Analyzing the Efficacy of Galectin-1 Ligand Inhibition in Adoptive T cell Therapy

描述(申请人提供)：Galectin-1(Galectin-1)，是一种与b-半乳糖有亲和力的碳水化合物结合蛋白。GAL-1参与细胞的黏附、迁移和凋亡。此外，Gal-1已被发现与多种癌症有关。几个研究小组已经证明，黑色素瘤细胞分泌大量的Gal-1。这一观察结果与Gal1‘S对辅助性T细胞众所周知的促凋亡和免疫调节作用相结合，导致了Gal-1通过诱导黑色素瘤抗原特异性CD8+T细胞的免疫抑制而引发肿瘤免疫逃逸机制的假设。该项目的长期目标是研究在过继T细胞治疗环境中消除Gal-1配体对黑色素瘤抗原特异性CD8+细胞毒性T细胞(CTL)的效果。目前，过继的CD8+T细胞疗法在晚期黑色素瘤患者中显示出适度的改善。因此，在过继免疫治疗之前，阻断抗原特异性CTL上的Gal-1-Gal-1配体的相互作用，可以帮助避免目前存在的缺点，如T细胞对肿瘤来源的Gal-1的敏感性，从而增强抗肿瘤效果。为此，我的目标是确定缺乏Gal-1配体的CTL是否可以在自体过继T细胞转移环境中有效地抑制肿瘤生长。这些研究的结果将极大地影响改进过继免疫疗法治疗癌症的努力。具体目的1.已发表的研究表明Gal-1对T辅助细胞具有免疫调节作用。然而，关于Gal-1在CD8+T细胞上的作用的研究很少发表。我的初步数据表明，CTL表达Gal-1配体，并经历Gal-1介导的调节。此外，我们还发现Gal-1的表达与疾病进展和无效的CD8+T细胞反应有关。这些数据导致了这样的假设，即Gal-1抑制了有助于肿瘤免疫逃逸的CTL。为了验证这一假设，我将分析Gal-1介导的细胞因子表达、增殖能力和凋亡活性对抗原特异性CTL的影响。具体目的2.本实验室的研究表明，Gal-1配体抑制剂4-F-GlcNAc治疗B16肿瘤小鼠具有抗肿瘤活性2。4-F-GlcNAc对多种免疫细胞有抗碳水化合物的作用，所以我计划设计一种更有针对性的治疗方法。我将在体外用4-F-GlcNAc处理CTL，然后将细胞转移到荷瘤小鼠体内。我推测，经4-F-GlcNAc处理的CTL将免于Gal-1介导的细胞死亡和抑制，从而提高CTL水平，增强抗肿瘤活性。为了验证这一假设，我将使用FACS和ELISA法分析4-F-GlcNAc处理和未处理的CTL的细胞活性和细胞因子的表达。我还将研究转移的CD8+T细胞对黑色素瘤小鼠的抗肿瘤效果。总体而言，我的数据将探索4-F-GlcNAc治疗的CTL在过继T细胞免疫治疗中的治疗作用。

DESCRIPTION (provided by applicant): Galectin-1 (Gal-1), is a carbohydrate-binding protein with affinity for b-galactose. Gal-1 is involved in cell adhesion, migration, and apoptosis. In addition, Gal-1 has been found to be associated with a variety of cancers. Several groups have demonstrated that melanoma cells secrete an abundance of Gal-1. This observation taken together with Gal-1's well-known pro-apoptotic and immunoregulatory effects on helper T cells has led to the hypothesis that Gal-1 elicits tumor-immune escape mechanisms by inducing immunosuppression on melanoma antigen-specific CD8+ T cells. The long-term goal of this project is to study the efficacy of eliminating Gal-1 ligands on melanoma antigen-specific CD8+ cytotoxic T cells (CTLs) in an adoptive T cell therapy setting. Currently, adoptive CD8+ T cell therapy has shown modest improvements in patients with advanced melanoma. Thus, blocking Gal-1-Gal-1 ligand interactions on antigen-specific CTLs, prior to adoptive immunotherapy, could help avoid current shortcomings, such as T cell susceptibility to tumor-derived Gal-1, thereby enhancing anti-tumor efficacy. To this end, my goals are to determine whether CTLs lacking Gal-1 ligands can effectively attenuate tumor growth in an autologous adoptive T cell transfer setting. Results from these studies will greatly impact efforts to improve adoptive immunotherapy for treating cancer. Specific Aim 1. Published studies demonstrate that Gal-1 has immunoregulatory effects on T helper cells. However, few studies have been published about the role of Gal-1 on CD8+ T cells. My preliminary data establish that CTLs express Gal-1 ligands and undergo Gal-1-mediated modulation. Furthermore, we show that Gal-1 expression is correlated to disease progression and ineffective CD8+ T cell responses. These data has led to the hypothesis that Gal-1 suppresses CTLs contributing to tumor immune escape. To test this hypothesis, I will analyze the effects of Gal-1-mediated cytokine expression, proliferative potential and apoptotic activity on antigen-specific CTLs. Specific Aim 2. Studies in my lab have demonstrated that treatment of B16 tumor-bearing mice with Gal-1 ligand inhibitor, 4-F-GlcNAc, and results in anti-tumor activity2. 4-F-GlcNAc has anti-carbohydrate effects on many types of immune cells, so I plan to devise a more focused treatment. I will treat CTLs ex vivo with 4-F-GlcNAc and then transfer the cells into tumor-bearing mice. I hypothesize that CTLs treated with 4-F-GlcNAc will be spared from Gal-1-mediated cell death and suppression, thus increasing CTL levels and enhancing anti-tumor activity. To test this hypothesis, I will analyze for cell viability and cytokine expression using FACS and ELISA in 4-F-GlcNAc treated and untreated CTLs. I will also study the anti-tumor efficacy of the transferred CD8+ T cells in melanoma-bearing mice. Overall, my data will explore the therapeutic utility of CTLs treated with 4-F-GlcNAc in adoptive T cell immunotherapy.