreover, in rat liver transplantation model we will confirm our proposed theory by blocking the granzyme B in vivo and in vitro. Therefore, we hope to provide a new way for clinical therapy for patients with organ transplantation.

调节性B 细胞（Breg）在器官移植术后免疫耐受的形成过程中发挥重要调节作用。2006年有研究首次报道淋巴瘤的B细胞可分泌颗粒酶B（GrB），我们前期研究发现健康人外周血的Breg也能分泌GrB，有免疫调节作用；进一步研究发现 Breg能直接抑制移植受体内效应性CD4+T细胞的活化。临床上肝移植患者容易诱导免疫耐受，而Breg在肝移植免疫耐受形成中的作用机理并不清楚。据研究报道TCR ζ链是GrB的作用底物，因此我们提出假说：在临床长期存活肝移植患者外周血中，Breg通过GrB与CD4+T效应细胞的TCR ζ链相互作用，抑制效应性T细胞活化增殖并诱导凋亡。本项目拟通过细胞免疫学和分子生物学技术，明确肝移植患者外周血中分泌GrB的Breg表型及上游信号通路，探明Breg抑制效应性T细胞的机理；再通过大鼠肝移植模型，阻断GrB信号通路，验证上述结论，为临床诱导免疫耐受奠定理论基础。

背景：在临床上，肝脏移植容易诱导免疫耐受，因此研究肝移植患者长期存活的免疫调节机制，能够给临床免疫耐受的诱导，提供有力的支持和证据。目前研究发现B调节性细胞在自身免疫性疾病以及肾移植患者免疫调节方面发挥重要作用，肝移植中的作用还不清楚，然而其作用的B细胞亚群及机制并不明确。.内容：首先明确肝移植术后长期存活患者外周血产生GrB的调节性B细胞亚群及表型特点，再探讨研究调节性B细胞产生GrB的上游信号通路及下游抑制效应性T细胞发挥免疫调节的机制，最后通过肝移植模型，验证分泌GrB的调节性B细胞亚群的调节机制。.结果：1. 肝移植患者外周血B细胞经刺激后可以产生GrB和IL10，不能产生GrA和Perforin。不同药物刺激后B细胞产生的GrB的效果不同，与空白对照相比，CpG作用效果最强。IL21和IL2能显著增强B细胞活化产生GrB的效果。而产生GrB的B细胞不同于产生IL10的B细胞。.2. 与健康对照比较，长期肝移植患者外周血B细胞经刺激后高表达CD38，CD27，CD138，CD5。Western blotting实验，发现肝移植患者外周血激活GrB+Breg后其JAK1/STAT3表达升高。PCR检测发现IgG+IgM+IL21 与空白对照相比，mRNA水平明显增加。.3. 将肝移植患者外周血纯化的B细胞刺激后与健康人群的CD25-CD4+T细胞做共培养，提示T细胞增殖程度受到抑制，通过GrB发挥作用。CpG: n=7, 32.6 ±18.8%; CpG+Inhibitor: n=7, 13.7 ±14.05%; CpG+IL-2: n=17, 49.84±12.2%; CpG+IL-2+ Inhibitor: n=17, 29.59 ±12.9%; CD40L: n=5, 35.4 ±25.6%; CD40L+ Inhibitor: n=5, 23.57 ±16.0%; CpG+IL-21: n=13, 19.4 ±15.0%; CpG+IL-21+Inhibitor: n=13, 8.4 ±8.9% （p<0.05）.科学意义：通过研究发现，与健康对照比较，长期存活的肝移植患者体内存在表达高表达GrB的调节性B细胞，可以通过GrB抑制T细胞发挥免疫调节作用，有可能是诱导免疫耐受的一个方向。.