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

描述（由申请人提供）：我们已经启动了一个肿瘤浸润淋巴细胞（TIL）治疗项目，目前已经治疗了70多名转移性黑色素瘤患者，临床反应率为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TIL中与临床反应相关的特定淋巴细胞亚群。在最近对超过31名接受治疗的患者进行的基于facs的表型筛选中，我们发现表达B-和T-淋巴细胞衰减剂（BTLA）的分化CD3+CD8+ cd45ra - cd62l - cd27效应记忆（EM）细胞亚群与临床反应最密切相关。其他EM标记物，如CD27、CD28、CD25和PD-1没有预测作用。虽然BTLA传统上被定义为激活T细胞的负共刺激分子，但最近的数据表明，它也是CD8+ CTL分化程序中的新标记物，并且表达BTLA的黑色素瘤特异性CD8+ T细胞在体内具有分化程度较低，功能更多功能的表型。我们实验室的初步数据也发现，分选的CD8+BTLA+黑色素瘤TIL对IL-2有较高的增殖反应，而BTLA-对应物则反应较低，容易发生凋亡。此外，我们发现，一旦BTLA在T细胞上丢失，它就不能重新表达。这些观察结果使我们假设BTLA+CD8+ TIL亚群代表了一个分化程度较低的多功能亚群，其在体内的持久性导致对黑色素瘤过继性T细胞治疗的反应性增强。为了解决这一假设，我们建议进一步阐明CD8+BTLA+黑色素瘤TIL亚群在过继细胞治疗中的作用。在目标#1中，我们将对分选的BTLA+和BTLA- TIL进行一系列体外实验，研究它们的EM特性（增殖、存活、对TCR和细胞因子刺激的反应）及其抗肿瘤效应活性。在Aim #2中，使用高通量TCR V？通过CDR3区域测序，我们将从输注到患者体内的CD8+BTLA+或CD8+BTLA-亚群中追踪体内t细胞克隆的长期命运。在Aim #3中，我们将更多地关注CD8+BTLA+与BTLA-亚群在NOD-SCID x ？c-/- (NSG)小鼠，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是否能增强肿瘤控制，以解决一个必然的假设，即尽管BTLA是功能更强的T细胞的标记物，但它仍然通过与黑色素瘤细胞上表达的疱疹病毒进入介质（HVEM）的相互作用作为负共刺激分子。该项目具有创新性，因为它将在过继t细胞治疗中描述一种新的生物标志物，这种生物标志物的缺失可能会确定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.