Restifo N P, Marincola F M, Kawakami Y, Taubenberger J, Yannelli J R, Rosenberg S A
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Natl Cancer Inst. 1996 Jan 17;88(2):100-8. doi: 10.1093/jnci/88.2.100.
In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8+ T cells) can cause the regression of even large tumors. These antitumor CD8+ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the alpha chain and a noncovalently associated, soluble protein called beta2-microglobulin (beta 2m). Loss of beta 2m generally eliminates antigen recognition by antitumor CD8+ T cells.
We studied the loss of beta 2m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy.
We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 40 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry.
After one melanoma cell line (1074mel) was found not to express functional beta 2m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of alpha-chain mRNA but variable levels of beta 2m mRNA. In addition, no immunoreactive beta 2m protein was detected by western blot analysis. When human beta 2m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be beta 2m positive. This result was consistent with the hypothesis that loss of beta 2m resulted from immunotherapy.
These data suggest that the loss of beta 2m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.
在一部分转移性黑色素瘤患者中，带有细胞表面标志物CD8的T淋巴细胞（CD8 + T细胞）甚至能使大肿瘤消退。这些抗肿瘤CD8 + T细胞识别由主要组织相容性复合体（MHC）I类分子呈递在肿瘤细胞表面的肽抗原。MHC I类分子是一种异二聚体，由一个称为α链的整合膜糖蛋白和一个非共价结合的可溶性蛋白β2微球蛋白（β2m）组成。β2m的缺失通常会消除抗肿瘤CD8 + T细胞的抗原识别。
我们研究了β2m的缺失作为接受免疫治疗的一组患者中肿瘤逃避免疫识别的一种潜在机制。
我们从40例连续接受转移性黑色素瘤免疫治疗且有肿瘤标本的患者队列中的13例患者的肿瘤标本中成功培养出13个独立的肿瘤细胞培养物。这些细胞系，以及另一个从细胞因子基因治疗研究中的一名患者的肿瘤中获得的黑色素瘤细胞系（称为1074mel），分别通过体外细胞荧光法检测MHC I类表达，通过Northern和Western印迹分析检测信使RNA（mRNA）和蛋白质表达，并通过免疫组织化学进行离体检测。
通过细胞荧光分析发现一个黑色素瘤细胞系（1074mel）不表达功能性β2m后，在13个新建立的黑色素瘤细胞系中有4个（31%）被发现完全缺乏功能性MHC I类表达。对从5个无功能性MHC I类表达的细胞系中提取的RNA进行Northern印迹分析表明，这些细胞含有正常水平的α链mRNA，但β2m mRNA水平各不相同。此外，Western印迹分析未检测到免疫反应性β2m蛋白。当使用重组痘苗病毒瞬时表达人β2m时，细胞表面MHC I类表达得以重建，并且这5个细胞系呈递内源性抗原的能力得以恢复。肿瘤切片的免疫组织化学染色显示体内缺乏免疫反应性MHC I类，支持体外观察结果并非人为因素造成的观点。此外，从3例患者免疫治疗前获取的存档肿瘤切片显示为β2m阳性。这一结果与β2m缺失是由免疫治疗导致的假设一致。
这些数据表明β2m的缺失可能是肿瘤细胞获得免疫抗性的一种机制。本研究首次对接受免疫治疗的一组患者中肿瘤逃避免疫识别的分子途径进行了表征。
