Unit of Immuno-Biotherapy of Melanoma and Solid Tumors, Division of Molecular Oncology, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy.
Cancer Immunol Immunother. 2012 Aug;61(8):1169-82. doi: 10.1007/s00262-011-1179-z. Epub 2011 Dec 30.
Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.
肿瘤浸润淋巴细胞 (TILs) 已成功用于过继细胞转移 (ACT) 免疫治疗；然而，由于其数量稀少，这种治疗方法只能在有限的一部分皮肤黑色素瘤患者中进行。我们评估了是否可以确定一种从外周血单核细胞 (PBMC) 体外扩增 T 细胞的有效方案，该方案适用于皮肤和眼黑色素瘤患者的 ACT。从皮肤和眼黑色素瘤患者的 PBMC 中，在 IL-2 和 IL-15 的存在下，用自体照射的黑色素瘤细胞（混合淋巴细胞肿瘤细胞培养物；MLTCs）体外刺激，然后进行快速扩增方案（REP）。这些 T 淋巴细胞的功能活性进行了表征，并与 TILs 进行了比较。此外，还分析了眼黑色素瘤病变的体内免疫浸润。从 7 例皮肤和眼转移性黑色素瘤患者获得的所有 PBMC 样本中，均实现了高效的体外 MLTC 扩增黑色素瘤反应性 T 细胞。当将 REP 方案应用于这些 MLTC 时，可以获得大量的黑色素瘤特异性 T 细胞。大多数 MLTC 富含均匀表达共刺激分子（例如 NKG2D、CD28、CD134、CD137）的非终末分化 T(EM)细胞。从同一患者分离的短期体外培养的 TILs 检测到类似的抗肿瘤活性，同时共刺激分子的表达也存在差异。在这些眼黑色素瘤患者中，我们观察到具有抑制特征的免疫浸润和低比例的体外生长 TILs（我们的病例中有 28.5%）。我们的 MLTC 方案克服了这一限制，即使在这些患者中，也允许分离具有效应功能的 T 淋巴细胞。因此，我们的新型体外富集方案可从黑色素瘤患者中有效分离抗肿瘤循环 PBMC 衍生的 T 细胞。该方案似乎适用于皮肤和眼黑色素瘤患者的 ACT 研究。
