Labarrière Nathalie, Gervois Nadine, Bonnin Annabelle, Bouquié Régis, Jotereau Francine, Lang François
INSERM U601, 44093 Nantes, France.
Cancer Immunol Immunother. 2008 Feb;57(2):185-95. doi: 10.1007/s00262-007-0361-9. Epub 2007 Jul 24.
Choosing a reliable source of tumor-specific T lymphocytes and an efficient method to isolate these cells still remains a critical issue in adoptive cellular therapy (ACT). In this study, we assessed the capacity of MHC/peptide based immunomagnetic sorting followed by polyclonal T cell expansion to derive pure polyclonal and tumor-reactive Melan-A specific T cell populations from melanoma patient's PBMC and TIL. We first demonstrated that this approach was extremely efficient and reproducible. We then used this procedure to compare PBMC and TIL-derived cells from three melanoma patients in terms of avidity for Melan-A A27L analog, Melan-A(26-35)and Melan-A(27-35), tumor reactivity (lysis and cytokine production) and repertoire. Regardless of their origin, i.e., fresh PBMC, peptide stimulated PBMC or TIL, all sorted populations (from the three patients) were cytotoxic against HLA-A2+ melanoma cell lines expressing Melan-A. Although some variability in peptide avidity, lytic activity and cytokine production was observed between populations of different origins in a given patient, it differed from one patient to another and thus no correlation could be drawn between T cell source and reactivity. Analysis of Vbeta usage within the sorted populations showed the recurrence of Vbeta3 and Vbeta14 subfamilies in the three patients but differences in the rest of the Melan-A repertoire. In addition, in two patients, we observed major repertoire differences between populations sorted from the three sources. We especially documented that in vitro peptide stimulation of PBMC, used to facilitate the sort by enriching in specific T lymphocytes, could significantly alter their repertoire and reactivity towards tumor cells. We conclude that PBMC which are easily obtained from all melanoma patients, can be as good a source as TIL to derive high amounts of tumor-reactive Melan-A specific T cells, with this selection/amplification procedure. However, the conditions of peptide stimulation should be improved to prevent a possible loss of reactive clonotypes.
在过继性细胞疗法（ACT）中，选择可靠的肿瘤特异性T淋巴细胞来源以及高效的细胞分离方法仍然是一个关键问题。在本研究中，我们评估了基于MHC/肽的免疫磁珠分选结合多克隆T细胞扩增从黑色素瘤患者外周血单核细胞（PBMC）和肿瘤浸润淋巴细胞（TIL）中获得纯多克隆且具有肿瘤反应性的Melan - A特异性T细胞群体的能力。我们首先证明了这种方法极其高效且可重复。然后，我们使用该程序比较了来自三名黑色素瘤患者的PBMC和TIL来源的细胞对Melan - A A27L类似物、Melan - A(26 - 35)和Melan - A(27 - 35)的亲和力、肿瘤反应性（细胞裂解和细胞因子产生）以及T细胞受体库。无论其来源如何，即新鲜PBMC、肽刺激的PBMC或TIL，所有分选群体（来自三名患者）对表达Melan - A的HLA - A2 +黑色素瘤细胞系均具有细胞毒性。尽管在给定患者中不同来源的群体之间观察到肽亲和力、裂解活性和细胞因子产生存在一些差异，但患者之间存在差异，因此无法得出T细胞来源与反应性之间的相关性。对分选群体中Vβ使用情况的分析表明，三名患者中Vβ3和Vβ14亚家族反复出现，但Melan - A T细胞受体库的其余部分存在差异。此外，在两名患者中，我们观察到从三种来源分选的群体之间存在主要的T细胞受体库差异。我们特别记录到，用于通过富集特异性T淋巴细胞来促进分选的PBMC体外肽刺激，可显著改变其T细胞受体库以及对肿瘤细胞的反应性。我们得出结论，通过这种分选/扩增程序，从所有黑色素瘤患者中容易获得的PBMC可以作为与TIL一样好的来源，用于获得大量具有肿瘤反应性的Melan - A特异性T细胞。然而，应改善肽刺激条件以防止反应性克隆型的可能丢失。
