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

描述（由申请人提供）：我们已经启动了一项肿瘤浸润淋巴细胞（TIL）治疗计划，目前已治疗了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，以确定与临床反应相关的输注TIL中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-淋巴细胞衰减因子（BTLA）的分化的CD 3 + CD 8 + CD 45 RA-CD 62 L-CD 27-效应记忆（EM）细胞亚群与临床应答最强相关。其他EM标志物，如CD 27、CD 28、CD 25和PD-1均无预测性。虽然BTLA被经典地定义为活化T细胞上的负共刺激分子，但最近的数据表明它也是CD 8 + CTL分化程序中的新标记物，并且表达BTLA的黑素瘤特异性CD 8 + T细胞在体内具有分化程度较低、多功能的表型。我们实验室的初步数据还发现，分选的CD 8 +BTLA+黑色素瘤TIL对IL-2具有高增殖反应，而它们的BTLA-对应物是低反应的并且易于凋亡。此外，我们发现一旦BTLA在T细胞上丢失，它就不能再表达。这些观察结果使我们假设BTLA+ CD 8 + TIL亚群代表分化程度较低的多功能亚群，其在体内的持续性导致对黑素瘤中过继性T细胞疗法的反应性增强。为了解决这一假设，我们建议进一步阐明这种CD 8 +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进行一系列体外测定，研究它们的EM特性（增殖、存活、对TCR和细胞因子刺激的响应）和它们的抗肿瘤效应物活性。在目标#2，使用高通量TCR V？通过CDR 3区测序，我们将追踪来自输注到患者中的CD 8 +BTLA+或CD 8 +BTLA-亚群的T细胞克隆的体内长期命运。在目标#3中，我们将更多地关注CD 8 +BTLA+与BTLA-亚群在NOD-SCID x？c-/-（NSG）小鼠，我们将研究这些子集的相对持久性，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，以解决一个推论假设，即尽管BTLA是功能更高的T细胞的标志物，但它仍然通过与疱疹病毒的相互作用作为一种负性共刺激分子黑色素瘤细胞上表达的进入介体（HVEM）。该项目具有创新性，因为它将表征过继性T细胞治疗中的一种新型生物标志物，其丢失可能会识别CD 8 + CTL向衰老状态分化的关键转折点。

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.