Functional Attributes of a CD8+BTLA+ T cell subset in Adoptive T Cell Therapy

描述(申请人提供)：我们已经启动了肿瘤浸润性淋巴细胞(TIL)治疗计划，目前已经治疗了70多名转移性黑色素瘤患者，临床有效率为45%-50%。同时，我们正在进行相关的生物标志物研究，以确定输注的TIL中与临床反应相关的特定淋巴细胞亚群。在最近对31名接受治疗的患者进行的基于FACS的表型筛查中，我们发现分化的CD3+CD8+CD45RA-CD62L-CD27-效应记忆(EM)细胞亚群表达B-和T-淋巴细胞衰减器(BTLA)与临床反应最相关。其他EM标志物，如CD27、CD28、CD25和PD-1不能预测。尽管BTLA被经典地定义为激活的T细胞上的负共刺激分子，但最近的数据表明，它也是CD8+CTL分化程序中的一个新标记物，体内表达BTLA的黑色素瘤特异性CD8+T细胞具有分化较少、功能更多功能的表型。我们实验室的初步数据还发现，分选的CD8+BTLA+黑色素瘤TIL对IL-2有较高的增殖反应，而BTLA-TIL反应较弱，容易发生凋亡。此外，我们还发现，一旦BTLA在T细胞上丢失，它就不能重新表达。这些观察使我们假设BTLA+CD8+TIL亚群代表了一个分化较少的多功能亚群，其在体内的持久性导致了对黑色素瘤过继T细胞治疗的增强反应。为了解决这一假设，我们建议进一步揭示CD8+BTLA+黑色素瘤TIL亚群在过继细胞治疗中的作用。在第一个目标中，我们将对分离的BTLA+和BTLA-TIL进行一系列的体外检测，研究它们的EM特性(增殖、存活、对TCR和细胞因子刺激的反应)以及它们的抗肿瘤效应。在目标2中，使用高通量TCR V？通过CDR3区域测序，我们将跟踪体内从CD8+BTLA+或CD8+BTLA-亚群注入患者的T细胞克隆的长期命运。在目标3中，我们将更多地关注CD8+BTLA+与BTLA-亚群在NOD-SCID x？C-/-(NSG)小鼠中的过继转移实验中的机制作用，其中我们将研究这些亚群的相对持久性、肿瘤浸润和抗肿瘤效应功能。这些实验还将测试使用抗BTLA抗体阻断BTLA是否增强了肿瘤控制，以解决一个推论，即虽然BTLA是功能更高的T细胞的标志，但它仍然通过与黑色素瘤细胞上表达的疱疹病毒进入介体(HVEM)相互作用而起到负共刺激分子的作用。这个项目是创新的，因为它将表征过继T细胞治疗中的一种新的生物标记物，其丢失可能识别CD8+CTL分化向衰老状态的关键转折点。

DESCRIPTION (provided by applicant): We have initiated a tumor-infiltrating lymphocyte (TIL) therapy program that has now treated over 70 metastatic melanoma patients with an ongoing 45-50% clinical response rate. In parallel, we are performing correlative biomarker studies to identify specific lymphocyte subsets within infused TIL associated with clinical responses. In a recent FACS-based phenotypic screen on over 31 treated patients, we found that a subset of differentiated CD3+CD8+CD45RA-CD62L-CD27-effector-memory (EM) cells expressing B- and T- lymphocyte attenuator (BTLA) stood out to be most strongly associated with clinical response. Other EM markers, such as CD27, CD28, CD25, and PD-1 were not predictive. Although BTLA is classically defined as a negative co-stimulatory molecule on activated T cells, recent data suggests it is also a new marker in the CD8+ CTL differentiation program and that melanoma-specific CD8+ T cells expressing BTLA have a less differentiated, more polyfunctional phenotype in vivo. Preliminary data in our lab has also found that sorted CD8+BTLA+ melanoma TIL have a high proliferative response to IL-2, while their BTLA- counterparts were hyporesponsive and prone to apoptosis. In addition, we have found that once BTLA is lost on T cells, it cannot be re-expressed. These observations have led us to hypothesize that the BTLA+CD8+ TIL subset represents a less differentiated, polyfunctional subset whose persistence in vivo results in enhanced responsiveness to adoptive T cell therapy in melanoma. To address this hypothesis, we propose to further unravel the role of this CD8+BTLA+ melanoma TIL subset in adoptive cell therapy. In Aim #1, we will perform a series of in vitro assays on sorted BTLA+ and BTLA- TIL studying their EM properties (proliferation, survival, response to TCR and cytokine stimulation) and their anti-tumor effector activity. In Aim #2, using high-throughput TCR V? CDR3 region sequencing, we will track the long-term fate of T-cell clones in vivo from the CD8+BTLA+ or CD8+BTLA- sub-populations infused into patients. In Aim #3, we will focus in more on the mechanistic role of the CD8+BTLA+ versus the BTLA- subset in a set of adoptive transfer experiments in NOD-SCID x ?c-/- (NSG) mice, where we will study the relative persistence, tumor infiltration, and anti-tumor effector function of these subsets. These experiments will also test whether blockade of BTLA, using anti-BTLA antibodies, enhances tumor control to address a corollary hypothesis that although BTLA is a marker for more highly functional T cells, it still serves as a negative costimulatory molecule through its interaction with the herpes virus entry mediator (HVEM) expressed on melanoma cells. This project is innovative as it will characterize a novel biomarker in adoptive T-cell therapy whose loss may identify a key turning point in CD8+ CTL differentiation towards a senescent state.