Centre de recherche de l'Hôpital Maisonneuve-Rosemont, Montreal, Quebec, Canada.
Division of Hematology-Oncology, Hôpital Maisonneuve-Rosemont and Department of Medicine, University of Montréal, Montreal, Quebec, Canada.
J Transl Med. 2015 Apr 19;13:123. doi: 10.1186/s12967-015-0495-z.
Adoptive transfer of minor histocompatibility antigen (MiHA)-specific T cells is a promising therapy for patients with hematological cancers. However, the efficacy of the transferred cells is hampered by the acquisition of terminal effector differentiation and exhaustion features during expansion in vitro thus preventing their function and persistence in vivo. Yet, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined and are likely to vary depending on the method used for expansion.
Using the clinically relevant HLA-A0201-restricted MiHA HA-1 as well as reagents and procedures that are readily transferable to a clinical environment, we designed a novel culture protocol and defined how exhaustion features appeared in function of time. The optimal time points for the expansion of "fit" MiHA-specific T cells were delineated using phenotypic and functional assessments including KLRG-1 and PD-1 surface markers as well as Ki67 staining and cytokine secretion assays.
Following a priming phase, an enrichment step and a rapid expansion stage, our method generates MiHA-specific T-cell lines. Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases. While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression. A similar pattern was also observed after stimulating a virus-specific memory repertoire, except for the more rapid acquisition of exhaustion features upon repeated antigen exposure.
Our results offer novel insights on the impact of culture duration on the acquisition of T-cell exhaustion features. Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.
过继转移次要组织相容性抗原（MiHA）特异性 T 细胞是治疗血液系统恶性肿瘤患者的一种很有前途的疗法。然而，在体外扩增过程中，转移细胞获得终末效应分化和衰竭特征，从而阻止其在体内的功能和持久性，这限制了转输细胞的疗效。然而，在培养过程中诱导 T 细胞分化和功能损伤的因素仍未得到很好的定义，并且可能因扩增方法的不同而有所不同。
使用临床上相关的 HLA-A0201 限制性 MiHA HA-1 以及易于在临床环境中转用的试剂和程序，我们设计了一种新的培养方案，并确定了衰竭特征是如何随着时间的推移而出现的。通过表型和功能评估（包括 KLRG-1 和 PD-1 表面标志物以及 Ki67 染色和细胞因子分泌测定），确定了“合适”MiHA 特异性 T 细胞扩增的最佳时间点。
在一个初始阶段、一个富集阶段和一个快速扩增阶段之后，我们的方法产生了 MiHA 特异性 T 细胞系。随着培养时间的延长，表型和功能障碍的证据开始出现，但在延长初始或快速扩增阶段后，其特征有所不同。虽然在初始阶段反复暴露于抗原会导致抗原特异性群体的下降和抗原特异性 CD8+T 细胞上 PD-1 和 KLRG-1 的表达，但延长无抗原的扩增阶段会导致增殖停滞和抗原特异性细胞的相对丢失，而不会损害多效性细胞因子的分泌或诱导 PD-1 和 KLRG-1 的表达。在刺激病毒特异性记忆库时也观察到类似的模式，除了在反复暴露于抗原时更快地获得衰竭特征。
我们的研究结果提供了关于培养时间对 T 细胞衰竭特征获得影响的新见解。使用新的临床兼容方案，我们定义了在过继转移前在培养中最佳分化和扩增 MiHA 特异性 T 细胞时需要监测的关键参数。
