Abstract
Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates that often share structural similarities to conventional secondary lymphoid organs. In a variety of solid cancers, the presence of these structures commonly correlates with high densities of tumor-infiltrating T lymphocytes and prolonged patient survival. These observations suggest that TLS act as sites for the development of beneficial antitumor immune responses. However, few murine tumor models have been described that could enable a more comprehensive understanding of the functionality of TLS in solid cancers. We previously reported that murine B16-F1 melanoma or Lewis lung carcinoma cells transfected to express the model antigen ovalbumin form intratumoral TLS after implantation into the peritoneal cavity of C57BL/6 mice. In this chapter, we describe immunofluorescent microscopy and flow cytometry approaches for identifying and characterizing intratumoral TLS. Additionally, we describe an adoptive transfer method for demonstrating the infiltration of naïve T cells into B16-OVA melanoma tumors via the lymph node-like vasculature, which is an essential functional feature of tumor-associated TLS.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Vesely M, Kershaw M, Schreiber R, Smyth M (2011) Natural innate and adaptive immunity to cancer. Annu Rev Immunol 29:235–271
Fridman WH, Pagès F, Sautès-Fridman C, Galon J (2012) The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12:298–306. https://doi.org/10.1038/nrc3245
Galon J, Angell HK, Bedognetti D, Marincola FM (2013) The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity 39:11–26. https://doi.org/10.1016/j.immuni.2013.07.008
Rosenberg SA, Restifo NP, Yang JC et al (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 8:299–308. https://doi.org/10.1038/nrc2355
Klebanoff CA, Acquavella N, Yu Z, Restifo NP (2011) Therapeutic cancer vaccines: are we there yet? Immunol Rev 239:27–44. https://doi.org/10.1111/j.1600-065X.2010.00979.x
Lee S, Margolin K (2011) Cytokines in cancer immunotherapy. Cancers 3:3856–3893. https://doi.org/10.3390/cancers3043856
Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12:252–264. https://doi.org/10.1038/nrc3239
Gajewski TF, Louahed J, Brichard VG (2010) Gene signature in melanoma associated with clinical activity. Cancer J 16:399–403. https://doi.org/10.1097/PPO.0b013e3181eacbd8
Ji R-R, Chasalow SD, Wang L et al (2012) An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother 61:1019–1031. https://doi.org/10.1007/s00262-011-1172-6
Gajewski TF, Schreiber H, Fu Y-X (2013) Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol 14:1014–1022. https://doi.org/10.1038/ni.2703
Pitzalis C, Jones GW, Bombardieri M, Jones SA (2014) Ectopic lymphoid-like structures in infection, cancer and autoimmunity. Nat Rev Immunol 14:447–462. https://doi.org/10.1038/nri3700
Kratz A, Campos-Neto A, Hanson MS, Ruddle NH (1996) Chronic inflammation caused by lymphotoxin is lymphoid neogenesis. J Exp Med 183:1461–1472. https://doi.org/10.1084/jem.183.4.1461
Buckley CD, Barone F, Nayar S et al (2015) Stromal cells in chronic inflammation and tertiary lymphoid organ formation. Annu Rev Immunol 33:715–745. https://doi.org/10.1146/annurev-immunol-032713-120252
Ruddle NH (2016) High endothelial venules and lymphatic vessels in tertiary lymphoid organs: characteristics, functions, and regulation. Front Immunol 7:491. https://doi.org/10.3389/fimmu.2016.00491
Randall TD, Mebius RE (2014) The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms. Mucosal Immunol 7:455–466. https://doi.org/10.1038/mi.2014.11
Cruz-Migoni S, Caamaño J (2016) Fat-associated lymphoid clusters in inflammation and immunity. Front Immunol 7:612. https://doi.org/10.3389/fimmu.2016.00612
Dieu-Nosjean M-C, Goc J, Giraldo NA et al (2014) Tertiary lymphoid structures in cancer and beyond. Trends Immunol 35:571–580. https://doi.org/10.1016/j.it.2014.09.006
Goc J, Fridman W-H, Sautès-Fridman C, Dieu-Nosjean M-C (2013) Characteristics of tertiary lymphoid structures in primary cancers. OncoImmunology 2:e26836. https://doi.org/10.4161/onci.26836
Behr DS, Peitsch WK, Hametner C et al (2014) Prognostic value of immune cell infiltration, tertiary lymphoid structures and PD-L1 expression in Merkel cell carcinomas. Int J Clin Exp Pathol 7:7610–7621
Caro GD, Bergomas F, Grizzi F et al (2014) Occurrence of tertiary lymphoid tissue is associated with T-cell infiltration and predicts better prognosis in early-stage colorectal cancers. Clin Cancer Res 20:2147–2158. https://doi.org/10.1158/1078-0432.CCR-13-2590
Goc J, Germain C, Vo-Bourgais TKD et al (2014) Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells. Cancer Res 74:705–715. https://doi.org/10.1158/0008-5472.CAN-13-1342
Thompson ED, Enriquez HL, Fu Y-X, Engelhard VH (2010) Tumor masses support naive T cell infiltration, activation, and differentiation into effectors. J Exp Med 207:1791–1804. https://doi.org/10.1084/jem.20092454
Peske JD, Thompson ED, Gemta L et al (2015) Effector lymphocyte-induced lymph node-like vasculature enables naive T-cell entry into tumours and enhanced anti-tumour immunity. Nat Commun 6:7114. https://doi.org/10.1038/ncomms8114
Hargadon KM, Brinkman CC, Sheasley-O’Neill SL et al (2006) Incomplete differentiation of tumor-specific CD8+ T cells in tumor-draining lymph nodes. J Immunol 177:6081–6090
Palazón A, Teijeira A, Martínez-Forero I et al (2011) Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes. Cancer Res 71:801–811. https://doi.org/10.1158/0008-5472.CAN-10-1733
Sasaki K, Zhu X, Vasquez C et al (2007) Preferential expression of very late antigen-4 on type 1 CTL cells plays a critical role in trafficking into central nervous system tumors. Cancer Res 67:6451–6458. https://doi.org/10.1158/0008-5472.CAN-06-3280
Scimone ML, Aifantis I, Apostolou I et al (2006) A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus. Proc Natl Acad Sci 103:7006–7011. https://doi.org/10.1073/pnas.0602024103
Walch JM, Zeng Q, Li Q et al (2013) Cognate antigen directs CD8+ T cell migration to vascularized transplants. J Clin Invest 123:2663–2671. https://doi.org/10.1172/JCI66722
Funding
This work was supported by the United States Public Health Service (USPHS) grants R01 CA78400 and R01 CA181794 (V.H.E.). Additional support was provided by USPHS grant P30 CA0044579 to the University of Virginia Cancer Center. A.B.R. was supported by USPHS training grant AI007496 and the Wagner Fellowship. J.D.P. was supported by USPHS training grant GM007276 and the Farrow Fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rodriguez, A.B., Peske, J.D., Engelhard, V.H. (2018). Identification and Characterization of Tertiary Lymphoid Structures in Murine Melanoma. In: Dieu-Nosjean, MC. (eds) Tertiary Lymphoid Structures. Methods in Molecular Biology, vol 1845. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8709-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8709-2_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8708-5
Online ISBN: 978-1-4939-8709-2
eBook Packages: Springer Protocols