The role of immunosuppressive cells in metastasis

免疫抑制细胞在转移中的作用

• 批准号: 7732243
• 负责人: Arya Biragyn
• 金额: $ 23.8万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732243
• 关键词: Adam11 gene; Affect; Antibodies; Antitumor Response; Autoantigens; B-Lymphocytes; Binding Proteins; Biological; Brain; Breast Cancer Cell; CCL17 gene; CCL22 gene; CD8B1 gene; Carcinoma; Cell Count; Cell Death Induction; Cells; Cytotoxic T-Lymphocytes; Data; Disease; Disease Outcome; Distant; Drug Formulations; Flow Cytometry; Galectin 1; Growth; Hand; Heterophile Antigens; Human; Immune; Immune response; Immunosuppressive Agents; In Vitro; Inbred BALB C Mice; Interleukin-2; Lead; Lectin; Ligands; Liver; Lung; Maintenance; Mammary gland; Mediating; Memory; Monoclonal Antibody HuM291; Muromonab-CD3; Mus; Myelogenous; NK Cell Activation; Natural Killer Cells; Neoplasm Metastasis; Outcome; Pathway interactions; Patients; Peripheral; Peripheral Blood Lymphocyte; Play; Primary Neoplasm; Process; Production; Progressive Disease; Proteins; Regulation; Reporting; Resistance; Role; Signal Transduction; Site; Staging; Supporting Cell; Surface; T-Cell Proliferation; T-Lymphocyte; Zip Code; anergy; beta-galactoside; bone; cancer cell; cell killing; chemokine; chemokine receptor; chlorambucil/dactinomycin/methotrexate protocol; cytotoxic; implantation; improved; killings; macrophage; malignant breast neoplasm; neoplastic cell; novel; peripheral blood; prevent; receptor; response; tumor

Here we have hypothesized that breast cancer cells utilize a migratory pathway of immune cells, such as CCR4-expressing T cells, to metastasize into inflamed lungs producing TARC and MDC. Interestingly, primary tumor growing at distant site induced production of TARC/CCL17 (a chemokine ligand for CCR4) in lungs of mice presumably to facilitate recruitment of CCR4-expressing tumor cells. Thus, to demonstrate this, we have asked question whether 4T1 tumor cells express and utilize CCR4 for lung metastasis. However, our flow cytometry analysis has only detected a weak expression of CCR4 on the surface of 4T1 cells, suggesting that a proportion of the cells may indeed express the receptor. In support, the cells were also able to chemotax in vitro to TARC, indicating that the receptor is functionally active. To confirm this, 4T1 cells were treated with CCR4-targeting chemotoxin, a novel formulation developed by us to specifically kill cells through unique chemokine receptors. As a result, the treatment generated resistant to chemotoxin cells (designated 4T1wt-PE and 4T1.2-PE). Unlike parental 4T1 tumor cells, 4T1wt-PE and 4T1.2-PE cells did not chemotax to TARC and could not metastasize in to lungs. Our data have clearly demonstrated that CCR4 was expressed on proportion of 4T1 tumor cells, and only CCR4-expressing cells metastasized into lungs. However and surprisingly, this inherent capability alone was not sufficient to establish lung metastasis. Lung metastasis also required an active participation of CCR4+ Tregs. We have found that Tregs facilitated lung metastasis by regulating or even killing NK cells. This presumably explains our finding that NK cell counts were significantly reduced in peripheral blood (PB) of both tumor-bearing mice and human patients with an advanced stage IV breast cancer. Taken together, we propose that CCR4 is a lung metastasis Zip code utilized for dissemination of tumors together with their protector immune cells, Tregs. Strategies that abrogate any part of this process, such as targeted inactivation of CCR4+ cells or direct depletion of Tregs, would be expected to improve the outcome of the disease. This in turn would activate both antitumor innate and adaptive immune responses through activation of NK cells and cytolytic T cells. Indeed, the treatment of tumor -bearing mice with TARC-chemotoxin significantly reduced lung metastasis of 4T1 cells through depletion of Tregs and metastasizing tumors and activating NK cells. Our data clearly indicate the important role of Tregs in regulation of antitumor responses. Despite significant efforts, practically very little is known about mechanisms of suppressive activity exerted by Tregs. Recently, we have demonstrated that Tregs in human PBL consisted of at least two distinct subsets, memory-type CCR4+Tregs and nave-type CCR4- Tregs (Baatar et al., 2007). While freshly isolated CCR4+Tregs presumably represent natural Tregs and appear to be primed to readily suppress T cell proliferation, CCR4-Tregs require TCR-mediated activation to render them fully active. It was reported by others that activated Tregs could kill target cells, including CD4+Tcells, CD8+ T cells, utilizing both GZ-A and GZ-B. However, we did not detect GZ-A expression by either Treg subsets regardless of anti-CD3/CD28 or IL-2 stimulation. In our hands, GZ-B was not detected in non-activated T cells, and Tregs do not kill target T cells. In addition, upon activation, GZ-B was exclusively expressed in CCR4- T cells (both Tregs and non-Treg cells), but not in CCR4+Tregs, and did not correlate with their suppressive activities. Thus, GZ-B may not be a primary mechanism of regulation, although we cannot rule out its utilization by activated CCR4-Tregs, for example, when used at significantly higher cell concentrations. On the other hand, we have found that Tregs regulate T cell proliferation through a cell contact-dependent process involving FasL/Fas signaling. Thus, we have first hypothesized that Tregs may also regulate NK cells utilizing FasL/Fas signaling. Our study indicates that the process is independent of FasL/Fas signaling, as Tregs retained ability to regulate NK cells in the presence of neutralizing Fas antibodies. In contrast, we have found that Tregs express and utilize lectin-type proteins, such as beta-galactoside-binding protein (bGBP). bGBP and its dimeric lectin form Galectin-1 are immunosuppressive proteins expressed by activated immune cells, such as T cells, B cells and macrophages. Interestingly, bGBP actively participated in regulation of CD8+ T cells. It was used by Tregs to regulate TCR signaling of CD8+ T cells without induction of cell death, although bGBP is known to be cytotoxic for activated T cells. We have demonstrated that the mechanism of this process is in the capacity of bGBP to induce limited (non-processive) TCR signaling in target T cells; as it only activates Zap70, but not downstream molecules, such as ERK, Ras and PI3K. Although non-processive TCR signaling can lead to anergy, bGBP does not induce anergy of CD8+ T cells. This presumably allows Tregs to transiently prevent activation of CD8+ T cells by self-antigens, while keeping responses to xenogeneic antigens unaffected, indicating the important biological role of bGBP in the maintenance and control of peripheral tolerance.

在这里，我们假设乳腺癌细胞利用免疫细胞（例如表达 CCR4 的 T 细胞）的迁移途径转移到产生 TARC 和 MDC 的发炎肺部。有趣的是，远处生长的原发肿瘤诱导小鼠肺部产生 TARC/CCL17（CCR4 的趋化因子配体），这可能是为了促进表达 CCR4 的肿瘤细胞的募集。因此，为了证明这一点，我们提出了 4T1 肿瘤细胞是否表达并利用 CCR4 进行肺转移的问题。然而，我们的流式细胞术分析仅在4T1细胞表面检测到CCR4的微弱表达，这表明部分细胞可能确实表达该受体。作为支持，细胞还能够在体外对 TARC 趋化，表明该受体具有功能活性。为了证实这一点，我们用靶向 CCR4 的趋化毒素处理 4T1 细胞，这是我们开发的一种新制剂，可通过独特的趋化因子受体特异性杀死细胞。结果，该治疗产生了对趋化毒素细胞的抗性（称为 4T1wt-PE 和 4T1.2-PE）。与亲代4T1肿瘤细胞不同，4T1wt-PE和4T1.2-PE细胞不会趋化至TARC并且不能转移至肺部。 我们的数据清楚地表明CCR4在4T1肿瘤细胞中表达，并且只有表达CCR4的细胞转移到肺部。然而，令人惊讶的是，仅这种固有能力不足以确定肺转移。肺转移也需要 CCR4+ Tregs 的积极参与。我们发现Tregs通过调节甚至杀死NK细胞来促进肺转移。这大概解释了我们的发现：荷瘤小鼠和患有晚期 IV 期乳腺癌的人类患者的外周血 (PB) 中 NK 细胞计数显着减少。 综上所述，我们认为 CCR4 是一种肺转移邮政编码，用于肿瘤及其保护性免疫细胞 Tregs 的传播。废除该过程任何部分的策略，例如 CCR4+ 细胞的定向灭活或直接耗竭 Tregs，有望改善疾病的结果。这反过来又会通过激活 NK 细胞和溶细胞 T 细胞来激活抗肿瘤先天免疫反应和适应性免疫反应。事实上，用 TARC-趋化毒素治疗荷瘤小鼠可通过消除 Tregs、转移肿瘤和激活 NK 细胞，显着减少 4T1 细胞的肺转移。 我们的数据清楚地表明 Tregs 在调节抗肿瘤反应中的重要作用。尽管付出了巨大的努力，但实际上人们对 Tregs 的抑制活性机制知之甚少。最近，我们证明人类 PBL 中的 Tregs 至少由两个不同的子集组成：记忆型 CCR4+Treg 和幼稚型 CCR4-Treg（Baatar 等，2007）。虽然新鲜分离的 CCR4+Treg 可能代表天然的 Tregs，并且似乎已准备好抑制 T 细胞增殖，但 CCR4-Treg 需要 TCR 介导的激活才能使其完全活跃。据报道，激活的Tregs可以利用GZ-A和GZ-B杀死靶细胞，包括CD4+T细胞、CD8+T细胞。然而，无论抗 CD3/CD28 或 IL-2 刺激如何，我们都没有检测到任一 Treg 亚群的 GZ-A 表达。在我们手中，未激活的T细胞中未检测到GZ-B，并且Tregs不会杀死目标T细胞。此外，激活后，GZ-B仅在CCR4-T细胞（Treg细胞和非Treg细胞）中表达，但在CCR4+Treg细胞中不表达，并且与其抑制活性不相关。因此，GZ-B 可能不是主要的调节机制，尽管我们不能排除它被激活的 CCR4-Treg 所利用，例如，当在显着较高的细胞浓度下使用时。另一方面，我们发现 Tregs 通过涉及 FasL/Fas 信号传导的细胞接触依赖性过程来调节 T 细胞增殖。因此，我们首先假设Tregs也可能利用FasL/Fas信号传导调节NK细胞。我们的研究表明，该过程独立于 FasL/Fas 信号传导，因为 Tregs 保留了在中和 Fas 抗体存在的情况下调节 NK 细胞的能力。相比之下，我们发现 Tregs 表达并利用凝集素型蛋白，例如 β-半乳糖苷结合蛋白 (bGBP)。 bGBP 及其二聚体凝集素形式 Galectin-1 是由激活的免疫细胞（例如 T 细胞、B 细胞和巨噬细胞）表达的免疫抑制蛋白。有趣的是，bGBP 积极参与 CD8+ T 细胞的调节。尽管已知 bGBP 对活化的 T 细胞具有细胞毒性，但 Tregs 使用它来调节 CD8+ T 细胞的 TCR 信号传导而不诱导细胞死亡。我们已经证明，该过程的机制在于 bGBP 能够在靶 T 细胞中诱导有限（非进行性）TCR 信号传导；因为它只激活 Zap70，而不激活下游分子，例如 ERK、Ras 和 PI3K。尽管非进行性 TCR 信号传导可导致无反应性，但 bGBP 不会诱导 CD8+ T 细胞无反应性。这可能使得 Tregs 能够暂时阻止自身抗原激活 CD8+ T 细胞，同时保持对异种抗原的反应不受影响，这表明 bGBP 在维持和控制外周耐受中的重要生物学作用。