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Tertiary Lymphoid Structures pp 47–69Cite as

Development of Tools for the Selective Visualization and Quantification of TLS-Immune Cells on Tissue Sections

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1845))

Abstract

Tertiary lymphoid structures (TLS) are considered as genuine markers of inflammation. Their presence within inflamed tissues or within the tumor microenvironment has been associated with the local development of an active immune response. While high densities of TLS are correlated with disease severity in autoimmune diseases or during graft rejection, it has been associated with longer patient survival in many cancer types. Their efficient visualization and quantification within human tissues may represent new tools for helping clinicians in adjusting their therapeutic strategy. Some immunohistochemistry (IHC) protocols are already used in the clinic to appreciate the level of immune infiltration in formalin-fixed, paraffin-embedded (FFPE) tissues. However, the use of two or more markers may sometimes be useful to better characterize this immune infiltrate, especially in the case of TLS. Besides the growing development of multiplex labeling approaches, imaging can also be used to overcome some technical difficulties encountered during the immunolabeling of tissues with several markers.

This chapter describes IHC methods to visualize in a human tissue (tumoral or not) the presence of TLS. These methods are based on the immunostaining of four TLS-associated immune cell populations, namely follicular B cells, follicular dendritic cells (FDCs), mature dendritic cells (mDCs), and follicular helper T cells (TFH), together with non-TFH T cells. Methodologies for subsequent quantification of TLS density are also proposed, as well as a virtual multiplexing method based on image registration using the open-source software ImageJ (IJ), aiming at co-localizing several immune cell populations from different IHC stainings performed on serial tissue sections.

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Abbreviations

AID:

Activation-induced cytidine deaminase

AP:

Alkaline phosphatase

APAAP:

Alkaline phosphatase anti-alkaline phosphatase

Bcl6:

B-cell lymphoma 6

CSR:

Class-switch recombination

FDC:

Follicular dendritic cell

FFPE:

Formalin-fixed, paraffin-embedded

GC:

Germinal center

HRP:

Horseradish peroxidase

HS:

Human serum

IF:

Immunofluorescence

IHC:

Immunohistochemistry

IJ:

ImageJ

mDC:

Mature dendritic cell

NSCLC:

Non-small cell lung cancer

RA:

Rheumatoid arthritis

ROA:

Region of analysis

ROI:

Region of interest

SHM:

Somatic hypermutation

SIFT:

Scale invariant feature transform

SLO:

Secondary lymphoid organ

TFH :

Follicular helper T cell

TLS:

Tertiary lymphoid structure

WSI:

Whole-slide image

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Funding

This work was supported by the “Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne University, Paris-Descartes University, the Labex Immuno-Oncology (LAXE62_9UMRS872 Fridman), CARPEM (Cancer Research for PErsonalized Medicine), Fondation ARC pour la Recherche sur le Cancer. Priyanka Devi-Marulkar was supported by a grant from the Fondation ARC pour la Recherche sur le Cancer. Claire Germain was supported by a grant from MedImmune LLC.

Author information

Author notes

  1. Priyanka Devi-Marulkar

    Present address: “Cytokine Signaling” Unit, Department of Immunology, Institut Pasteur, INSERM U1221, Paris, France

  2. Claire Germain

    Present address: Laboratory “Immune Intervention and Biotherapies”, UPMC UMRS CR7—Inserm U1135—CNRS ERL 8255, Centre d’Immunologie et des Maladies Infectieuses (CIMI), Groupe hospitalier Pitié-Salpêtrière, Paris Cedex 13, France

Authors and Affiliations

  1. Center of Cellular Imaging and Cytometry, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS 1138, Cordeliers Research Center, Paris, France

    Christophe Klein

  2. Sorbonne University, UMRS 1138, Cordeliers Research Center, Paris, France

    Christophe Klein, Priyanka Devi-Marulkar, Marie-Caroline Dieu-Nosjean & Claire Germain

  3. Paris Descartes University, Sorbonne Paris Cité, UMRS 1138, Cordeliers Research Center, Paris, France

    Christophe Klein, Priyanka Devi-Marulkar, Marie-Caroline Dieu-Nosjean & Claire Germain

  4. Laboratory “Cancer, immune control and Escape”, INSERM, UMRS 1138, Cordeliers Research Center, Paris, France

    Priyanka Devi-Marulkar, Marie-Caroline Dieu-Nosjean & Claire Germain

Authors
  1. Christophe Klein
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  2. Priyanka Devi-Marulkar
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  3. Marie-Caroline Dieu-Nosjean
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  4. Claire Germain
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Corresponding author

Correspondence to Claire Germain .

Editor information

Editors and Affiliations

  1. Laboratory “Cancer, Immune Control and Escape”, UMRS 1138 INSERM, Cordeliers Research Center, Sorbonne University, UMR 1138, Paris Descartes University, Sorbonne Paris City, UMR 1138, Paris, France

    Marie-Caroline Dieu-Nosjean

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Klein, C., Devi-Marulkar, P., Dieu-Nosjean, MC., Germain, C. (2018). Development of Tools for the Selective Visualization and Quantification of TLS-Immune Cells on Tissue Sections. 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_4

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  • DOI: https://doi.org/10.1007/978-1-4939-8709-2_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8708-5

  • Online ISBN: 978-1-4939-8709-2

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