Mitchell Malcolm S, Lund Teri A, Sewell Andrew K, Marincola Francesco M, Paul Elyse, Schroder Kim, Wilson Darcy B, Kan-Mitchell June
Department of Medicine, Wayne State University School of Medicine, Detroit, MI, 48201, USA.
Cancer Immunol Immunother. 2007 Mar;56(3):287-301. doi: 10.1007/s00262-006-0191-1. Epub 2006 Jul 28.
The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (10(9)) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2(+) MUC1(+) MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were "consensus" agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-alpha, IFN-gamma, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.
黏蛋白MUC1分子在多种腺癌中过表达，因此是免疫治疗的潜在靶点。在与HLA - A*0201（A2）结合的MUC1肽中，来自信号序列的M1.2（LLLLTVLTV）似乎在人类中具有最强的免疫原性。在此我们表明，大量（10⁹）与四聚体结合的M1.2特异性细胞毒性T淋巴细胞（CTL）可在体外从健康成年人的循环前体中产生。然而，所需共刺激信号的水平存在显著的个体差异。与四聚体结合的细胞在培养中也需要成熟，才能成为HLA - A2(+) MUC1(+) MCF7细胞系的高效杀伤细胞，已知该细胞系内源性加工的M1.2数量较少。然而，与针对HIV - 1表位的CTL相比，M1.2特异性CTL的功能亲和力较低。尽管亲和力较低，但M1.2特异性CTL具有多功能性，在与抗原识别脱颗粒时会分泌多种细胞因子。为了鉴定可能是更优免疫原的潜在激动剂肽，使用M1.2特异性CTL培养物筛选了一个大型九聚体组合肽库。在54个预测肽中，有4个是“共识”激动剂，因为它们被来自其他两个供体的CTL识别。当加载到用野生型HLA - A2转染的C1R靶细胞上时，两种激动剂p29（LLPWTVLTV）和p15（VLLWTVLTV）具有同等的刺激作用。两种激动剂均诱导M1.2特异性CTL产生IL - 2、TNF - α、IFN - γ并脱颗粒。相比之下，当用加载到用携带消除与CD8共受体结合突变的HLA - A2分子转染的C1R细胞上的肽刺激CTL时，这些通过T细胞受体特异性激活而受到严格调控的细胞因子的产生受到限制。因此，M1.2及其激动剂的激活均依赖于CD8，表明共受体的补偿对于人类T细胞对M1.2的反应是必要的。
