使用造血细胞因子接种肿瘤的实验性黑色素瘤免疫治疗模型
Summary
该协议提出了一种癌症免疫治疗模型，使用表达Flt3L的B16-F10黑色素瘤进行基于细胞的肿瘤疫苗接种。该协议演示了程序，包括培养的肿瘤细胞的制备，肿瘤植入，细胞照射，肿瘤生长的测量，肿瘤内免疫细胞的分离和流式细胞术分析。
Abstract
Fms样酪氨酸激酶3配体（Flt3L）是一种造血细胞因子，可促进树突状细胞（DC）的存活和分化。它已被用于肿瘤疫苗中，以激活先天免疫并增强抗肿瘤反应。该协议展示了使用基于细胞的肿瘤疫苗的治疗模型，该疫苗由表达Flt3L的B16-F10黑色素瘤细胞以及肿瘤微环境（TME）中免疫细胞的表型和功能分析组成。描述了培养的肿瘤细胞制备、肿瘤植入、细胞照射、肿瘤大小测量、肿瘤内免疫细胞分离和流式细胞术分析的程序。该方案的总体目标是提供一个临床前实体瘤免疫治疗模型，以及一个研究肿瘤细胞与浸润免疫细胞之间关系的研究平台。这里描述的免疫治疗方案可以与其他治疗方式相结合，例如免疫检查点阻断（抗CTLA-4，抗PD-1，抗PD-L1抗体）或化疗，以提高黑色素瘤的癌症治疗效果。
Introduction
癌症免疫疗法因其毒性副作用较小和反应更持久而被认为是一种有前途的治疗策略。已经开发了几种类型的免疫疗法，包括溶瘤病毒疗法、癌症疫苗、细胞因子疗法、单克隆抗体、过继细胞转移（CAR-T 细胞或 CAR-NK）和免疫检查点阻断1。
对于癌症疫苗，有不同形式的治疗性疫苗，例如基于全细胞的疫苗，蛋白质或肽疫苗以及RNA或DNA疫苗。疫苗接种依赖于抗原呈递细胞（APC）处理肿瘤抗原（包括肿瘤特异性抗原）并将其以免疫原性形式呈递给T细胞的能力。树突状细胞（DC）已知是最有效的APC，被认为在抗肿瘤免疫中起重要作用2，3。这些细胞吸收并处理肿瘤抗原，然后迁移到引流淋巴结（dLN），通过T细胞受体（TCR）和共刺激分子的参与来启动和激活肿瘤特异性T效应（Teff）细胞。这导致肿瘤特异性细胞毒性T细胞（CTL）的分化和扩增，CTL浸润肿瘤并杀死肿瘤细胞4。因此，DC的活化和成熟代表了刺激肿瘤抗原免疫的有吸引力的策略。
已知 Flt3L 可促进表达 MHC II 类、CD11c、DEC205 和 CD86 蛋白的功能成熟 DC 的成熟和扩增5。肿瘤内而非静脉内给药含有 Flt3L 基因 （Adv-Flt3L） 的腺病毒载体已被证明可促进针对口位肿瘤的免疫治疗活性6。Flt3L还被用于基于肿瘤细胞的疫苗，该疫苗由稳定表达逆转录病毒转导的Flt3L的辐照B16-F10细胞组成，作为增强DC对肿瘤抗原交叉呈递的一种方式，从而增加抗肿瘤反应。这里描述的B16-Flt3L肿瘤疫苗接种方案基于James Allison博士的第7组发表的一项研究。在本文中，他们报告说，B16-Flt3L疫苗与CTLA-4阻断相结合，协同诱导对已建立的黑色素瘤的排斥反应，从而提高生存率。
该协议的目标是为黑色素瘤提供临床前免疫治疗模型。这里描述了如何制备和植入肿瘤疫苗的详细程序，以及如何分析实体瘤肿瘤内免疫细胞的组成和功能。
Protocol
Representative Results
Discussion
这里描述的协议是基于Allison小组的研究。他们证明，B16-Flt3L疫苗与CTLA-4阻断的组合对存活率和肿瘤生长显示出协同作用，而在单独接受B16-Flt3L疫苗或抗CTLA-4抗体治疗的小鼠中没有观察到肿瘤生长减少7。最近的研究揭示了一种新型的Treg内在CTLA4-PKCη信号通路，该通路在调节Treg11的接触依赖性抑制活性中起着重要的强制性作用。单独使用B16-Flt3L疫苗治疗或具有Treg?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Stephen Schoenberger博士提供B16-Flt3L细胞，并感谢LJI动物和流式细胞术设施的工作人员提供出色的支持。
Materials
| 0.25% trypsin-EDTA | Gibco | 25200-056 | |
| 10% heat-inactivated FBS | Omega Scientific | FB-02 | Lot# 209018 |
| 30G needle | BD Biosciences | 305106 | |
| 96 well V-shape-bottom plate | SARSTEDT | 83.3926.500 | |
| B16 cell line expressing Fms-like tyrosine kinase 3 ligand (B16-Flt3L) | Gift of Dr. Stephen Schoenberger, LJI | Flt3L cDNAs were cloned into the pMG-Lyt2 retroviral vector, as in refernce 5, Supplemental Figure 1 | |
| B16-F10 cell lines | ATCC | CRL-6475 | |
| Centrifuge 5810R | Eppendorf | ||
| Cytofix fixation buffer | BD Biosciences | BDB554655 | Cell fixation buffer (4.2% PFA) |
| Cytofix/Cytoperm kit | BD Biosciences | 554714 | Fixation/Permeabilization Solution Kit |
| DNase I | Sigma | 11284932001 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Corning | 10013CV | |
| Electronic digital caliper | Fisherbrand | 14-648-17 | |
| FlowJo software | Tree Star | Flow cytometer data analysis | |
| GolgiStop (protein transport inhibitor) | BD Biosciences | 554724 | 1:1500 dilution |
| HEPES (1M) | Gibco | 15630-080 | |
| Ionomycin | Sigma | I0634 | |
| Iscove’s modified Dulbecco’s medium (IMDM) | Thermo Fisher | 12440053 | |
| LSR-II cytometers | BD Biosciences | Flow cytometer | |
| MEM nonessential amino acids | Gibco | 11140-050 | |
| penicillin and streptomycin | Gibco | 15140-122 | |
| Percoll | GE Healthcare Life Sciences | GE17-0891-02 | density gradient specific medium |
| PMA | Sigma | P1585 | |
| Red Blood Cell Lysing Buffer Hybri-Max liquid | Sigma | R7757-100ML | |
| RPMI 1640 medium | Corning | 10-040-CV | |
| RS2000 X-ray Irradiator | Rad Source Technologies | ||
| sodium pyruvate | Gibco | 11360-070 | |
| Sterile cell strainer 40 μm | Fisherbrand | 22-363-547 | |
| Sterile cell strainer 70 μm | Fisherbrand | 22-363-548 | |
| TL Liberase | Roche | 477530 | |
| Zombie Aqua fixable viability kit | BioLegend | 423101 | |
| Antibodies | |||
| Anti-mCD45 | BioLegend | 103135 | Clone: 30-F11 Fluorophore: BV570 Dilution: 1:200 |
| Anti-mCD3ε | BioLegend | 100327 | Clone: 145-2C11 Fluorophore: PerCP-Cy5.5 Dilution: 1:200 |
| Anti-mCD8 | BioLegend | 100730 100724 |
Clone: 53-6.7 Fluorophore: Alexa Fluor 700, Alexa Fluor 647 Dilution: 1:200 |
| Anti-mCD4 | BioLegend | 100414 | Clone: GK1.5 Fluorophore: APC-Cy7 Dilution: 1:200 |
| Anti-mFoxp3 | Thermo Fisher Scientific | 11577382 | Clone: FJK-16s Fluorophore: FITC Dilution: 1:100 |
| Anti-m/hGzmB | BioLegend | 372208 | Clone: QA16A02 Fluorophore: PE Dilution: 1:100 |
| Anti-mIFNg | BioLegend | 505826 | Clone: XMG1.2 Fluorophore: PE-Cy7 Dilution: 1:100 |
| Anti-mCD19 | BioLegend | 115543 | Clone: 6D5 Fluorophore: BV785 Dilution: 1:100 |
| Anti-mGr1 | BioLegend | 108423 | Clone: RB6-8C5 Fluorophore: APC/Cy7 Dilution: 1:200 |
| Anti-mCD11b | BioLegend | 101223 | Clone: M1/70 Fluorophore: Pacific blue Dilution: 1:100 |
| Anti-mF4/80 | BioLegend | 123114 | Clone: BM8 Fluorophore: PECy7 Dilution: 1:100 |
| Anti-mCD11c | BioLegend | 117328 | Clone: N418 Fluorophore: PerCP Cy5.5 Dilution: 1:100 |
| Anti-mMHCII | BioLegend | 107622 | Clone: M5/114.15.2 Fluorophore: AF700 Dilution: 1:400 |
| Anti-mCD103 | BioLegend | 121410 | Clone: 2E7 Fluorophore: Alexa Fluor 647 Dilution: 1:200 |
| Anti-mCD86 | BioLegend | 105007 | Clone: GL-1 Fluorophore: PE Dilution: 1:200 |
| FC-blocker (Rat anti-mouse CD16/CD32) | BD Biosciences | 553141 | Clone: 2.4G2 Dilution: 1:200 |
References
- Zhang, Y., Zhang, Z. The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications. Cell & Molecular Immunology. 17 (8), 807-821 (2020).
- Banchereau, J., Steinman, R. M. Dendritic cells and the control of immunity. Nature. 392 (6673), 245-252 (1998).
- Banchereau, J., et al. Immunobiology of dendritic cells. Annual Review of Immunology. 18, 767-811 (2000).
- Martinez-Lostao, L., Anel, A., Pardo, J. How do cytotoxic lymphocytes kill cancer cells. Clinical Cancer Research. 21 (22), 5047-5056 (2015).
- Maraskovsky, E., et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. Journal of Experimental Medicine. 184 (5), 1953-1962 (1996).
- Talmadge, J. E., et al. Intratumoral, injection of adenoviral Flt3 ligand has therapeutic activity in association with increased intratumoral levels of T cells but not dendritic cells. Blood. 104 (11), 5280 (2004).
- Curran, M. A., Allison, J. P. Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors. American Association for Cancer Research. 69 (19), 7747-7755 (2009).
- Simon, S. R., Ershler, W. B. Hormonal influences on growth of B16 murine melanoma. Journal of the National Cancer Institute. 74 (5), 1085-1088 (1985).
- Broz, M. L., et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell. 26 (6), 938 (2014).
- Salmon, H., et al. Expansion and activation of CD103(+) dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition. Immunity. 44 (4), 924-938 (2016).
- Liu, H. Y., et al. Leveraging the Treg-intrinsic CTLA4-PKCeta signaling pathway for cancer immunotherapy. Journal for Immunotherapy Cancer. 9 (9), 002792 (2021).
- Kong, K. F., et al. Protein kinase C-eta controls CTLA-4-mediated regulatory T cell function. Nature Immunology. 15 (5), 465-472 (2014).
