Institut National de la Santé et de la Recherche Médicale, Unité 1102, Institut de Cancérologie de l’Ouest, Nantes-Saint Herblain, France.
Haematologica. 2013 Feb;98(2):316-22. doi: 10.3324/haematol.2012.071712. Epub 2012 Aug 8.
Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T(H) lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.
通过体外引发产生肿瘤抗原特异性 CD4(+) T 辅助细胞 (T(H)) 系对于癌症的过继细胞治疗具有重要意义，但由于缺乏合适的工具来鉴定和分离低频率的肿瘤抗原特异性 CD4(+) T 细胞，该方法的发展受到限制。在这里，我们使用了最近开发的 MHC Ⅱ类/肽四聚体，该四聚体包含来自 NY-ESO-1（ESO）的免疫显性肽，ESO 是一种在不同的人类实体瘤和血液恶性肿瘤中经常表达的肿瘤抗原，来实施体外引发平台，从而生成 ESO 特异性 T(H) 系。我们通过流式细胞术分选从循环淋巴细胞中分离出表型定义的 CD4(+) T 细胞亚群，并用肽 ESO(119-143)、自体 APC 和 IL-2 进行体外刺激。我们通过用 DR52b/ESO(119-143)四聚体（ESO-四聚体）染色来评估培养物中 ESO 特异性细胞的频率，并通过用 TCR 可变β链（BV）特异性抗体共同染色来评估 ESO-四聚体(+)细胞的 TCR 库。我们通过流式细胞术分选分离 ESO-四聚体(+)细胞，并通过 PHA、APC 和 IL-2 进行扩展，以生成 ESO 特异性 T(H) 系。我们通过用 ESO 肽或重组蛋白刺激来鉴定这些系的抗原识别，通过用特异性抗体进行细胞内染色来鉴定细胞因子产生，通过用同种异体 APC 刺激来鉴定同种异体反应性。使用这种方法，我们可以从常规的 CD4(+)CD25(-)幼稚和中央记忆群体中一致地生成 ESO-四聚体(+) T(H) 系，但不能从效应记忆群体或 CD4(+)CD25(+)Treg 中生成。体外引发的 T(H) 系识别 ESO 的亲和力与用 ESO 疫苗免疫的患者中的 ESO-四聚体(+)细胞相当，并使用相似的 TCR 库。在这项研究中，我们使用 MHC Ⅱ类/ESO 四聚体，实施了体外引发平台，从而能够从非免疫个体中生成 ESO 单特异性多克隆 T(H) 系。这是一种对于过继细胞治疗携带 ESO 表达肿瘤的患者具有潜在意义的方法。
