Luscher M A, Newton B L, Barber B H
Department of Immunology, University of Toronto, Ontario, Canada.
J Immunol. 1994 Dec 1;153(11):5068-81.
W6/32 is a monomorphic anti-HLA class I Ab that cross-reacts with the product of the exchange of murine beta 2-microglobulin (beta 2-m) with human or bovine beta 2-m on H-2 Db. Using W6/32 we have developed a simple and rapid flow cytometric method to measure the beta 2-m exchange kinetics for Db at the surface of intact H-2b cell lines. We find that 10 to 25% of the Db heavy chains exchange bound beta 2-m for soluble beta 2-m with a t1/2 of 10 to 15 min. The kinetics of the exchange are consistent with a mechanism that includes a free heavy chain intermediate on the reaction coordinate. The remainder of Db molecules appear to be refractory to beta 2-m exchange even after long-term culture with an exogenous source of beta 2-m. The exchange process proceeds at a similar rate on wild-type cells that bear a diverse complement of autologous peptides on their class I molecules, and on mutant RMA-S cells having class I molecules primarily occupied with a single, defined, high affinity synthetic peptide Ag. The t1/2 for dissociation of a radiolabeled analogue of a high affinity naturally presented peptide Ag is 10 h or more, implying that the free heavy chain intermediate in the beta 2-m exchange mechanism retains bound peptides. Further, we find that synthetic peptide Ags added to non-mutant cells bind both to class I molecules that subsequently exchange bound beta 2-m, and to molecules that retain endogenous beta 2-m.
W6/32是一种单克隆抗HLA I类抗体，它能与小鼠β2-微球蛋白（β2-m）与人类或牛β2-m在H-2 Db上交换的产物发生交叉反应。利用W6/32，我们开发了一种简单快速的流式细胞术方法，用于测量完整H-2b细胞系表面Db的β2-m交换动力学。我们发现，10%至25%的Db重链会将结合的β2-m与可溶性β2-m进行交换，半衰期为10至15分钟。交换动力学与一种机制一致，该机制在反应坐标上包括一个游离重链中间体。即使在用外源β2-m长期培养后，其余的Db分子似乎也难以进行β2-m交换。在野生型细胞（其I类分子上带有多种自体肽）和突变的RMA-S细胞（其I类分子主要被单一、明确的高亲和力合成肽抗原占据）上，交换过程以相似的速率进行。高亲和力天然呈递肽抗原的放射性标记类似物的解离半衰期为10小时或更长，这意味着β2-m交换机制中的游离重链中间体保留了结合的肽。此外，我们发现添加到非突变细胞中的合成肽抗原既能与随后交换结合的β2-m的I类分子结合，也能与保留内源性β2-m的分子结合。
