Developing new immunotherapies based of CD4+ T cells

开发基于 CD4 T 细胞的新免疫疗法

基本信息

批准号：
8552948
负责人：
Nicholas Restifo
金额：
$ 76.68万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8552948
关键词：
Adoptive Cell Transfers Adoptive Transfer Advanced Malignant Neoplasm Amino Acids Animals Antigens Apoptotic Autoimmune Process Breeding C57BL/6 Mouse CCL20 gene CCL23 gene CD4 Positive T Lymphocytes CD8B1 gene Cell Culture Techniques Cells Clinical Trials Collaborations Cultured Cells DNA Sequence Rearrangement Development Differentiation Antigens Employee Strikes Engineering Enzyme-Linked Immunosorbent Assay Epitopes Evolution Exposure to Female Gene Expression Profile Goals Growth Human Immunologics Immunotherapy In Vitro Interferon Type II Interleukin 2 Receptor Gamma Interleukin-10 Interleukin-17 Interleukin-2 Interleukin-6 Interleukin-9 Link Lymphocyte MHC Class II Genes MHC class II transactivator protein Malignant Neoplasms Mature T-Lymphocyte Mediating Messenger RNA Metastatic Neoplasm to the Lung Microarray Analysis Modeling Mus Patients Phenotype Play Population Prevention Relapse Research Personnel Role STAT3 gene Scientist Severities Signal Transduction Splenocyte Staining method Stains T cell differentiation T-Lymphocyte T-Lymphocyte Subsets Testing Th1 Cells Transforming Growth Factor beta Transgenes Transgenic Animals Transgenic Mice Transgenic Model Transgenic Organisms Translating Tumor Cell Line Tumor Immunity Tumor Necrosis Factor-alpha Tyrosinase related protein-1 Up-Regulation Vaccinia virus Vitiligo Y Chromosome base cancer cell cancer therapy cell growth cytokine design human TNF protein human disease immunogenic in vivo interferon gamma receptor interleukin-22 interleukin-23 melanocyte melanoma mouse model neutralizing antibody polarized cell programs response tumor tumor growth

项目摘要

The roles of CD4+ cells in anti-tumor immunity remains controversial and poorly understood. CD4+ T cells can differentiate in diverse subsets, but these T cell subsets have not been comprehensively studied in tumor-bearing mice. Although several mouse models have been previously described, they often involve the prevention of cancer cells modified to express potentially highly-immunogenic foreign or surrogate antigens (e.g. OVA or H-Y in female hosts) or their treatment as unrealistically small, non-vascularized pulmonary metastases. To more closely mimic human disease, we created transgenic mice expressing a class II-restricted TCR recognizing an endogenous melanocyte differentiation antigen called tyrosinase-related protein 1 (TRP-1 or gp75). We had indirect evidence that a recombinant vaccinia virus (rVV) encoding TRP-1 elicited a Th-dependent autoimmune vitiligo, but attempts to clone CD4+ cells reactive to TRP-1 were unsuccessful, perhaps due to tolerance-related mechanisms. We successfully generated B16/CIITA-specific CD4+ cells from antigen-negative Bw mice, identified the TRP-1 epitope they recognize corresponding to amino acids 113-127 (CRPGWRGAACNQKI) and cloned their TCR (Vbeta14/Valpha3.2). A founder with Y-chromosome-linked transgene was designated TRP-1 TCR transgenic and bred onto a RAG1-/- background to eliminate the rearrangement of endogenous TCR. To evaluate the degree of protection against the growth of melanoma conveyed by TRP-1 cells, we inoculated BwRAG1-/-TRP-1 Tg mice with B16 cells. Despite the presence of a large population of melanoma-specific T cells, the growth rate and lethality of the B16 tumor was only minimally delayed in TCR transgenic animals. A similar lack of protection against the tumor has been demonstrated previously in other TCR transgenic models and attributed to immunologic ignorance and lack of co-stimulatory signaling. We also found that adoptive transfer of cells cultured in IL-2 had minimal impact on tumor growth.We generated different TRP-1 T helper subsets (Th1, Th17 and Th0) by culturing the cells under strictly defined polarizing conditions. The degree of expansion of both Th1 and Th17 was similar and higher in comparison to the neutral (Th0) condition. To assess subset commitment, we analyzed the phenotype, gene expression patterns and cytokine secretion profiles. Th1 cells produced high quantities of IFN-gamma, TNF-alpha, IL-10 and lower amounts of IL-2 upon antigen stimulation. As expected, only Th17-skewed cells secreted significant high quantities of IL-17A and CCL20 (MIP3) as well as IL-2, IL-6 and IL-21 and produced smaller but amounts of IFN-gamma and TNF-alpha. Non-polarized Th0 cells secreted IFN-gamma at intermediate levels but did not produce IL-17A. Recognition was stronger upon exposure to B16 engineered to express the MHC class II transactivator CIITA, but there was no release of cytokines upon incubation with TRP-1-negative tumor cell lines MCA205 and EL-4. Intracellular staining upon restimulation demonstrated that virtually all Th1 cells produced IFN-gamma, while only those cells programmed in TGF-beta and IL-6 contained a significant percentage of IL-17A-secreting lymphocytes were also capable of producing IFN-gamma. Microarray analysis of in vitro polarized cells populations showed a striking up-regulation of IL-17A (105-fold difference) and CCL20/MIP3 (95-fold difference). mRNAs encoding IL-17F and IL-22, which are additional markers of Th17 polarization, were also elevated. As suggested by ELISA results, mRNA levels for IL-2 and IL-21 as well as another common gamma-chain cytokine, IL-9, were higher in Th17-polarized population. We treated C57BL/6 mice bearing 10-12-day-established tumors with adoptive transfer of Th0, Th1 or Th17 cells. Surprisingly, only Th17-skewed cells mediated a significant (p=0.001 vs.Th0 and Th1-treated groups) tumor regressionleading to a complete cure and the long-term survival (Fig. 39). Despite initial tumor shrinkage, all animals injected with Th0 or Th1 cells relapsed and eventually had to be sacrificed because of melanoma progression. Long-term surviving mice developed vitiligo in both Th1 and Th17-treated groups, but the severity of this autoimmune manifestation was far greater in the Th17-treated animals. In addition, the absolute numbers of Vbeta14+CD4+ splenocytes recovered after transfer from Th17-treated animals were consistently the highest, indicating persistence and/or proliferation advantage of cells polarized with TGF-beta and IL-6 over the other subtypes.To test the roles of cytokines produced by Th17 cells, we used neutralizing antibodies to IL-17A, IFN-gamma or IL-23, which is known to support the survival of Th17 T lymphocytes. Unexpectedly, tumor rejection was completely inhibited only by anti-IFN-gamma treatment while the effects of in vivo neutralization of IL-17A and IL-23 did not reach statistical significance (p>0.05 vs. Th17 isotype control) (Fig. 40). In exploring this further, we found that therapy with Th17-polarized cells was equally effective in both WT (C57BL/6) and IFN-gamma-/- hosts, indicating that host IFN-gamma did not play a major role. In sharp contrast, the ability of hosts to receive the IFN-gamma signal was critical because Th17-skewed cells were essentially ineffective in IFN-gammaR-/- mice. It is possible that Th17 phenotype is not stable and undergoes evolution into type 1 response in vivo after adoptive cell transfer in the TRP-1 model. Many cytokines involved (IL-6, IL-21, IL-23) signal via STAT3. STAT3-mediated signaling has been implicated in cancer development and has been associated with anti-apoptotic and pro-survival effects. While STAT3 may have a role in de novo cancer formation, it is also possible that in mature T cells it might have some effects that are beneficial and allow for better survival upon adoptive cell transfer. Finally, the impact of distinct cytokines on lineage commitment decisions is better established in CD4+ than CD8+ T cells, but it seems likely that similar differentiation plasticity might occur in CD8+ T cells, as shown in a hypothetical model of CD8+ T cell differentiation that we have developed in the lab.

CD4+细胞在抗肿瘤免疫中的作用仍然存在争议且知之甚少。 CD4+ T 细胞可以分化为不同的亚群，但这些 T 细胞亚群尚未在荷瘤小鼠中进行全面研究。尽管之前已经描述了几种小鼠模型，但它们通常涉及预防经过修饰以表达潜在的高免疫原性外来或替代抗原（例如女性宿主中的 OVA 或 H-Y）的癌细胞，或治疗不切实际的小非血管化肺转移瘤。为了更接近地模拟人类疾病，我们创建了表达 II 类限制性 TCR 的转基因小鼠，该 TCR 识别称为酪氨酸酶相关蛋白 1（TRP-1 或 gp75）的内源性黑素细胞分化抗原。我们有间接证据表明，编码 TRP-1 的重组牛痘病毒 (rVV) 会引发 Th 依赖性自身免疫性白癜风，但尝试克隆对 TRP-1 具有反应性的 CD4+ 细胞并未成功，这可能是由于耐受相关机制所致。我们成功地从抗原阴性 Bw 小鼠中产生了 B16/CIITA 特异性 CD4+ 细胞，鉴定了它们识别的对应于氨基酸 113-127 (CRPGWRGAACNQKI) 的 TRP-1 表位，并克隆了它们的 TCR (Vbeta14/Valpha3.2)。具有 Y 染色体连锁转基因的创始人被指定为 TRP-1 TCR 转基因，并在 RAG1-/- 背景上培育，以消除内源 TCR 的重排。为了评估 TRP-1 细胞对黑色素瘤生长的保护程度，我们给 BwRAG1-/-TRP-1 Tg 小鼠接种了 B16 细胞。尽管存在大量黑色素瘤特异性 T 细胞，但 TCR 转基因动物中 B16 肿瘤的生长速度和致死率仅略有延迟。先前在其他 TCR 转基因模型中也证明了类似的缺乏针对肿瘤的保护作用，并归因于免疫学无知和缺乏共刺激信号传导。我们还发现，在 IL-2 中培养的细胞的过继转移对肿瘤生长的影响极小。我们通过在严格定义的极化条件下培养细胞，产生了不同的 TRP-1 T 辅助亚群（Th1、Th17 和 Th0）。与中性（Th0）条件相比，Th1 和 Th17 的扩张程度相似且更高。为了评估子集承诺，我们分析了表型、基因表达模式和细胞因子分泌谱。 Th1 细胞在抗原刺激后产生大量的 IFN-γ、TNF-α、IL-10 和少量的 IL-2。正如预期的那样，只有 Th17 倾向的细胞分泌大量的 IL-17A 和 CCL20 (MIP3) 以及 IL-2、IL-6 和 IL-21，并产生数量较少但数量较多的 IFN-γ 和 TNF-α。非极化 Th0 细胞分泌中等水平的 IFN-γ，但不产生 IL-17A。暴露于表达 MHC II 类反式激活因子 CIITA 的 B16 后，识别能力更强，但与 TRP-1 阴性肿瘤细胞系 MCA205 和 EL-4 一起孵育后，没有释放细胞因子。再刺激时的细胞内染色表明，几乎所有 Th1 细胞都产生 IFN-γ，而只有那些在 TGF-β 和 IL-6 中编程的细胞含有显着百分比的 IL-17A 分泌淋巴细胞也能够产生 IFN-γ。体外极化细胞群的微阵列分析显示 IL-17A（105 倍差异）和 CCL20/MIP3（95 倍差异）显着上调。编码 IL-17F 和 IL-22（Th17 极化的附加标记）的 mRNA 也升高。 ELISA 结果表明，Th17 极化人群中 IL-2 和 IL-21 以及另一种常见的 γ 链细胞因子 IL-9 的 mRNA 水平较高。我们通过 Th0、Th1 或 Th17 细胞的过继转移治疗携带 10-12 天肿瘤的 C57BL/6 小鼠。令人惊讶的是，只有Th17偏向细胞介导显着的（相对于Th0和Th1治疗组p=0.001）肿瘤消退，导致完全治愈和长期存活（图39）。尽管最初肿瘤缩小，但所有注射 Th0 或 Th1 细胞的动物都会复发，并最终因黑色素瘤进展而不得不被处死。长期存活的小鼠在 Th1 和 Th17 治疗组中都出现了白癜风，但这种自身免疫表现在 Th17 治疗组中严重得多。此外，从 Th17 处理的动物转移后回收的 Vbeta14+CD4+ 脾细胞的绝对数量始终是最高的，表明用 TGF-β 和 IL-6 极化的细胞相对于其他亚型具有持久性和/或增殖优势。为了了解 Th17 细胞产生的细胞因子的作用，我们使用了 IL-17A、IFN-γ 或 IL-23 的中和抗体，已知这些抗体可以支持 Th17 T 淋巴细胞的存活。出乎意料的是，仅通过抗IFN-γ治疗就完全抑制了肿瘤排斥，而体内中和IL-17A和IL-23的效果并未达到统计学显着性（与Th17同种型对照相比，p>0.05）（图40）。在进一步探索中，我们发现 Th17 极化细胞治疗在 WT (C57BL/6) 和 IFN-gamma-/- 宿主中同样有效，表明宿主 IFN-gamma 并未发挥主要作用。与此形成鲜明对比的是，宿主接收 IFN-gamma 信号的能力至关重要，因为 Th17 倾斜细胞在 IFN-gammaR-/- 小鼠中基本上无效。 Th17表型可能不稳定，在TRP-1模型中过继细胞转移后在体内进化为1型反应。许多细胞因子（IL-6、IL-21、IL-23）通过 STAT3 发出信号。 STAT3 介导的信号传导与癌症的发展有关，并与抗凋亡和促生存作用相关。虽然 STAT3 可能在癌症从头形成中发挥作用，但它也有可能在成熟 T 细胞中产生一些有益的作用，并允许在过继细胞转移后更好地存活。最后，不同细胞因子对谱系定型决策的影响在 CD4+ 中比 CD8+ T 细胞中更好地确定，但在 CD8+ T 细胞中似乎可能出现类似的分化可塑性，如我们的 CD8+ T 细胞分化的假设模型所示。在实验室开发的。