T CELL TRAFFICKING IN ADOPTIVE CELLULAR IMMUNOTHERAPY

免疫细胞免疫疗法（ACI）治疗播散性癌症， 离体扩增的肿瘤反应性成淋巴细胞。 这些细胞 靶病灶植入不良，导致观察到的结果不一 在治疗试验中。 这项赠款的重点是机制和 白细胞募集到转移性淋巴结转移部位的临床后果 ACI期间的疾病，疫苗致敏，离体扩增的淋巴结 细胞 新的初步研究表明，单剂量的TNF-α， 在输注前4小时腹膜内给药，增加了 将输注的疫苗致敏细胞募集到肺中， 微转移这种治疗通过以下方式促进肿瘤生长的抑制： 这些细胞与用于离体扩增的方案无关。 单独用TNF-α预处理在所用浓度下无活性 (oneμ g/动物，单次给药），并且没有产生可见的症状。 新的初步研究还表明， 负责抑制肿瘤生长（Tc/h-1亚群）的可能是 与抑制这种反应的那些（Tc/h-2）不同， 亚群）基于高水平的选择素-配体表达。 的PI 提出选择素介导的Tc/h-1细胞募集到 转移性疾病的位点对于抑制肿瘤生长是必需的。 因此，血管系统上选择素的瞬时诱导 与转移以及Tc/h-1亚群富集相关 在输注细胞的群体中，应该改善临床结果。 此外，基于选择素的疫苗-引发和扩增的分级分离可以在体外进行。 细胞应该既富集Tc/h-1亚群，又耗尽Tc/h-2亚群， 子集 具体目标1研究所需的粘附受体 用于募集肿瘤引流淋巴结细胞（TDLN）和宿主 白细胞进入肺部，3天内建立肺微转移。 用稳定的荧光染料或同类标记物标记的TDLN， 受体特异性抗体和选择素基因被破坏的小鼠将 用于这些实验。 TDLN的相对贡献和 还将确定宿主细胞对临床结果募集。 具体目标2研究TDLN的粘附受体表型， VPLN（目前在临床试验中的人类对应物）分泌高水平的 IFN-γ和GM-CSF水平（Tc/h-1）和高水平IL-4， IL-10（Tc/h-2）。 此外，基于选择素的分级分离程序 将开发分离Tc/h-1和Tc/h-2亚组的方法。 的 将分次群体与起始群体进行比较， 彼此在体外和体内针对恶性细胞的活性。 特异性目的3确定是否诱导内皮粘附 肿瘤相关血管上的受体增加TDLN募集， 抑制肿瘤生长。 上述新的初步研究支持 对于这个假设。 促炎细胞因子，如TNF-α，将 在输注TDLN之前给药。 实体就雇员 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） 将建立。对TDLN活性的影响 免疫原性或大肿瘤，对所需的细胞数量， 抑制转移和全身IL-2的剂量或持续时间 将对治疗进行评估。 粘附受体的作用， 然后将确定募集（TDLN和宿主细胞）， 改善临床有效性的准备方案。 的 大幅修订的补助金纠正了最初发现的弱点， 审查. 新的初步研究表明， 直接和直接的好处。 许多试剂和 补助金中使用的技术是PI最近开发的， 目前在其他地方可用。 该项目补充了受资助的 在密歇根大学进行的ACI临床试验（CA 69102）， 提高该项目的科学回报。 最后 PI和co-PI之间的合作将提高生产力， 确保信息从临床前快速转移到 临床设置。

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.