Manoury-Schwartz B, Chiocchia G, Lotteau V, Fournier C
INSERM U283, Université René Descartes, Hôpital Cochin, Paris.
Int Immunol. 1997 Apr;9(4):581-9. doi: 10.1093/intimm/9.4.581.
Type II collagen (CII) is an arthritogenic self antigen in DBA/1 (H-2q) mice. To analyze the intracellular processing of this fibrillar protein in the context of I-Aq molecules, we have generated hybrid antigen-presenting cells (APC) by fusion of B lymphoma (A20 and M12) cells with CII-primed spleen cells from DBA/1 mice. Efficient presentation of CII by these APC to specific T cell hybridomas required prior cleavage of the antigen and intracellular handling of the peptides. Inhibition of protein transport by brefeldin A prevented the presentation of CII peptides to T cell hybridomas, indicating that the intracellular presentation of CII was dependent on neo-synthesis of I-Aq molecules. In contrast, exposure of hybrid B lymphomas to leupeptin, a protease inhibitor, induced a dose-dependent increase of CII-specific T cell response, while abrogating the I-Aq-restricted presentation of ovalbumin. The enhancing effect of leupeptin was also observed when immune B cells were used as APC. In contrast, leupeptin inhibited the presentation of CII peptides by macrophages or total spleen cells. Pulse-chase analysis of metabolically labeled hybrid APC and immunoprecipitation with antibodies specific for class II molecules or invariant (li) chain revealed that leupeptin did not affect the li chain processing or the formation of stable class II dimers. The stimulatory effect of leupeptin observed on CII presentation suggests that leupeptin protects CII epitopes by interfering with proteases involved in the intracellular degradation of CII.
II型胶原蛋白（CII）是DBA/1（H-2q）小鼠中的一种致关节炎自身抗原。为了在I-Aq分子的背景下分析这种纤维状蛋白的细胞内加工过程，我们通过将B淋巴瘤（A20和M12）细胞与来自DBA/1小鼠的经CII致敏的脾细胞融合，生成了杂交抗原呈递细胞（APC）。这些APC将CII有效呈递给特异性T细胞杂交瘤需要抗原预先裂解以及肽的细胞内处理。布雷菲德菌素A对蛋白质转运的抑制作用阻止了CII肽向T细胞杂交瘤的呈递，表明CII的细胞内呈递依赖于I-Aq分子的重新合成。相比之下，将杂交B淋巴瘤暴露于蛋白酶抑制剂亮抑酶肽，可诱导CII特异性T细胞反应呈剂量依赖性增加，同时消除卵清蛋白的I-Aq限制性呈递。当使用免疫B细胞作为APC时，也观察到了亮抑酶肽的增强作用。相反，亮抑酶肽抑制巨噬细胞或全脾细胞对CII肽的呈递。对代谢标记的杂交APC进行脉冲追踪分析，并用针对II类分子或恒定链（li）的特异性抗体进行免疫沉淀，结果表明亮抑酶肽不影响li链的加工或稳定II类二聚体的形成。观察到亮抑酶肽对CII呈递的刺激作用表明，亮抑酶肽通过干扰参与CII细胞内降解的蛋白酶来保护CII表位。
