Lymph node-like vasculature and naive T cell infiltration into tumors

描述(由申请人提供)：肿瘤内免疫细胞的有限表达是肿瘤控制的主要障碍，不同于免疫抑制障碍。了解并克服这一限制将使我们能够将许多免疫疗法的有效性扩展到更广泛的癌症患者。所有的肿瘤浸润性淋巴细胞都被认为是在肿瘤引流淋巴结(LN)中分化的效应器。 然后进入肿瘤。然而，我们发现原始的CD8T细胞(TCD8)直接进入肿瘤，在那里它们分化为功能效应器。单纯的TCD8侵袭依赖于L-选择素和CCR7，以及表达周围结节寻址(PNAD)和趋化因子CCL21的肿瘤血管的形成和类似LN的高内皮小静脉。我们还发现有组织的第三级淋巴组织(TLO)与这种LN样血管并列。PNAD LN样血管构筑在人类实体肿瘤中与TLO样组织相关，并与乳腺癌患者更长时间的无转移、无疾病和总生存期相关。这些数据表明，LN样血管的诱导可能通过增强naive TCD8的渗透和原位激活来增强抗肿瘤免疫。LN中高内皮微静脉的诱导受淋巴毒素受体信号控制。然而，我们发现CCL21的表达依赖于TCD8效应器分泌的干扰素γ，而PNAD的表达受淋巴毒素-α3的控制。这表明早期的效应器的渗透诱导了LN样的血管形成，这反过来又支持了在肿瘤内分化的幼稚T细胞的自我维持的募集。该应用的目的是：(1)确定肿瘤坏死因子受体配体如何调节肿瘤血管上PNAD的表达。我们将评估哪些支架蛋白和酶对肿瘤相关的PNAD负责。我们还将确定肿瘤坏死因子α和淋巴毒素-CCL3是否冗余地诱导皮下肿瘤中PNAD的表达，因为两者都是肿瘤坏死因子受体1和2的配体；(2)确定α在肿瘤中的表达是如何调节的。我们将确定干扰素γ如何通过gp38CD31neg细胞和血管内皮细胞在IP肿瘤中调控CCL21的表达。我们还将确定在SC肿瘤中控制CCL21表达的机制；(3)评估与肿瘤中LN样血管形成相关的TLO样结构的组织和功能。我们将评估gp38 CD31neg细胞在肿瘤中作为TLO组织者的作用，以及影响其活性的因素。我们还将确定这些TLO是否作为抗肿瘤免疫的积极或消极调节的基因位点。作为一个R21应用，我们的目的是进行实验，区分控制这些分子表达的不同新机制，使未来R01应用的开发能够研究如何操纵它们来增强抗肿瘤免疫和癌症控制。

DESCRIPTION (provided by applicant): Limited representation of intratumoral immune cells is a major barrier to tumor control distinct from the barrier of immunosuppression. Understanding and overcoming this limitation will enable us to extend the effectiveness of many immunotherapies to a broader cross-section of cancer patients. It has been assumed that all tumor- infiltrating lymphocytes are effectors that differentiate in tumor-draining lymph nodes (LN) and subsequently enter the tumor. However, we showed that naive CD8 T cells (TCD8) enter tumors directly, where they differentiate into functional effectors. Naive TCD8 infiltration depends on L-selectin and CCR7, and on the development of tumor blood vessels that express peripheral node addressing (PNAd) and the chemokine CCL21 and resemble LN high endothelial venules. We have also identified organized tertiary lymphoid tissue (TLO) in juxtaposition to this LN-like vasculature. PNAd+ LN-like vasculature has been reported in human solid tumors in association with TLO-like tissue, and correlated with longer metastasis-free, disease-free, and overall survival in breast cancer patients. These data suggest that induction of LN-like vasculature could bolster anti- tumor immunity by enhancing the infiltration and in situ activation of naive TCD8. Induction of high endothelial venules in LN is controlled by lymphotoxin-ß receptor signaling. However, we found that CCL21 expression depends on IFNγ secreted by TCD8 effectors, while PNAd expression is controlled by lymphotoxin-α3. This suggests that early infiltration of effectors induces LN-like vasculature, which in turn supports a self-sustaining recruitment of naïve T cells that differentiate within the tumor. The aims of this application are: (1) to determine how TNF receptor ligands regulate the expression of PNAd on tumor vasculature. We will evaluate which scaffolding proteins and enzymes are responsible for tumor associated PNAd. We will also determine if TNFα and lymphotoxin-α3 act redundantly to induce PNAd expression in subcutaneous tumors, since both are ligands for TNF receptors 1 and 2; (2) to determine how the expression of CCL21 in tumors is regulated. We will determine how IFNγ controls the expression of CCL21 by gp38+CD31neg cell and endothelial cells in IP tumors. We will also identify mechanisms controlling CCL21 expression in SC tumors; (3) to evaluate the organization and function of TLO-like structures that form in association with LN-like vasculature in tumors. We will evaluate the role of gp38+CD31neg cells as organizers of TLO in tumors, and the factors that influence their activity. We will also determine whether these TLO serve as a locus for positive or negative regulation of anti- tumor immunity. As an R21 application, our purpose is to conduct experiments that will distinguish among different novel mechanisms that control expression of these molecules, enabling development of a future R01 application to investigate means to manipulate them to enhance anti-tumor immunity and cancer control.