Service d'Anatomie et Cytologie Pathologiques, Centre Hospitalier Universitaire Hôtel Dieu, Nantes, France.
CRCINA, INSERM, Université d'Angers, Université de Nantes, Nantes, France.
Mod Pathol. 2020 Mar;33(3):468-482. doi: 10.1038/s41379-019-0322-9. Epub 2019 Aug 13.
We previously demonstrated that HLA-E/β2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, β2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/β2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94 tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/β2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/β2m colorectal cancers were significantly enriched in CD94 intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94 tumor-infiltrating lymphocytes mostly corresponded to CD8 αβ T cells, and to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94 intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/β2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.
我们之前的研究表明，结直肠癌中肿瘤细胞 HLA-E/β2m 的过度表达与预后不良有关。然而，上皮内肿瘤浸润淋巴细胞中其特异性受体 CD94/NKG2 的表达、其确切表型和功能，以及与结直肠癌的分子状态和预后的关系尚不清楚。基于 234 例结直肠癌患者的回顾性队列，我们通过组织微阵列免疫组化评估 HLA-E、β2m、CD94、CD8 和 NKp46 的表达。肿瘤细胞 HLA-E/β2m 的表达谱和肿瘤浸润淋巴细胞的密度与临床病理和分子特征（微卫星状态、BRAF 和 RAS 突变）相关。然后，从 27 例前瞻性结直肠癌的原发性肿瘤中，我们通过多参数流式细胞术对分离出的 CD94 肿瘤浸润淋巴细胞的性质（T 和/或 NK 细胞）及其抑制性 NKG2A 或激活性 NKG2C 链的共表达进行了特征描述。使用体外定向细胞溶解活性测定法确定了它们的生物学功能。我们的结果表明，与微卫星稳定型相比，微卫星不稳定型 HLA-E/β2m 优先过度表达（分别为 45%和 19%，p<0.0001），而与 RAS 或 BRAF 突变状态无关。然而，HLA-E/β2m 结直肠癌在微卫星不稳定和微卫星稳定的肿瘤中明显富含 CD94 上皮内肿瘤浸润淋巴细胞。这些 CD94 肿瘤浸润淋巴细胞主要对应于 CD8αβ T 细胞，其次是 NK 细胞，并且主要共表达功能性抑制性 NKG2A 链。最后，与肿瘤细胞密切接触的高数量 CD94 上皮内肿瘤浸润淋巴细胞与总生存期较差独立相关。总之，这些发现强烈表明，HLA-E/β2m-CD94/NKG2A 代表了一种新的可药物治疗的抑制性免疫检查点，优先在微卫星不稳定的肿瘤中表达，但也在微卫星稳定的肿瘤亚群中表达，为结直肠癌免疫治疗提供了新的机会。
