Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.

人类白细胞抗原I类（HLA class I, HLA-I）与人类白细胞抗原II类（HLA class II, HLA-II）复合物介导的抗原呈递，需依赖各自通路特异性的蛋白实现。恒定链（invariant chain, Ii）衍生的肽段CLIP，可通过与不同HLA-II肽结合槽发生瞬时且宽泛的结合，在抗原装载前稳定HLA-II分子，进而参与HLA-II介导的抗原呈递过程。本研究证实，CLIP可与白血病细胞表面的HLA-I分子发生非常规结合。在HLA-II阴性的急性髓系白血病（acute myeloid leukemia, AML）细胞中，我们检测到CLIP肽段在细胞膜表面的呈递。对AML细胞进行Ii基因沉默后，其细胞表面HLA-I的呈递水平显著降低，这表明CLIP在HLA-I抗原呈递通路中发挥直接作用。从B淋巴母细胞样细胞系（B-lymphoblastoid cell lines, B-LCL）的HLA-I特异性肽洗脱物中，我们共鉴定出5种Ii衍生肽段，其中2种来源于CLIP区域。体外肽结合实验结果显示，洗脱获得的CLIP肽段RMATPLLMQALPM可高效结合4种不同的HLA-I超型：-A2、-B7、-A3和-B40。此外，该CLIP肽段的截短变体同样可结合这4种HLA-I超型，尽管计算机模拟算法仅预测其可与HLA-A2或HLA-B7结合。用这些肽段免疫HLA-A2转基因小鼠后，未诱导出细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）应答。综上，本研究数据表明CLIP具有极强的结合宽泛性，可与多种不同的HLA-I分子结合。CLIP参与HLA-I抗原呈递通路这一发现，既可能反映了白血病细胞中的异常机制，亦可为阐明全新的抗原加工通路或免疫逃逸机制提供新的研究思路。