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

  • Christophe Klein
  • Priyanka Devi-Marulkar
  • Marie-Caroline Dieu-Nosjean
  • Claire GermainEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Tertiary lymphoid structure Double immunohistochemistry Virtual multiplexing Image registration CD20 DC-LAMP PD-1 B-cell follicle Mature dendritic cell Follicular helper T cell Open-source software ImageJ 

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

Notes

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.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Christophe Klein
    • 1
    • 2
    • 3
  • Priyanka Devi-Marulkar
    • 2
    • 3
    • 4
    • 5
  • Marie-Caroline Dieu-Nosjean
    • 2
    • 3
    • 4
  • Claire Germain
    • 2
    • 3
    • 4
    • 6
    Email author
  1. 1.Center of Cellular Imaging and CytometryInstitut National de la Santé et de la Recherche Médicale (INSERM) UMRS 1138, Cordeliers Research CenterParisFrance
  2. 2.Sorbonne University, UMRS 1138, Cordeliers Research CenterParisFrance
  3. 3.Paris Descartes University, Sorbonne Paris Cité, UMRS 1138, Cordeliers Research CenterParisFrance
  4. 4.Laboratory “Cancer, immune control and Escape”INSERM, UMRS 1138, Cordeliers Research CenterParisFrance
  5. 5.“Cytokine Signaling” Unit, Department of ImmunologyInstitut Pasteur, INSERM U1221ParisFrance
  6. 6.Laboratory “Immune Intervention and Biotherapies”UPMC UMRS CR7—Inserm U1135—CNRS ERL 8255, Centre d’Immunologie et des Maladies Infectieuses (CIMI), Groupe hospitalier Pitié-SalpêtrièreParis Cedex 13France

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