从胰腺癌患者中扩增肿瘤反应性T细胞。

Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.

作者信息

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

机构信息

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.

出版信息

J Immunother. 2016 Feb-Mar;39(2):81-9. doi: 10.1097/CJI.0000000000000111.

DOI:10.1097/CJI.0000000000000111

PMID:26849077

Abstract

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.

摘要

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。