The Clonal Analysis of Effector Mechanism in Graft Rejection

极限稀释分析（LDA）是一种高精度的定量方法，是阐明排斥机制和耐受性的重要手段。通过计算Tcp的频率[f (Tcp)]，可以估计Tcp （T细胞毒性前体）的绝对克隆大小。实验模型为大鼠异位心脏移植。Buffalo （BUF: RT-1^b）大鼠作为心脏供体，Wistar-Furth （WFu: RT-1^u）大鼠作为受体。未处理大鼠脾脏T细胞的f （Tcp）为1:2343<±>591，而排斥前的f （Tcp）高达1/195<±>123，约为未处理大鼠的12倍。然而，同种异体移植的WFu大鼠在排斥反应后f （Tcp）降低了1∶696<正负>243。因此，本研究集中于移植后第5天持续排斥反应中移植物浸润细胞（GICs）的f （Tcp）。同种异体GICs的f （Tcp）比同种异体GICs的f （Tcp）高1∶113，而同种异体GICs的f （Tcp）比同种异体GICs的f （Tcp）高1∶8584。同种异体移植物获得的GICs数量为（2.1<±>1.1）x10^7/块。这是从等移植物中分离出来的10倍。对GICs-T进行FACS分析，OX19^+ (T)、OX8^+ (Tc/s)和W3/25^+ (Th)细胞的百分数分别为70.9%、64.5%和25.9%。分别。移植后第5天，脾脏和淋巴结T细胞的f (Tcp)s分别为1∶195<正负>123、1∶731<正负>81。然而，GICs的f （Tcp）最高，为1:84<正负>53。因此，在移植后第5天，大约1.2%（1/84）的GICs-T细胞是供体特异性的Tcp。另一方面，GICs也有能够以供体特异性的方式抑制混合淋巴细胞培养（MLC）增殖反应的T细胞。我们可以在移植的第5天从GICs中克隆出一些克隆体。这些无性系的表面标记为Ox19^+ (T)、W3/25^+ (Th)、OX8^- (Ts/c)和OX22^+ (LCA)。这些克隆能够以供体特异性的方式抑制MLC反应。因此，提示在移植后第5天的持续排斥反应中，可能存在T细胞抑制供体抗原特异性Tc细胞无限制增殖的调节机制。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