Meng Qingda, Liu Zhenjiang, Rangelova Elena, Poiret Thomas, Ambati Aditya, Rane Lalit, Xie Shanshan, Verbeke Caroline, Dodoo Ernest, Del Chiaro Marco, Löhr Matthias, Segersvärd Ralf, Maeurer Markus J
Departments of *Laboratory Medicine, Division of Therapeutic Immunology ‡Microbiology, Tumor and Cell Biology, Karolinska Institute Departments of †Clinical Science, Intervention and Technology §Laboratory Medicine, Division of Pathology ∥Neurosurgery ¶CAST, Center for Allogeneic Stem Cell Transplantation, Karolinska Hospital, Stockholm, Sweden.
J Immunother. 2016 Feb-Mar;39(2):81-9. doi: 10.1097/CJI.0000000000000111.
Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-[gamma] production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V[beta] was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8(+) T cells. CD3(+)CD8(+), CD3(+)CD4(+), and CD3(+)CD4(-)CD8(-)[double-negative (DN) T cells] resided predominantly in central (CD45RA(-)CCR7(+)) and effector (CD45RA-CCR7-) memory subsets. CD8(+) TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4(+) TILs showed only up to 12% LAG-3(+) staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8(+)) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-[alpha] and IFN-[gamma] production. Twelve of 17 of CD8(+) TILs showed preferential expansion of certain TCR V[beta] families (eg, 99.2% V[beta]13.2 in CD8(+) TILs, 77% in the V[beta]1, 65.9% in the V[beta]22, and 63.3% in the V[beta]14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8(+) V[beta]13.2) reacting strongly against autologous tumor defined by INF-[gamma] production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.
产生对癌症具有反应性的T淋巴细胞是有效的过继性细胞疗法的先决条件。我们建立了一种从胰腺导管腺癌患者中获取肿瘤浸润淋巴细胞（TILs）的方案。将来自17例胰腺癌标本的肿瘤样本与细胞因子（IL-2、IL-15和IL-21）一起培养以扩增TILs。培养10天后，用抗CD3抗体（OKT3）和辐照的同种异体外周血单核细胞刺激TILs。通过流式细胞术检测细胞内细胞因子产生来检测TILs对肿瘤相关抗原（间皮素、生存素或NY-ESO-1）的反应性。使用铬51释放试验测量细胞毒性，并通过INF-γ产生（ELISA）检测TILs对自体肿瘤细胞的反应性。通过CD45RA、CCR7、4-1BB、LAG-3、PD-1、TIM3和CTLA-4标记分析来检测TIL组成。通过流式细胞术确定TCR Vβ，并通过PCR分析测量CDR3区域长度并随后测序来评估TCR克隆性。我们能够从17/17例患者中可靠地获得TILs，其中大多数是CD8(+) T细胞。CD3(+)CD8(+)、CD3(+)CD4(+)和CD3(+)CD4(-)CD8(-)[双阴性（DN）T细胞]主要存在于中央（CD45RA(-)CCR7(+)）和效应（CD45RA-CCR7-）记忆亚群中。CD8(+) TILs对LAG-3检测均呈阳性（约100%），而CD4(+) TILs仅显示高达12%的LAG-3(+)染色，并且PD-1在来自不同患者的TILs中呈现广泛的表达模式。通过ICS确定，来自个体患者的TILs对NY-ESO-1有强烈识别（CD8(+)中高达11.9%和8.2%），或分别通过TNF-α和IFN-γ产生确定对间皮素有强烈识别。17例CD8(+) TILs中有12例显示某些TCR Vβ家族的优先扩增（例如，CD8(+) TILs中99.2%的Vβ13.2、Vβ1中77%、Vβ22中65.9%和Vβ14家族中63.3%）。TCR CDR3分析显示单克隆或寡克隆TCR，其中一些（例如，CD8(+) Vβ13.2）通过INF-γ产生或细胞毒性对自体肿瘤有强烈反应。我们已经优化了生成胰腺癌特异性TILs的方法，这些TILs可用于胰腺癌患者的过继性细胞治疗。
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