Developing new immunotherapies based of CD4+ T cells

开发基于 CD4 T 细胞的新免疫疗法

• 批准号: 8763317
• 负责人: Nicholas Restifo
• 金额: $ 58.16万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8763317
• 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

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 regression leading 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，而且人们对其知之甚少。CD4+ T细胞可以分化成不同的亚群，但这些T细胞亚群尚未在荷瘤小鼠中得到全面的研究。尽管先前已经描述了几种小鼠模型，但它们通常涉及预防被修饰为表达潜在的高免疫原性外源或替代抗原（例如雌性宿主中的OVA或H-Y）的癌细胞，或者将其作为不切实际的小的、无血管化的肺转移瘤进行治疗。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识别内源性黑素细胞分化抗原酪氨酸酶相关蛋白1 （TRP-1或gp75）。我们有间接证据表明，编码TRP-1的重组痘苗病毒（rVV）引发了th依赖性自身免疫性白癜风，但试图克隆对TRP-1反应的CD4+细胞未能成功，可能是由于耐受性相关机制。我们成功地从抗原阴性的Bw小鼠中生成了B16/ ciita特异性CD4+细胞，鉴定了它们识别的TRP-1表位对应的氨基酸113-127 (CRPGWRGAACNQKI)，并克隆了它们的TCR （Vbeta14/Valpha3.2）。将具有y染色体连锁转基因的创始人基因指定为TRP-1 TCR转基因，并将其培育到RAG1-/-背景上，以消除内源性TCR的重排。为了评估TRP-1细胞对黑色素瘤生长的保护程度，我们用B16细胞接种BwRAG1-/-TRP-1 Tg小鼠。尽管存在大量黑色素瘤特异性T细胞，但在TCR转基因动物中，B16肿瘤的生长速度和致死率仅被轻微延迟。先前在其他TCR转基因模型中已经证明了类似的对肿瘤缺乏保护的情况，这归因于免疫无知和缺乏共刺激信号。我们还发现在IL-2中培养的细胞过继转移对肿瘤生长的影响很小。我们在严格定义的极化条件下培养细胞，产生不同的TRP-1 T辅助亚群（Th1， Th17和Th0）。与中性（Th0）条件相比，Th1和Th17的扩增程度相似且更高。为了评估亚群承诺，我们分析了表型、基因表达模式和细胞因子分泌谱。Th1细胞在抗原刺激下产生大量的ifn - γ、tnf - α、IL-10和少量的IL-2。正如预期的那样，只有th17倾斜的细胞分泌大量的IL-17A和CCL20 （MIP3）以及IL-2、IL-6和IL-21，并产生少量的ifn - γ和tnf - α。非极化Th0细胞分泌ifn - γ中等水平，但不产生IL-17A。暴露于表达MHC II类反激活因子CIITA的B16后，识别能力增强，但与trp -1阴性肿瘤细胞系MCA205和EL-4孵养后，细胞因子没有释放。再刺激后的细胞内染色表明，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著比例的分泌il - 17a的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（差异105倍）和CCL20/MIP3（差异95倍）显著上调。编码IL-17F和IL-22的mrna （Th17极化的额外标记物）也升高。ELISA结果表明，在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。我们用Th0、Th1或Th17细胞过继转移治疗10-12天肿瘤的C57BL/6小鼠。令人惊讶的是，只有th17倾斜的细胞介导了显著的肿瘤消退（p=0.001，与th0和th1治疗组相比），导致完全治愈和长期生存（图39）。尽管最初肿瘤缩小，但所有注射Th0或Th1细胞的动物都复发了，最终由于黑色素瘤的进展不得不牺牲。在Th1和th17治疗组中，长期存活的小鼠都出现了白癜风，但在th17治疗组中，这种自身免疫表现的严重程度要大得多。此外，从th17处理动物移植后恢复的Vbeta14+CD4+脾细胞的绝对数量始终是最高的，这表明与tgf - β和IL-6极化的细胞比其他亚型具有持久性和/或增殖优势。为了测试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，已知这些抗体支持Th17 T淋巴细胞的存活。出乎意料的是，只有抗ifn - γ治疗才能完全抑制肿瘤排斥反应，而体内中和IL-17A和IL-23的效果没有达到统计学意义（p>0.05 vs. Th17同型对照）（图40）。在进一步研究中，我们发现用th17极化细胞治疗在WT （C57BL/6）和ifn - γ -/-宿主中同样有效，这表明宿主ifn - γ没有发挥主要作用。与之形成鲜明对比的是，宿主接收ifn - γ信号的能力至关重要，因为th17倾斜的细胞在ifn - γ -/-小鼠中基本上是无效的。在TRP-1模型中过继细胞转移后，Th17的表型可能不稳定，在体内进化为1型反应。许多细胞因子通过STAT3参与（IL-6, IL-21, IL-23）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有关。虽然STAT3可能在新生癌症形成中起作用，但在成熟的T细胞中，它也可能有一些有益的作用，并允许在过继细胞转移时更好地存活。最后，与CD8+ T细胞相比，不同细胞因子对谱系承诺决定的影响在CD4+细胞中得到了更好的确立，但似乎类似的分化可塑性可能发生在CD8+ T细胞中，正如我们在实验室开发的CD8+ T细胞分化的假设模型所示。