Nielsen M B, Marincola F M
Surgery Branch, National Cancer Institute, Bethesda, MD 20893-1502, USA.
Cancer Chemother Pharmacol. 2000;46 Suppl:S62-6. doi: 10.1007/pl00014052.
In recent years significant progress in the understanding of the immune biology of melanoma has evolved from the identification of melanoma antigens (MAs) recognized by T cells. MAs consist of intracellular proteins that are expressed on the surface of cancer cells in association with human leukocyte antigen (HLA) class I molecules and therefore are suitable targets for cytotoxic T lymphocytes (CTLs). Several new monitoring strategies have been implemented to evaluate the status of activation and localization of vaccine-induced T cells in the peripheral circulation as well as the tumor site, including limiting dilution, in vitro sensitization, and ELISPOT. Previous studies aimed at monitoring patients receiving vaccination have utilized mainly those three methods. These methods have demonstrated that antigen-specific vaccination can elicit immune responses detectable in the peripheral blood of immunized patients. These assays, however, have been faulted by their requirement for in vitro expansion of T cells (limiting dilution or in vitro sensitization) or for limited sensitivity (ELISPOT). More recently, the use of soluble HLA/peptide complex tetramers, intracellular fluorescence-activated cell sorting (FACS) analysis, and real-time polymerase chain reaction (PCR) has been proposed for the monitoring of vaccine trials. These methods have the appeal of allowing direct enumeration of T cells specific for a particular epitope within relevant samples such as peripheral blood lymphocytes, lymph nodes, and tumors. We are evaluating whether utilizing a combination of HLA/peptide tetramer (tHLA) together with Taqman-based real-time reverse-transcription (RT)-PCR and intracellular FACS analysis could establish a direct and comprehensive strategy for the assessment of epitope-specific immune response in vivo. In conditions close to those of the tumor microenviroment or in peripheral blood lymphocytes, however, a different status of T cell activation can be expected due to a direct stimulation of T cells by tumor or antigen-presenting cells. We observed that activated T cells can easily be detected in the peripheral blood of patients who have received MA-specific vaccines. However, when T cells are stimulated with their relevant epitope, a high level of T cell receptor downregulation occurs that does not allow identification of vaccine-specific T cells directly with tHLA. Thus evaluation of epitope-specific T cells at the tumor site, where they might be exposed to stimulation by interaction with tumor cells and/or in bulk peripheral blood mononuclear cells, might be more efficiently analyzed with functional methods such as intracellular FACS and Taqman-based real time RT-PCR.
近年来，对黑色素瘤免疫生物学的认识取得了重大进展，这源于对T细胞识别的黑色素瘤抗原（MA）的鉴定。MA由细胞内蛋白质组成，这些蛋白质与人类白细胞抗原（HLA）I类分子结合后在癌细胞表面表达，因此是细胞毒性T淋巴细胞（CTL）的合适靶点。已经实施了几种新的监测策略来评估疫苗诱导的T细胞在外周循环以及肿瘤部位的激活和定位状态，包括有限稀释法、体外致敏法和酶联免疫斑点法（ELISPOT）。以往旨在监测接受疫苗接种患者的研究主要采用了这三种方法。这些方法表明，抗原特异性疫苗接种可引发免疫反应，在免疫患者的外周血中可检测到。然而，这些检测方法存在缺陷，因为它们需要体外扩增T细胞（有限稀释法或体外致敏法）或灵敏度有限（ELISPOT）。最近，有人提出使用可溶性HLA/肽复合物四聚体、细胞内荧光激活细胞分选（FACS）分析和实时聚合酶链反应（PCR）来监测疫苗试验。这些方法的吸引力在于能够直接计数相关样本（如外周血淋巴细胞、淋巴结和肿瘤）中对特定表位特异的T细胞。我们正在评估将HLA/肽四聚体（tHLA）与基于Taqman的实时逆转录（RT）-PCR和细胞内FACS分析相结合，是否能够建立一种直接且全面的策略来评估体内表位特异性免疫反应。然而，在接近肿瘤微环境的条件下或在外周血淋巴细胞中，由于肿瘤或抗原呈递细胞对T细胞的直接刺激，可能会出现不同的T细胞激活状态。我们观察到，在接受MA特异性疫苗的患者外周血中很容易检测到激活的T细胞。然而，当T细胞受到其相关表位刺激时，会发生高水平的T细胞受体下调，这使得无法直接用tHLA鉴定疫苗特异性T细胞。因此，在肿瘤部位评估表位特异性T细胞可能更有效地通过功能方法进行分析，如细胞内FACS和基于Taqman的实时RT-PCR，在肿瘤部位，它们可能通过与肿瘤细胞和/或大量外周血单核细胞相互作用而受到刺激。
