T CELL TRAFFICKING IN ADOPTIVE CELLULAR IMMUNOTHERAPY

过继细胞免疫疗法(ACI)治疗播散性癌症 体外扩增的肿瘤反应性淋巴母细胞。这些细胞贩卖 对靶病变的不良影响导致观察到的结果好坏参半 在治疗试验中。这笔赠款的重点是机制和 白细胞重新聚集到转移部位的临床后果 疫苗预置的体外扩张淋巴结行ACI期间的疾病 细胞。新的初步研究表明，单剂肿瘤坏死因子-α， 在输液前4小时内给药，增加了 已建立的注入疫苗的细胞在肺内的招募 微转移。这种治疗方法通过以下方式促进抑制肿瘤生长 这些细胞无论采用何种方案进行体外扩增。 在所使用的浓度下，单独使用肿瘤坏死因子-α是无效的。 (1毫克/只动物，单剂)，没有产生明显的症状。 新的初步研究还表明，接种疫苗的细胞 负责抑制肿瘤生长的(TC/h-1亚群)可以 有别于抑制这种反应的那些(Tc/h-2 亚集)基于高水平的选择素配体表达。《少年派》 认为选择素介导的TC/h-1细胞募集到 转移灶对于抑制肿瘤生长是必不可少的。 因此，选择素在血管系统上的瞬时诱导 与TC/h-1亚群的转移和浓集有关 在人群中输注的细胞应该会改善临床结果。 此外，基于选择素的疫苗分级-启动和扩展 细胞既应富含T_c/h-1亚群，又应耗尽T_c/h-2 子集。特定目标1研究所需的黏附受体 用于肿瘤引流淋巴细胞(TDLN)和宿主的招募 白细胞进入肺内，3天后确定肺微转移。 用稳定的荧光染料或同源标记标记TDLN， 受体特异性抗体和选择素基因中断的小鼠将 在这些实验中使用。TDLN和TDLN的相对贡献 宿主细胞募集对临床结果的影响也将被确定。 特异性目的2研究TDLN和TDLN的黏附受体表型 VPLN(目前正在进行临床试验的人类同行)分泌高水平的 干扰素-γ和粒-巨噬细胞集落刺激因子(TC/h-1)水平及高水平IL-4和 IL-10(TC/h-2)。此外，基于选择素的分级程序 将开发分离T_c/h-1和T_c/h-2子集的方法。这个 分割后的种群将与起始种群进行比较，并 彼此在体外和体内对肿瘤细胞的活性。 特定目标3确定是否诱导内皮细胞黏附 肿瘤相关血管上的受体增强TDLN募集和 抑制肿瘤生长。上面引用的新的初步研究支持 对于这一假设。促炎细胞因子，如肿瘤坏死因子-α，将 在输注TDLN之前给药。这些因素的影响 制备方案对标准和分级TDLN行为的影响 (TC/Th-1和TC/Th-2亚群；高和低选择素配体表达) 将会建立起来。对TDLN活动的影响 免疫原性或大型肿瘤，对所需的细胞数量 对转移的抑制和全身IL-2的剂量或持续时间 将对治疗进行评估。黏附受体及其受体的作用 然后将确定招募(TDLN和宿主细胞) 提高临床疗效的预备性方案。这个 广泛修订的补助金纠正了最初的 复习一下。新的初步研究表明， 在ACI中直接和直接的好处。许多试剂和 赠款中使用的技术是由PI最近开发的，而不是 目前在其他地方可以买到。该项目是对已资助项目的补充 密歇根大学ACI的临床试验(CA69102)和Will 提高这一项目的科学回报。最后， 主计长和共同主计长之间的合作将提高生产力和 确保将信息从临床前快速传输到 临床环境。

Adoptive cellular immunotherapy (ACI) treats disseminated cancers with ex-vivo expanded tumor-reactive lymphoblasts. These cells traffick poorly into target lesions contributing to the mixed results observed in therapeutic trials. This grant focuses on the mechanisms and clinical consequences of leukocyte recruitment into sites of metastic disease during ACI with vaccine-primed, ex-vivo expanded lymph node cells. New preliminary studies show that a single-dose of TNF-alpha, administered intraperitoneally 4 hours prior to infusion, increased the recruitment of infused, vaccine-primed cells into lungs with established micrometastases. This treatment promoted inhibition of tumor-growth by these cells regardless of the protocol used for ex-vivo expansion. Pretreatment with TNF-alpha alone was inactive at the concentration used (one mu g/animal, single dose) and did not produce visible symptoms. New preliminary studies also suggest that the vaccine-primed cells responsible for inhibition of tumor growth (the Tc/h-1 subsets) can be distinguished from those that suppress this response (the Tc/h-2 subsets) based on high levels of selectin-ligand expression. The PI proposed that selectin-mediated recruitment of the Tc/h-1 cells into sites of metastatic disease is essential for inhibition of tumor growth. Consequently, transient induction of selectins on the vasculature associated with metastases as well as enrichment of the Tc/h-1 subset in the population of infused cells should improve clinical outcome. Furthermore, selectin-based fractionation of vaccine-primed and expanded cells should both enrich the Tc/h-1 subsets and deplete the Tc/h-2 subsets. Specific Aim 1 investigates the adhesion receptors required for the recruitment of tumor-draining lymph node cells (TDLN) and host leukocytes into lungs with 3-day established pulmonary micrometastases. TDLNs labeled with stable fluorescent dyes or congenic markers, receptor-specific antibodies and mice with disrupted selectin-genes will be used in these experiments. The relative contributions of TDLN and host cell recruitment to clinical outcome will also be determined. Specific aim 2 investigates the adhesion receptor phenotype of TDLN and VPLN (human-counterparts currently in clinical trials) that secrete high levels of IFN-gamma and GM-CSF (Tc/h-1) and high levels of IL-4 and IL-10(Tc/h-2). In addition, selectin-based fractionation procedures will be developed that separate the Tc/h-1 and Tc/h-2 subsets. The fractionated populations will be compared to the starting population and each other for activity against malignant cells in vitro and in vivo. Specific aim 3 determines whether induction of endothelial adhesion receptors on tumor-associated vessels augments TDLN recruitment and inhibits tumor-growth. The new preliminary studies cited above support for this hypothesis. Proinflammatory cytokines, such as TNF-alpha, will be administered prior to infusion of TDLNs. The impact of these preparative regimens on the behavior of standard and fractionated TDLN (Tc/Th-1 and Tc/Th-2 subsets; high and low selectin-ligand expression) will be established. The impact on TDLN activity against poorly immunogenic or large neoplasms, on the number of cells required for inhibition of metastases and on the dose or duration of systemic IL-2 therapy will be assessed. The role of adhesion receptors and recruitment (TDLN and host cells) will then be determined for preparative regimens that improve clinical effectiveness. The extensively revised grant rectifies the weakness detected by the initial review. The new preliminary studies demonstrate the potential for direct and immediate benefits in ACI. Many of the reagents and technologies used in the grant are recently developed by the PI and not currently available elsewhere. The project complements the funded clinical trial of ACI at the University of Michigan (CA69102) and will enhance the scientific return from this project. Finally, the collaboration between the PI and co-PI will enhance productivity and ensure the rapid transfer of information from a pre-clinical to a clinical setting.