肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学
Summary
在这里，我们提出了一种肿瘤移植方案，用于表征小鼠肿瘤模型中肿瘤固有和外周来源的肿瘤浸润淋巴细胞。通过流式细胞术特异性追踪受体来源的免疫细胞的流入，揭示了抗肿瘤免疫反应期间这些细胞表型和功能变化的动力学。
Abstract
T细胞介导的免疫在针对肿瘤的免疫反应中起着至关重要的作用，其中细胞毒性T淋巴细胞（CTL）在根除癌细胞中起主导作用。然而，肿瘤微环境（TME）内肿瘤抗原特异性CD8 + T细胞的起源和补充仍然模糊不清。该方案采用B16F10-OVA黑色素瘤细胞系，其稳定表达替代新抗原，卵清蛋白（OVA）和TCR转基因OT-I小鼠，其中超过90%的CD8 + T细胞特异性识别OVA衍生的肽OVA257-264（SIINFEKL）与I类主要组织相容性复合物（MHC）分子H2-Kb结合。这些特征使得能够在肿瘤发生期间研究抗原特异性T细胞反应。
将该模型与肿瘤移植手术相结合，将来自供体的肿瘤组织移植到肿瘤匹配的同源受体小鼠中，以精确追踪受体来源的免疫细胞流入移植的供体组织，从而可以分析肿瘤固有和外围起源的抗原特异性CD8 +的免疫应答。 T细胞。发现这两个种群之间发生了动态转变。总的来说，这种实验设计为精确研究TME中CD8 + T细胞的免疫反应提供了另一种方法，这将为肿瘤免疫学提供新的线索。
Introduction
CD8 + T细胞介导的免疫反应在控制肿瘤生长中起着关键作用。在肿瘤发生期间，幼稚的CD8 + T细胞在抗原识别时以MHC I类限制方式被激活，随后分化为效应细胞并浸润到肿瘤质量1，2中。然而，在肿瘤微环境（TME）内，长期的抗原暴露以及免疫抑制因素会驱动浸润的肿瘤特异性CD8 + T细胞进入低反应状态，称为“疲惫”3。疲惫的T细胞（Tex）与急性病毒感染中产生的效应或记忆T细胞不同，无论是转录还是表观遗传。这些Tex细胞的主要特征是一系列抑制性受体的持续和升高表达以及效应器功能的分层丧失。此外，耗尽的CD8 + T细胞的增殖能力受损导致肿瘤特异性T细胞的数量减少，使得TME内残留的CD8 + T细胞几乎不能提供足够的保护性免疫来对抗肿瘤进展3。因此，维持或加强肿瘤内抗原特异性CD8 + T细胞对于肿瘤抑制是必不可少的。
此外，免疫检查点阻断（ICB）疗法被认为通过增加T细胞浸润来重振肿瘤中的Tex，从而增加T细胞数量并恢复T细胞功能以增强肿瘤抑制。ICB治疗的广泛应用改变了癌症治疗的格局，很大一部分患者经历了持久的反应4，5，6。然而，大多数患者和癌症类型对ICB没有或只是暂时有反应。TME中T细胞浸润不足被认为是导致ICB耐药性的基本机制之一7，8。
几项研究已经证明了肿瘤浸润CD8 + T细胞（TILs）在患者和小鼠模型9，10，11，12中的异质性。已经证实，肿瘤肿块中表达T细胞因子-1（TCF1）的CD8 + T细胞亚群表现出干细胞样性质，这可能进一步产生终末耗尽的T细胞，并负责ICB治疗后的增殖爆发12，13，14，15，16，17，18，19，20，21，22.然而，已经证明，TME中仅存在一小部分抗原特异性TCF1 + CD8 + T细胞，并响应ICB 23，24，25，26产生扩展的分化后代池。该人群的有限规模是否足以确保细胞毒性T淋巴细胞（CTL）的持久性以控制肿瘤进展尚不清楚，并且是否需要从外围组织补充需要进一步研究。此外，最近的研究表明，在抗程序性细胞死亡蛋白1治疗后，预先存在的肿瘤特异性T细胞的重振能力不足，并且出现了新的，以前不存在的clonotypes。这表明T细胞对检查点阻断的反应可能是由于新涌入的T细胞克隆27的不同库。结合旁观者在TME中非肿瘤反应性细胞毒性T细胞组分的存在，这些发现促使建立了肿瘤同种异体移植模型来研究外围来源CD8 + T细胞11的作用。
到目前为止，几种肿瘤植入，以及免疫细胞过继转移，已广泛应用于肿瘤免疫学领域28。TILs，外周血单核细胞和来自其他组织的肿瘤反应性免疫细胞可以使用这些方法很好地表征。然而，在研究全身性和局部抗肿瘤免疫之间的相互作用时，这些模型似乎不足以检查来自外围的免疫细胞与TME之间的相互作用。在这里，将肿瘤组织从供体移植到肿瘤匹配的受体小鼠中，以精确追踪受体来源的免疫细胞的流入，并同时观察TME中的供体来源的细胞。
在这项研究中，用B16F10-OVA黑色素瘤细胞系建立了黑色素瘤的小鼠同源模型，该模型稳定地表达替代物新抗原卵清蛋白。TCR转基因OT-I小鼠，其中超过90%的CD8 + T细胞特异性识别与I类MHC分子H2-Kb结合的OVA衍生肽OVA 257-264（SIINFEKL），能够研究B16F10-OVA肿瘤模型中开发的抗原特异性T细胞反应。将此模型与肿瘤移植相结合，比较肿瘤固有和外围抗原特异性CD8 + T细胞的免疫反应，以揭示这两个群体之间的动态转变。总的来说，这种实验设计为精确研究TME中CD8 + T细胞的免疫反应提供了另一种方法，这为TME中肿瘤特异性T细胞免疫反应的动力学提供了新的线索。
Protocol
Representative Results
Discussion
T细胞介导的免疫在针对肿瘤的免疫反应中起着至关重要的作用，其中CTL在根除癌细胞中起主导作用。然而，TME内肿瘤抗原特异性CTL的起源尚未阐明30。这种肿瘤移植方案的使用提供了一个重要的线索，即尽管存在干细胞样TCF1 +祖细胞CD8 + T细胞，但肿瘤内抗原特异性CD8 + T细胞可能不会持续很长时间。值得注意的是，外周来源的肿瘤特异性CD8 + T细?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究由国家杰出青年自然科学基金（LY第31825011号）和国家自然科学基金（QH第31900643号，ZW 31900656号）资助。
Materials
| 0.22 μm filter | Millipore | SLGPR33RB | |
| 1 mL tuberculin syringe | KDL | BB000925 | |
| 1.5 mL centrifuge tube | KIRGEN | KG2211 | |
| 100 U insulin syringe | BD Biosciences | 320310 | |
| 15 mL conical tube | BEAVER | 43008 | |
| 2,2,2-Tribromoethanol (Avertin) | Sigma | T48402-25G | |
| 2-Methyl-2-butanol | Sigma | 240486-100ML | |
| 70 μm nylon cell strainer | BD Falcon | 352350 | |
| APC anti-mouse CD45.1 | BioLegend | 110714 | Clone:A20 |
| B16F10-OVA cell line | bluefbio | BFN607200447 | |
| BSA-V (bovine serum albumin) | Bioss | bs-0292P | |
| BV421 Mouse Anti-Mouse CD45.2 | BD Horizon | 562895 | Clone:104 |
| cell culture dish | BEAVER | 43701/43702/43703 | |
| centrifuge | Eppendorf | 5810R-A462/5424R | |
| cyclophosphamide | Sigma | C0768-25G | |
| Dulbecco's Modified Eagle Medium | Gibco | C11995500BT | |
| EasySep Mouse CD8+ T Cell Isolation Kit | Stemcell Technologies | 19853 | |
| EDTA | Sigma | EDS-500g | |
| FACS tubes | BD Falcon | 352052 | |
| fetal bovine serum | Gibco | 10270-106 | |
| flow cytometer | BD | FACSCanto II | |
| hemocytometer | PorLab Scientific | HM330 | |
| isoflurane | RWD life science | R510-22-16 | |
| KHCO3 | Sangon Biotech | A501195-0500 | |
| LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation | Life Technologies | L10199 | |
| needle carrier | RWD Life Science | F31034-14 | |
| NH4Cl | Sangon Biotech | A501569-0500 | |
| paraformaldehyde | Beyotime | P0099-500ml | |
| PE anti-mouse TCR Vα2 | BioLegend | 127808 | Clone:B20.1 |
| Pen Strep Glutamine (100x) | Gibco | 10378-016 | |
| PerCP/Cy5.5 anti-mouse CD8a | BioLegend | 100734 | Clone:53-6.7 |
| RPMI-1640 | Sigma | R8758-500ML | |
| sodium azide | Sigma | S2002 | |
| surgical forceps | RWD Life Science | F12005-10 | |
| surgical scissors | RWD Life Science | S12003-09 | |
| suture thread | RWD Life Science | F34004-30 | |
| trypsin-EDTA | Sigma | T4049-100ml |
Referências
- Blank, C. U., et al. Defining ‘T cell exhaustion. Nature Reviews Immunology. 19 (11), 665-674 (2019).
- Leko, V., Rosenberg, S. A. Identifying and targeting human tumor antigens for T cell-based immunotherapy of solid tumors. Cancer Cell. 38 (4), 454-472 (2020).
- McLane, L. M., Abdel-Hakeem, M. S., Wherry, E. J. CD8 T cell exhaustion during chronic viral infection and cancer. Annual Review of Immunology. 37, 457-495 (2019).
- Davis, M. M., Brodin, P. Rebooting human immunology. Annual Review of Immunology. 36, 843-864 (2018).
- Sharma, P., Allison, J. P. The future of immune checkpoint therapy. Science. 348 (6230), 56-61 (2015).
- Littman, D. R. Releasing the brakes on cancer immunotherapy. Cell. 373 (16), 1490-1492 (2015).
- Verma, V., et al. PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nature Immunology. 20, 1231-1243 (2019).
- Hashimoto, M., et al. CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annual Review of Medicine. 69, 301-318 (2018).
- Dammeijer, F., et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell. 38 (5), 685-700 (2020).
- Buchwald, Z. S., et al. Tumor-draining lymph node is important for a robust abscopal effect stimulated by radiotherapy. Journal for ImmunoTherapy of Cancer. 8 (2), 000867 (2020).
- Philip, M., Schietinger, A. Heterogeneity and fate choice: T cell exhaustion in cancer and chronic infections. Current Opinion in Immunology. 58, 98-103 (2019).
- Miller, B. C., et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nature Immunology. 20, 326-336 (2019).
- Wu, T. D., et al. Peripheral T cell expansion predicts tumour infiltration and clinical response. Nature. 579, 274-278 (2020).
- Im, S. J., Konieczny, B. T., Hudson, W. H., Masopust, D., Ahmed, R. PD-1+ stemlike CD8 T cells are resident in lymphoid tissues during persistent LCMV infection. Proceedings of the National Academy of Sciences of the United State of America. 117 (8), 4292-4299 (2020).
- Beltra, J. C., et al. Developmental relationships of four exhausted CD8(+) T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity. 52 (5), 825-841 (2020).
- Myers, L. M., et al. A functional subset of CD8(+) T cells during chronic exhaustion is defined by SIRPalpha expression. Nature Communications. 10 (1), 794 (2019).
- Jansen, C. S., et al. An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature. 576, 465-470 (2019).
- Jadhav, R. R., et al. Epigenetic signature of PD-1+ TCF1+ CD8 T cells that act as resource cells during chronic viral infection and respond to PD-1 blockade. Proceedings of the National Academy of Sciences of the United State of America. 116 (28), 14113-14118 (2019).
- Li, H., et al. Dysfunctional CD8 T cells form a proliferative, dynamically regulated compartment within human melanoma. Cell. 176 (4), 775-789 (2018).
- Kurtulus, S., et al. Checkpoint blockade immunotherapy induces dynamic changes in PD-1(-)CD8(+) tumor-infiltrating T cells. Immunity. 50 (1), 181-194 (2019).
- Fransen, M. F., et al. Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy. JCI Insight. 3 (23), 124507 (2018).
- E, J. F., et al. CD8(+)CXCR5(+) T cells in tumor-draining lymph nodes are highly activated and predict better prognosis in colorectal cancer. Human Immunology. 79 (6), 446-452 (2018).
- Snell, L. M., et al. CD8(+) T cell priming in established chronic viral infection preferentially directs differentiation of memory-like cells for sustained immunity. Immunity. 49 (4), 678-694 (2018).
- Siddiqui, I., et al. Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity. 50 (1), 195-211 (2019).
- Wang, Y., et al. The transcription factor TCF1 preserves the effector function of exhausted CD8 T cells during chronic viral infection. Frontiers in Immunology. 10, 169 (2019).
- Krishna, S., et al. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science. 370 (6522), 1328-1334 (2020).
- Yost, K. E., et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nature Medicine. 25, 1251-1259 (2019).
- Zitvogel, L., Pitt, J. M., Daillere, R., Smyth, M. J., Kroemer, G. Mouse models in oncoimmunology. Nature Reviews Cancer. 16 (12), 759-773 (2016).
- Li, Y., et al. Bcl6 preserves the suppressive function of regulatory T cells during tumorigenesis. Frontiers in Immunology. 11, 806 (2020).
- Yu, D., Ye, L. A portrait of CXCR5(+) follicular cytotoxic CD8(+) T cells. Trends in Immunology. 39 (12), 965-979 (2018).
- Bracci, L., et al. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clinical Cancer Research. 13 (2), 644-653 (2007).
- Salem, M. L., El-Naggar, S. A., Mahmoud, H. A., Elgharabawy, R. M., Bader, A. M. Cyclophosphamide eradicates murine immunogenic tumor coding for a non-self-antigen and induces antitumor immunity. International Journal of Immunopathology and Pharmacology. 32, 1-5 (2018).
- Thorsson, V., et al. The Immune landscape of cancer. Immunity. 48 (4), 812-830 (2018).
