The Clonal Analysis of Effector Mechanism in Graft Rejection

极限稀释分析(LDA)是一种准确度高的定量方法，是阐明排斥机制和耐受性不可缺少的手段。通过计算TcP[f(Tcp)]的频率，可以估计Tcp(T细胞毒前体)的绝对克隆大小。实验模型为大鼠异位心脏移植。水牛(BUF：RT-1^b)大鼠为心脏供体，Wistar-Furth(WFU：RT-1^u)大鼠为受体。未经治疗的大鼠脾T细胞的f(Tcp)为1：2343+-gt；591，但排斥前的f(Tcp)高达1/195&lt；+-gt；123，约为未治疗大鼠的12倍。然而，移植的WFU大鼠在移植排斥反应后，f(Tcp)降低了1：696和正负243。因此，本研究集中在移植后第5天持续排斥期间移植物浸润性细胞(GICs)的f(Tcp)。同种异体移植物中的GIC显示…的f(Tcp)非常高与同种异体的f(Tcp)1：8584相比，增加了1：113。从同种异体移植物中获得的GICs数为(2.1；+-1.1)×107个/片。这是从同种异体分离出来的数量的十倍。GICS-T的流式细胞术分析显示，OX19^+(T)、OX8^+(TC/S)和W3/25^+(Th)细胞的百分比分别为70.9%、64.5%和25.9%。移植后第5天，脾T细胞和淋巴结T细胞的f(Tcp)S分别为1：195和1：731。然而，GIC的f(Tcp)最高，为1：84&+-gt；53。因此，约1.2%(1/84)的GICs-T细胞是供体特异性Tcp。另一方面，GICs-T细胞也以供体特异性的方式抑制混合淋巴细胞培养(MLC)的增殖反应。我们可以在移植的第5天从GICs中克隆出一些细胞。这些无性系的表面标记分别为OX19^+(T)、W3/25^+(Th)、OX8^-(ts/c)和OX22^+(Lca)。这些克隆能以供体特异性方式抑制MLC反应。因此，在移植后第5天持续排斥期间，T细胞可能存在一种调节机制，可抑制供体抗原特异性TC细胞在移植物中的无限增殖。较少

Limiting Dilution Analysis (LDA) is a quantitative method with a high degree of accuracy and an indispensable way for the elucidation of rejection mechanism and tolerance. It is possible to estimate the absolute clonal size of Tcp (T cytotoxic precursor) by calculating the frequency of Tcp[ f (Tcp)]. The experimental model is a heterotopic heart transplantation in rats. Buffalo (BUF : RT-1^b) rats were used as heart donors and Wistar-Furth (WFu : RT-1^u) rats as recipients. The spleen T cells of untreated rats had a f (Tcp) of 1:2343<plus-minus>591, but the f (Tcp) just before rejecti on increased up to 1/195<plus-minus>123 which was about 12 times as high as that of the untreated rats. However, the allografted WFu rats had a decreased f (Tcp) of 1 : 696<plus-minus>243 after graft rejection.Thus, the present study concentrated upon the f (Tcp) of graft infiltrating cells (GICs) on day 5 postgrafting during ongoing rejection. The GICs in an allograft showed a remarkably high f (Tcp) of … More 1 : 113 in comparison with a f (Tcp) of 1 : 8584 seen in an isograft. The number of GICs obtained from allografts was (2.1<plus-minus>1.1)x10^7/graft. This was ten times as many as that isolated from isografts. The FACS analysis of GICs-T revealed that the number of percentage in OX19^+ (T), OX8^+ (Tc/s) and W3/25^+ (Th) cells were 70.9%, 64.5% and 25.9%. respectively.On day 5 postgrafting, the f (Tcp)s of spleen and lymphnode T cells were 1 : 195<plus-minus>123, 1:731<plus-minus>81, respectively. However, the GICs had the highest f (Tcp) of 1:84<plus-minus>53. Therefore, about 1.2% (1/84) of GICs-T cells was the donor specific Tcp on day5 postgrafting during ongoing rejeOn the other hand, the GICs also had T cells which could suppress the proliferative response of Mixed Lymphocyte Culture (MLC) in a donor specific manner. We could make some clones from the GICs on day 5 transplantation. The surface markers of these clones were Ox19^+ (T), W3/25^+ (Th),OX8^- (Ts/c), and OX22^+ (LCA). These clones could suppress the MLC response in a donor specific manner.Therefore, it is suggested that there might be a regulation mechanism by the T cells which could suppress the unlimited proliferation of donor antigen specific Tc cells in the graft during ongoing rejection on day 5 postgrafting. Less