Methods in molecular biology vol 1591 t cell trafficking methods and protocols

An appreciation of lymphocyte migration was first visualized in 1896 by Saxer who described “wandering lym-phocytes” in emergent lymph nodes, a cell type later identified Later work by A

T-Cell

Traffi cking

George Edward Rainger

Helen M Mcgettrick Editors

Methods and Protocols

Second Edition

Methods in

Molecular Biology 1591

Me t h o d s i n Mo l e c u l a r Bi o l o g y

Series Editor

John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

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T-Cell Trafficking

Methods and Protocols Second Edition

Edited by

George Edward Rainger

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, UK

Helen M Mcgettrick

Institute of Inflammation and Ageing, College of Medicine and Dental Sciences,

University of Birmingham, Birmingham, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology

ISBN 978-1-4939-6929-6 ISBN 978-1-4939-6931-9 (eBook)

DOI 10.1007/978-1-4939-6931-9

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Editors

George Edward Rainger

Institute of Cardiovascular Sciences

University of Birmingham

Birmingham, UK

Helen M Mcgettrick Institute of Inflammation and Ageing College of Medicine and Dental Sciences University of Birmingham

Birmingham, UK

Welcome to the second edition of Methods and Protocols for assessing T cell Trafficking,

in the Methods in Molecular Biology series The trafficking of T cells is relevant in ous contexts It occurs during population and maturation of T cells in the thymus and is required for dissemination of antigen nạve T cells to the secondary lymphatic organs where immune responses are initiated Indeed, T cell trafficking within lymph nodes plays an important role in the maturation of primary and secondary immune responses Antigen experienced effector T cells can undertake compartmentalized recirculation during immune surveillance In addition they are recruited to tissues during inflammation where they play important roles in the inflammatory response Importantly, we now recognize that inap-propriate or persistent trafficking of T cells into such sites makes a major contribution to the pathogenesis of immune-mediated inflammatory diseases which have an autoimmune

numer-or chronic inflammatnumer-ory component

Thus the trafficking of T cells has both physiological and pathological relevance and provides some challenging environments in which to make quantitative measurements This has seen the development of expertise which goes well beyond the standard laboratory methodologies which can be supported by commercially available kits and reagents The methods in this edition have been developed by experts in T cell trafficking, who have spent many years perfecting them Each chapter contains a step-by-step guide to conducting the assays, with useful hints to avoid common pitfalls The volume is organized into three sec-tions The first addresses homeostatic T cell trafficking during thymic maturation, followed

by the subsequent colonization of and egress from secondary lymphoid organs The second addresses T cell trafficking during “normal” inflammatory and immune responses Lastly,

we include a section on T cell trafficking in disease Each section is headed by an tive and accessible introduction written by experts who are actively investigating the regula-tion of T cell trafficking in these different scenarios We believe this will ensure that this book will become an essential point of reference for those new to the field of T cell traffick-ing, or to those looking to expand their technical capabilities

informa-We would like to thank all the authors for their invaluable contributions and willingness

to share their expertise Thanks also to Professor John Walker, the series editor, for ance in the process of compiling the book

guid-G Ed Rainger is generously supported by the British Heart Foundation of the UK.Helen M Mcgettrick is supported by generous funds from Arthritis Research UK

Helen M Mcgettrick

Preface

Contents

Preface v Contributors ix

1 Introduction to Homeostatic Migration 1

Mark C Coles

2 Analysis of Thymocyte Migration, Cellular Interactions, and Activation

by Multiphoton Fluorescence Microscopy of Live Thymic Slices 9

Jessica N Lancaster and Lauren I.R Ehrlich

3 Visualizing and Tracking T Cell Motility In Vivo 27

Robert A Benson, James M Brewer, and Paul Garside

4 Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular

Cell Network 43

Mario Novkovic, Lucas Onder, Gennady Bocharov,

and Burkhard Ludewig

5 Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes 59

Manisha Menon, Alexandre P Benechet, and Kamal M Khanna

6 Introduction: T Cell Trafficking in Inflammation and Immunity 73

Myriam Chimen, Bonita H.R Apta, and Helen M Mcgettrick

7 Leukocyte Adhesion Under Hemodynamic Flow Conditions 85

Charlotte Lawson, Marlene Rose, and Sabine Wolf

8 Endocrine Regulation of Lymphocyte Trafficking In Vitro 101

Bonita H.R Apta, Myriam Chimen, and Helen M Mcgettrick

9 Mesenchymal Stromal Cells as Active Regulators of Lymphocyte

Recruitment to Blood Vascular Endothelial Cells 121

Helen M Mcgettrick, Lewis S.C Ward, George Edward Rainger,

and Gerard B Nash

10 Monitoring RhoGTPase Activity in Leukocytes Using Classic

“Pull-Down” Assays 143

Marouan Zarrouk, David Killock, Izajur Rahman, Jessica Davies,

and Aleksandar Iveti ć

11 Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells

to Assess Lymphocyte-Endothelial Interactions 155

Jasmeet S Reyat, Michael G Tomlinson, and Peter J Noy

12 Introduction to Lymphocyte Trafficking in Disease 169

Patricia F Lalor and Elizabeth A Hepburn

13 Using Ex Vivo Liver Organ Cultures to Measure Lymphocyte Trafficking 177

Benjamin G Wiggins, Zania Stamataki, and Patricia F Lalor

14 In Vitro and Ex Vivo Models to Study T Cell Migration Through

the Human Liver Parenchyma 195

Benjamin G Wiggins, Konstantinos Aliazis, Scott P Davies,

Gideon Hirschfield, Patricia F Lalor, Gary Reynolds, and Zania Stamataki

15 Monitoring Migration of Activated T Cells to Antigen-Rich

Non-lymphoid Tissue 215

Eleanor Jayne Ward, Hongmei Fu, and Federica Marelli-Berg

16 Tissue Digestion for Stromal Cell and Leukocyte Isolation 225

Saba Nayar, Joana Campos, Nathalie Steinthal, and Francesca Barone

17 T Cell Response in the Lung Following Influenza Virus Infection 235

Robert A Benson, Jennifer C Lawton, and Megan K.L MacLeod

Index 249

Contents

Immunotherapy, University of Birmingham, Birmingham, UK

Sciences, University of Birmingham, Birmingham, UK

Inflammation and Ageing, College of Medical & Dental Sciences, University of

Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK

Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy

Inflammation, The University of Glasgow, Glasgow, UK

Barts and the London School of Medicine and Dentistry, Queen Mary University

of London, London, UK

Moscow, Russian Federation

Medical, Veterinary and Life Sciences, Glasgow Biomedical Research Centre,

University of Glasgow, Glasgow, UK

Inflammation and Ageing, College of Medical & Dental Sciences, University of

Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK

Sciences, University of Birmingham, Birmingham, UK

of York, North Yorkshire, UK

Immunotherapy, University of Birmingham, Birmingham, UK

Division, King’s College London, London, UK

and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

School of Medicine and Dentistry, Queen Mary University of London, London, UK

Veterinary and Life Sciences, Glasgow Biomedical Research Centre, Wellcome Trust Centre for Molecular Parasitology, Glasgow, UK

Hospital, Cheltenham, UK

Immunotherapy, University of Birmingham, Birmingham, UK

Contributors

Division, King’s College London, London, UK

Farmington, CT, USA

Division, King’s College London, London, UK

of Biomedical Research, University of Birmingham, Birmingham, UK

and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

London, UK

and Inflammation, The University of Glasgow, Glasgow, UK

Switzerland

and Inflammation, The University of Glasgow, Glasgow, UK

and Dental Sciences, University of Birmingham, Birmingham, UK

Farmington, CT, USA

Birmingham, UK

and Ageing, College of Medical & Dental Sciences, University of Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK

Division, King’s College London, London, UK

Birmingham, Birmingham, UK

JasMeet s Reyat • School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK

Immunotherapy, University of Birmingham, Birmingham, UK

of Biomedical Research, University of Birmingham, Birmingham, UK

Inflammation and Ageing, College of Medical & Dental Sciences, University of

Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK

Contributors

University of Birmingham, Birmingham, UK

London School of Medicine and Dentistry, Queen Mary University of London, London, UK

Birmingham, UK

Institute of Biomedical Research, University of Birmingham, Birmingham, UK

Division, King’s College London, London, UK

Contributors

George Edward Rainger and Helen M Mcgettrick (eds.), T-Cell Trafficking: Methods and Protocols, Methods in Molecular Biology,

vol 1591, DOI 10.1007/978-1-4939-6931-9_1, © Springer Science+Business Media LLC 2017

Key words Multiphoton, Modeling, 3D imaging, Migration, Thymus

interac-500 million years to provide a system that permits efficient tive immune responses to unknown pathogens, providing the capacity of very small numbers of lymphocytes to efficiently respond in localized lymphoid tissues and develop long-term memory to immunological challenges

adap-Over the last 120 years, immunologists have come to ate the role migration has in lymphoid tissue formation, immune cell development, and function using a range of technologies from light microscopy to radiolabeled cellular transfers in sheep and pigs

to lineage-specific fluorescent protein transgenic and knock-in mice permitting 3-dimensional (3D) imaging of immune homeo-stasis and function An appreciation of lymphocyte migration was first visualized in 1896 by Saxer who described “wandering lym-phocytes” in emergent lymph nodes, a cell type later identified

Later work by Alexandre Maximow identified three different ulations in lymph nodes, “wandering lymphocytes,” “resting wan-dering cells” (macrophages), and collagen-producing stromal

central principles of immune cell migration through blood vessels into both lymphoid tissues through specialized vessels, high endo-thelial venules (HEV), lymphocyte entry into peripheral tissues, and migration of immune cells from peripheral tissues to lymph nodes and subsequent entry into circulation through draining lym-

key insights into migration between tissues, the role of migration

in tissues was not well understood The emergence of multiphoton imaging in the early 2000s provided a new technological platform

to provide insights into the scale of lymphocyte migration and the

were found to very rapidly migrate within tissues and interact with antigen-presenting cells (T cell—dendritic cell, B cell -T cell, B cell—follicular dendritic cells (FDC)) in lymphoid tissues respond-ing to localized cues in their microenvironment produced by a 3D

cells (FRC), marginal reticular cells (MRC), and B cell zone stroma including FDCs are specialized stromal sets that support lympho-cyte homeostasis through the production of survival factors inter-leukin- 7 (IL-7) for T cells and BAFF for B cells; homeostatic chemokines CCL19, CCL21 (FRC, MRC), and CXCL13 (MRC, FDC); and signaling lipids (e.g., 7a,25 OH cholesterol) that con-

pro-cess permits very rare antigen-specific cells as low as one in million

to effectively respond to antigen in the highly organized stromal lymphoid tissue microenvironment

Despite the plethora of data from imaging and omics ogies, many questions remain to be addressed on how migration is regulated in lymphoid tissues through all stages of their develop-ment and function Understanding the molecular, biophysical, and cellular processes of immune cell migration is of clinical signifi-cance; targeting lymphocyte entry into (natalizumab: anti-VLA4) and exit (fingolimod: S1PR antagonism) from tissues has shown

vaccines in part work by stimulating tissue remodeling in B cell licles leading to germinal center reactions, an emergent behavior driven by active cross talk between innate immune cells, activated

fol-Mark C Coles

lymphocytes, and stroma Thus the development of new tics and vaccines that can enhance protective immune responses to pathogens, inhibit immune-mediated inflammatory disease pathol-ogy, and potentiate antitumor immune responses requires new insights into mechanisms regulating immune cell migration includ-ing entry and exit from tissues and migration within those tissues Increasingly it has become clear that modifying the kinetics of immune cell migration in lymphoid tissues is likely to be a potent method to selectively target immune function with less off-target effects Through the development of novel biologics, linked nucleic acids, small molecules, and adjuvants targeting receptors and sig-naling pathways that regulate immune cell movement it is possible

therapeu-to specifically target mechanisms of immune cell migration The scientific and clinical need to understand cellular migration has led

to the development of new methodologies described in subsequent chapters describing methodologies to image and quantify immune cell migration and stromal networks that support their migration

2 Migration in Immune Cell Development

The development of lymphocytes and immune tissues is a highly dynamic process involving migration between tissues (e.g., bone marrow to thymus) and the active movement and interactions of cells within primary and secondary lymphoid tissues where they undergo their development and maturation Lymphocytes arise from committed progenitors in the bone marrow, and B cells undergo their development and maturation in bone marrow migrating between specialized stromal niches that express IL-7 required for their survival and expansion and specialized galectin-1 expressing stromal niches that select for successful BCR rearrange-

further maturation into mature nạve B cells In contrast T cells undergo development in the thymus, a specialized organ that per-mits selection of restricted T cell repertoire with low affinity to self-MHC-peptide complex This process involves the active migra-tion and highly dynamic interactions of developing thymocytes in key anatomical niches in the thymus where they undergo a series of steps and check points in their development including pre-TCR

selection of CD4+CD8+ cells on cortical epithelium, and the tive selection of single-positive thymocytes by medullary microen-

production of chemokines by stromal and epithelial cells and actions with specialized dendritic cells and macrophages within the

Introduction to Homeostatic Migration

Migration not only dictates the development of immune cells;

it has an essential role in the formation of primary and secondary lymphoid tissues The development of the thymus involves not only infiltration of the developing thymus by lymphocyte progeni-tors but also the active migration of epithelium and neural crest-

and Peyer’s patch anlagen are active processes involving infiltration

by specialized lymphoid tissue initiator and inducer cells; their active migration and interactions with localized mesenchyme initi-ate the process of lymphoid stromal cell maturation into stromal subsets found in adult lymphoid tissues This process is regulated

by the same adhesion molecules (VCAM, ICAM) and chemokines (CXCL13, CCL19/21) that maintain the function of adult lym-phoid tissues [13]

3 Migration in Immune Cell Function: Entry to Exit

Under homeostatic conditions lymphocytes continually late, entering lymph nodes through HEVs and exiting through efferent lymphatics, with a transit time of 10–22 hrs dependent on

through the afferent lymphatics, with antigen either entering through conduit network (<70 kD) or actively transported by sub-capsular macrophages The migration of DCs is a CCR7-dependent process requiring formation of an active chemotactic gradient of CCL21 generated by expression of the atypical chemokine recep-

the entry and migration of T cells in the LN cortex are dependent

on localized expression of CCL19/21 by fibroblastic reticular cells and endothelial cells; in contrast B cells respond to CXCL13 expression by B cell stroma Expression of stromal chemokines leads to the phenomena of chemokinesis, where the migration rate

of lymphocytes is increased when in the presence of chemokine

the efficacy of immune responses as it is the chance encounter of lymphocyte with antigen-expressing DC that drives the efficacy of

T cell responses and capacity of B cells to encounter plement complexes on FDCs

antigen-com-The short transit times of nạve lymphocytes in LNs require active mechanisms controlling their egress, in contrast to nạve cells activated lymphocytes that are retained during the early stages

of an immune response in a CD69-dependent process This egress

of lymphocytes is dependent on sphingosine-1-phosphate (S1P) production, a small signaling lipid that activates S1P receptors expressed by lymphocytes Modulation of S1P1 (S1P receptor 1) expression through the synthetic pro-drug FTY720 that binds to

Mark C Coles

several S1P receptors drives receptor internalization and tion, blocks lymphocyte egress from LN and leads to lymphopenia

chemo-tactic gradient generation, localized S1P internalization and destruction lead to chemotactic gradient formation The expres-sion of the S1P transporter ABCC7 and S1P lyase SGPL1 by MRCs leads to the destruction of S1P in the marginal zone sur-rounding the LN lymphatics providing for the generation of very

4 Imaging Immune Responses: Multiphoton Microscopy

Although immune cells were known to migrate within tissues the dynamics of this process was not fully appreciated until the devel-opment of multiphoton microscopy (MP) This technology revo-lutionized immunology by permitting imaging of immune cells deep within tissues This was possible due to some unique features including deep tissue penetration of near-infrared light, lowered phototoxicity, and generation of second harmonics that permit visualization of the secondary structure of collagen Utilizing both tissue explants and in vivo imaging of lymph nodes the migration and behaviors of lymphocytes have been quantified under homeo-static conditions, antigenic stimulation, inflammation, pathogen infection using fluorescent dyes, and lineage-specific fluorescent protein mice in combination with mice where key pathways are inhibited using either tissue-specific gene knockout mice, inhibi-

approaches, the relative role of GPCR signaling pathways kine receptors, lipid-signaling molecules (EBI2, S1PR)), signaling pathways regulating lymphocyte migration in lymph nodes, and adhesion molecules in cell migration has been extensively studied

insights into the process of T cell migration in the thymus and interactions that drive positive and negative selection in the thymus [20, 21]

5 3-Dimensional Tissue Imaging

Immune homeostasis and antigen driven initiation of adaptive immune responses occur within the 3D structure of lymphoid tissues Although multiphoton imaging is a powerful technique

to study active immune cell migration it is inherently limited in the depth penetration of light into dense tissues like lymph nodes and the capacity to analyze more than four fluorescent signals is

Introduction to Homeostatic Migration

limited due to the design of most commercial microscope tems A number of techniques when combined with optical clear-ing technologies including optical projection tomography and light sheet microscopy have revolutionized the ability to quantify large volumes of tissues in 3D using multiple different antibodies

stromal topology and insights into pathogen-mediated LN

6 Quantifying Biology and the Emergence of Modeling Technologies

Through the quantification of multiphoton images, it is possible to obtain a large amount of quantitative information on immune cell migration and interactions including the velocity, meandering index, turning angles, duration and timing of interactions, and physiological outcomes of these interactions This has provided the data for modeling immune responses; this approach has provided new insights into the data and provided models for how immune

stromal networks Analysis of simultaneous multiphoton imaging

of stromal networks and lymphocytes provided evidence for a

This has been extended through the very elegant work by Burkhard

methodology to quantify 3D stromal networks in LNs using graph

driven experimentation as a methodology to understand

to understand how lymph nodes remodel during LCMV infection

new insights into mechanisms of immune cell migration and geting these processes to develop the next generations of immunotherapeutics

tar-7 Conclusions

The development of 3D imaging when combined with a tive approach is providing new insights into mechanisms regulat-ing immune cell migration The chapters that follow detail methodologies to image mouse lymph nodes and thymus in 3D and quantify the immune responses The application of whole- tissue clearing and imaging in these protocols provides the capacity

quantita-to generate 3D maps of immune cells within lymph nodes, and when combined with powerful mathematical techniques provides a platform to produce new insights into immune tissue function

Mark C Coles

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13 Veiga-Fernendes H, Coles MC, Foster KE,

Patel A, Williams A, Natarajan D, Barlow A,

Pachnis V, Kioussis D (2007) Tyrosine kinase

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E, Eller K, Chan L, Lucas B, Novitzky-Basso I, Nakamura K, Rulcke T, Nibbs RJ, Worbs T, Forster R, Rot A (2014) The atypical chemo- kine receptor CCRL1 shapes functional CCL21 gradients in lymph nodes Nat Immunol 15(7):623–630

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17 Cyster JG (2005) Chemokines, sphingosine- 1- phosphate, and cell migration in secondary lymphoid organs Annu Rev Immunol 23:127–159

18 Park SM, Angel CE, McIntosh JD, Books AES, Middleditch M, Chen CJJ, Ruggiero K, Cebon J, Dunbar PR (2014) Spingosine-1- phosphate lyase is expressed by CD68+ cells

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21 Le Borgne M, Ladi E, Dzhagalov I, Herzmark

P, Liao YF, Chakraborty AK, Robey EA (2009) The impact of negative selection on thymocyte migration in the medullar Nat Immunol 10(8):823–830

22 Richardson DS, Lichtman JW (2015) Clarifying tissue clearing Cell 162(2):246–257

23 Kumar V, Scandella E, Danuser R, Onder L, Nitschke M, Fukui Y, Halin C, Ludewig B, Stein JV (2010) Global lymphoid tissue remod- elling during a viral infection is orchestrated by

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Mark C Coles

George Edward Rainger and Helen M Mcgettrick (eds.), T-Cell Trafficking: Methods and Protocols, Methods in Molecular Biology,

vol 1591, DOI 10.1007/978-1-4939-6931-9_2, © Springer Science+Business Media LLC 2017

Chapter 2

Analysis of Thymocyte Migration, Cellular Interactions,

and Activation by Multiphoton Fluorescence Microscopy

of Live Thymic Slices

Jessica N Lancaster and Lauren I.R Ehrlich

Abstract

Thymocytes migrate through discrete compartments within the thymus, engaging in cellular interactions essential for their differentiation into functional and self-tolerant T cells Thus, understanding the tempo- ral and spatial behavior of thymocytes within an intact thymic microenvironment is critical for elucidating processes governing T cell development Towards this end, we describe methods for preparing thymic explant slices, in which the migration of thymocytes through three-dimensional space can be probed using time-lapse, multiphoton fluorescence microscopy Thymocytes, enriched for developmental subsets of interest, are labeled with cytoplasmic fluorescent dyes, and seeded onto live thymic slices that express an endogenous, stromal cell-specific fluorescent reporter In response to chemotactic cues produced by thymic stromal cells, the labeled thymocytes migrate within thymic microenvironments and engage in cellular interactions that recapitulate a physiological system, which can be readily imaged Here we describe specimen preparation that maintains the integrity of thymic structures We also describe imaging protocols for acquiring multiple fluorochrome channels to enable detection of thymocyte:stromal cell interactions and quantification of relative intracellular calcium levels to monitor T cell receptor activation Parameters for quantifying motility and interaction behaviors during data analysis are also briefly described The thy- mic slice is a versatile tool for probing live cell behaviors and developing novel hypotheses not readily apparent by static experimental methods.

Key words Multiphoton fluorescence microscopy, Thymocyte, Thymus, Migration, Cell–cell

interac-tion, Calcium flux, TCR activation

1 Introduction

Sequential stages of T cell maturation are coordinated with ordered

The diverse stromal cells within the thymus orchestrate the ization and timing of thymocyte movement through different thy-mic regions in order to provide signals for thymocyte survival,

stromal- derived migratory cues through the regulated expression

of chemokine receptors and integrins, and in turn engage stromal

Thus, describing the migratory behavior of thymocytes is essential for understanding T cell development Though the importance of thymocyte migration in distinct thymic microenvironments is well appreciated, the molecular cues that drive thymocyte localization and interactions with stromal cells are not completely understood Insights into these mechanisms are often inaccessible using stan-dard immunological techniques, which either offer snapshots of cellular processes or fail to recapitulate processes that occur only in organized, three-dimensional tissues Mechanisms governing thy-mocyte localization or interactions with stromal cells that promote thymocyte differentiation or selection can be queried by quantify-ing parameters of thymocyte migration as it occurs in situ

Two-photon/multiphoton microscopy enables the tion of fluorescent structures within tissues, due to a nonlinear excitation phenomenon that results in decreased light scattering

micros-copy is a valuable tool for observing fluorescently labeled live cells

as they migrate within intact tissue However, imaging within the thymus poses technical challenges: because the thymus is located next to the heart, intravital imaging is subject to motion artifacts

of intact thymic lobes, where they are often at the detection limit

techniques have been developed to generate live slices of thymus

introduction of both exogenous and genetically encoded cent markers into thymocyte and stromal cells in the system, the migration and behavior of thymocytes can be visualized by live cell

Although thymic slices are tissue explants, thymocytes within slices remain responsive to chemotactic cues released by thymic stroma, localize to the thymic microenvironments appropriate for distinct stages of differentiation [8], and retain motility comparable to thy-

responsiveness and thymocyte migration occur in both human and

thymo-cytes, which are critical checkpoints governing development of functional, self-tolerant T cells, are also supported on thymic slices

application of multiphoton microscopy to study the motility, ization, and stromal interactions of thymocyte subsets

local-Positive and negative selection are driven by activation of T cell receptors (TCRs) on thymocytes, which is induced by ligation of self-peptide:major histocompatibility complex molecules on thy-mic antigen presenting cells (APCs) The concentration of intracellular calcium, a secondary messenger downstream of TCR activation, has been shown to rapidly increase and fall within

Jessica N Lancaster and Lauren I.R Ehrlich

for TCR signaling With the availability of many calcium-sensitive fluorescent indicators, live cell tracking of intracellular calcium has

Cell-permeant dyes, such as Indo1 and Fluo-3/4/5, have altered fluorescence emission properties upon binding intracellular cal-

protein, whose fluorescence depends on calcium-dependent

as Indo-PE3, has been used in imaging thymic slices, due to ease

emission is ratiometric, with intensity at shorter wavelengths increasing at higher calcium concentrations, while intensity at lon-ger wavelengths decreases concomitantly, allowing it to serve as its own internal fluorescence control Previous studies that quantified intracellular calcium in thymocytes undergoing selection demon-strated that thymocytes exhibit reduced motility upon calcium flux [6] and form aggregates with elevated calcium levels [16] Further, the strength and frequency of calcium signaling have been used to

calcium fluxes, induced by relatively low avidity ligands, are

marked by sustained calcium elevation induced by high avidity

has advanced the study of thymocyte activation as it occurs in real time within the thymus

This chapter serves as a detailed protocol of thymic slice eration Here we describe how isolated thymocyte subsets can be labeled and introduced into thymic slices for the purpose of multi-photon imaging Multiphoton imaging of thymic slices can yield novel insights into the molecular and cellular mechanisms that govern real-time behavior of thymocyte migration, thymocyte:stromal cell interactions, and TCR activation

gen-2 Materials

1 Mice: Select strain based on thymocyte subset or molecule of

selection of thymocytes with a defined TCR specificity

5 Dissection instruments: surgical scissors (Roboz RS-5910), fine spring scissors (Roboz RS-5668), angled surgical scissors (Roboz RS-5918), forceps (Roboz 5), angled forceps (Roboz 5/45), curved forceps (Roboz RS-5135)

7 BrightLine hemacytometer

8 Trypan blue

9 Brightfield tissue culture microscope

10 Fluorescent probes: CellTracker dyes (Molecular Probes) are cytoplasmic loading dyes available in a wide range of colors; Indo1AM is a cell-permeant, leak-resistant ratiometric calcium indicator dye

11 Rat anti-mouse antibodies for depletion of hematopoietic eages, e.g., anti-B220 (clone RA3.3A1/6.1), anti-Ter119 (BE0183), anti-Gr1 (RB6-8C5), anti-CD11b (M1/70); or thymocyte subsets, e.g., anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD8 (53.6.72), anti-CD25 (PC-61.5.3)

beads

13 Dyna-Mag 15 magnet

14 DRPMI medium: powdered RPMI 1640 medium deficient in phenol red, sodium bicarbonate, and l-glutamine, supple-mented with 0.2 g/L sodium bicarbonate and 20 mM HEPES

15 DRPMI with 10% bovine calf serum (BCS)

16 Phosphate buffered saline (PBS)

17 PBS with 2% BCS

18 Complete RPMI medium: RPMI 1640 medium, mented with 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/

supple-mL streptomycin, and 10% (v/v) fetal bovine serum (FBS)

1 Mice: 3–4 weeks of age; select strain based on thymic vironment and endogenous fluorescent reporters of interest For example, in RIP-mOVA mice, medullary APCs express

knockin mice express green fluorescent protein (GFP) in AIRE+

2 Rodent anesthesia: Isoflurane (Southmedic, Ontario, CA) and vaporizer chamber (VetEquip)

3 Rodent guillotine (World Precision Instruments, Sarasota, FL, cat no DCAP or similar)

4 60 × 15 mm tissue culture dish

2.2 Generation

of Thymic Slices

Jessica N Lancaster and Lauren I.R Ehrlich

5 BioLite 35 mm tissue culture dish

6 PTFE-coated stainless steel double-edged razor blade

7 5 mL volume plastic specimen cups

8 Superglue

9 Tapered end micro spatula, spoon spatula

10 4% (w/v) low melting point agarose in PBS

11 T-type thermocouple with thin flexible probe

12 VT-1000 vibratome

inserts (EMD Millipore)

2 Silica grease

1 Upright fluorescence microscope with detector array, cence emission filter sets, water immersion objective lens, titanium:sapphire laser for multiphoton excitation, and laser scanning system Our setup employs the PrairieView Ultima

fluores-IV (Bruker, Billerica, MA), 20× NA 0.95 Plan Fluor water immersion objective (Olympus, Tokyo, Japan), photomulti-plier tube (PMT) detectors, 400/50, 480/40, 535/50, and 607/45 bandpass emission filters (Chroma Technology, Bellows Falls, VT), and two MaiTai HP lasers (SpectralPhysics, Santa Clara, CA)

2 Image acquisition software: microscope vendor specific, our setup employs PrairieView (Bruker)

3 Heated stage chamber RC-26GLP (Warner Instruments, Hamden, CT)

4 Inline perfusion heater SH-27B (Warner Instruments)

5 Nylon specimen harp SHD-26GH/2 (Warner Instruments)

6 Perfusion 300 mL IV set and flow regulator (Wolf Medical Supply, Sunrise, FL, cat no RF5600)

7 Bubbling stone and PVC tubing (Warner Instruments)

8 95% oxygen with 5% carbon dioxide

9 Imaging perfusion medium: powdered RPMI 1640 medium deficient in phenol red, sodium bicarbonate, and l-glutamine, supplemented with 2 g/L sodium bicarbonate, and 5 mM HEPES Adjust pH to 7.4, then add 0.85 mM calcium chlo-ride for a final calcium concentration of 1.25 mM

1 Imaging data analysis software: Analysis programs vary in accessibility of the user interface and price For our analysis we employ Fiji/ImageJ (National Institutes of Health, Bethesda, MD), Imaris version 8.2.0 (Bitplane, Concord, MA), and MATLAB version R2015a (Mathworks, Natick, MA)

3 Methods

secondary means such as cervical dislocation

2 Secure the mouse to the dissection board in the supine tion, with its abdomen exposed, by pinning down the append-ages Moisten fur with 10% ethanol to keep the fur clear of the dissection

3 Begin dissection of the mouse by lifting the skin up at the abdomen using curved forceps, and cut the skin up the midline from abdomen to throat using scissors

4 Make a perpendicular cut across the upper abdomen, and pull the skin away from the thin subdermal tissue layer Sliding the scissor blades between the skin and subdermal layer will dis-connect the fascia holding the tissues together

5 Lift the subdermal tissue using curved forceps and cut just below the ribs, taking care not to cut any internal organs, then extend the cut along the base of the ribs

6 Pierce the diaphragm with scissors, taking care not to cut the liver or lungs

7 Cut superiorly through the ribs on both lateral sides, ending near the axilla Using forceps lift up the front of the ribcage, exposing the heart and thymus above it

8 Use the curved forceps to pull the thymus out of the chest ity by pulling at its base

9 Gently rinse the thymus by dipping once a beaker of PBS to wash off residual red blood cells

10 Prepare a single cell suspension of thymocytes by mechanically dissociating thymic tissue through a cell strainer into 5 mL of DRPMI with 10% BCS

11 Count the live cells under a brightfield microscope by placing

a 1:1 mixture of cell suspension and Trypan Blue on a tometer slide Live cells will be round and transparent, while dead cells are stained blue Calculate the concentration of cells per the hemacytometer manufacturer instructions

BCS

2 Depending on which thymocyte subset is needed for the experiment, thymocytes are then incubated with purified rat anti-mouse antibodies specific for cell surface markers present

on lineages to be depleted For example, to enrich for CD4 single positive thymocytes, we incubate thymocytes with anti-

similarly, we enrich for CD8 single positive thymocytes with

3 Incubate the cells in antibodies on ice for 30 min, and then wash twice by resuspending in 5 mL of PBS with 2% BCS, pel-

leting the cells in swinging bucket centrifuge (5 min at 163 × g),

and discarding the supernatant

4 Prepare sheep anti-rat magnetic beads for use at a ratio of 2:1 cells:beads by washing the appropriate number of beads twice

in 1 mL of PBS with 2% BCS, separating the beads from the wash by attaching the tube to the magnetic holder and pipet-ting out the supernatant with a P1000 pipet Resuspend in 1

step k).

1 Distribute 106–107 cells each into 1.5 mL tubes, such that cells from one tube will be seeded onto one slice

2 Prepare a solution of the desired fluorescent dye by vigorously

thymocytes can be imaged in the 607 nm emission channel by

DRPMI prewarmed to 37 °C; calcium imaging can be recorded

in the 400 nm and 480 nm emission channels by staining with

3 Pellet the cells in a fixed angle centrifuge (468 × g), aspirate

supernatant, and resuspend the thymocytes thoroughly in 1.5

mL staining solution

4 Incubate at 37 °C in a water bath

5 After 30 min, pellet the cells using a fixed angle centrifuge

(468 × g), aspirate supernatant, and resuspend in 1.5 mL

com-plete RPMI medium

6 Incubate at 37 °C in a water bath for 30 min to allow excess dye to destain

3.1.3 Label Thymocytes

with a Fluorescent Probe

Multi-photon Microscopy of Thymocyte Migration

7 Pellet the cells using the fixed angle centrifuge, aspirate natant, and resuspend in 1.5 mL fresh complete RPMI medium

8 Keep cells at 37 °C in a water bath until thymic slices are prepared

1 Anesthetize mice using isoflurane in a vaporizer chamber, firm sedation by toe pinch, and euthanize mice with a rodent guillotine

2 Drain blood by blotting with paper towels, in order to obtain clean removal of the thymus

thy-mus in the chest cavity

4 Using angled scissors, excise the thymus with a single stroke at the base, while holding gently with forceps to avoid deforming the tissue

1 Gently pick up the thymus with curved forceps and rinse by dipping once in a beaker of PBS on ice

2 Transfer the thymus into a 60 × 15 mm tissue culture or similar large dish approximately half-full with cold PBS, so that the thymus is submerged

3 Carefully cut away any remaining connective tissue from the

4 Separate the two thymic lobes by first gently outlining the tum between the lobes with the blunt side of curved forceps, and then cutting the lobes apart with a razor blade

1 Microwave the 4% low melting point agarose until liquid, and then pour it into the specimen cups, one per lobe, until the cups are about half-full

2 Use the flexible thermocouple to stir the agarose while toring the temperature

3 Using the spoon spatula, transfer a lobe into the agarose once it’s cooled below 38 °C but before it sets at ~35 °C

4 Working quickly, manipulate the lobe using the tapered end spatula so that it remains upright, with the widest part of the lobe at the bottom of the specimen cup, and the convex and concave faces of the lobe oriented outwards towards the sides

of the cup (Fig 1) (see Note 3).

5 Once the agarose begins to set, transfer the cups to a small ice bath for at least 5 min so that the agarose solidifies completely

4 Prepare the vibratome by adding ice around the sectioning

Transfer and secure the specimen holder into the sectioning chamber according to the manufacturer’s instructions for the vibratome

1 Prepare the vibratome with a fresh razor blade and adjust the blade angle so the blade meets the agarose block at ~45 °C Set the vibratome frequency to 70 Hz, speed to 0.20 mm/s,

2 Section the agarose block containing the thymic lobe ously, collecting slices with the spoon spatula and transferring

3 Keep thymic slices in the dish on ice until ready to overlay labeled thymocytes

Fig 1 Embedding the thymic lobe within low melting point agarose Top view of

specimen cup; thymic lobe is oriented vertically within the agarose so that the broader end is directed towards the bottom of the cup Convex face of the lobe is oriented outwards, as labeled and outlined with dashed boundary

Multi-photon Microscopy of Thymocyte Migration

1 Place cell culture membrane inserts into small dishes with 1

mL prewarmed complete RPMI, enough to wet the membrane yet leave the culture insert at the air-liquid interface

2 Prepare a separate dish with 2 mL of complete RPMI

3 Pick up a thymic slice onto the spoon spatula, and wash by ping once into the complete RPMI dish

4 Place the thymic slice on top of the membrane at the air–liquid interface (Fig 3)

1 Using a P1000 pipet, gently aspirate excess medium from the surface of the thymic slices without contacting the tissue

2 Centrifuge the tubes containing dye-labeled thymocytes (fixed

remains with the cell pellet

3 Resuspend the cells by gently triturating with a P200 pipet, and transfer the thymocytes directly onto the surface of the thymic slice Application of a thin circle of silica grease around the border of the thymic slice can be used to further confine the thymocytes

Transfer the dish containing thymic slices newly overlaid with

the start of imaging

Varies with microscope platform, generally includes the following steps:

1 Warm up laser systems and tune to the excitation wavelength

2 Mount the objective lens

3 Assemble specimen holder onto the microscope stage

4 Initialize data acquisition software

the Imaging System

Fig 2 Orientation of agarose block on specimen holder for vibratome

section-ing (a) Top view of specimen holder; agarose is trimmed with a razor so that

the convex face of the lobe is aligned with the front (b) Complete assembly of

the vibratome setup, with the sectioning blade meeting the front of the rose block

aga-Jessica N Lancaster and Lauren I.R Ehrlich

1 Mount the bottle containing perfusion medium approximately 2–4 ft above the stage, and gravity fed to the stage inlet through the drip set

2 Prime the perfusion line with a 20 cc or other large syringe, and circulate the perfusion medium to the specimen chamber with the regulator flow rate at ~100 mL/h, or ~1 drop/s Adjust the outlet flow so that the medium can cover a thymic slice but does not overflow the chamber

3 Connect the bubbling stone to the 95% oxygen with 5% bon dioxide source, and feed gas into the perfusion medium so that it bubbles vigorously

4 Heat the microscope stage and inline perfusion heater, toring to ensure the medium is at 37 °C

1 Lifting the thymic slice from the petri dish using a tapered end spatula, use a P1000 pipet to gently rinse the top of the slice with warmed RPMI medium, to remove any cells that did not enter the slice

2 Transfer the thymic slice into the warmed perfusion medium

of the imaging specimen holder, so that the side of the slice

on which labeled thymocytes were introduced faces the objective lens

3.4.2 Setup Stage

Perfusion

3.4.3 Secure the Thymic

Slice onto the Stage

Fig 3 Thymic slice preparation for tissue culture Sectioned thymic slices are

placed at the air-medium interface on cell culture membrane inserts Multiple slices can be placed together for labeled thymocyte overlay The thymic slices and seeded thymocytes are incubated at 37 °C, 5% CO2 for at least 30 min before the start of imaging

Multi-photon Microscopy of Thymocyte Migration

3 Using forceps, carefully secure the thymic slice by placing the nylon specimen harp onto the specimen holder without dam-aging the tissue

4 Once in place, lower the objective lens to immerse in the medium

5 If binocular view is available on the microscope, align the objective and roughly focus on the edge of the thymic slice, to assist in fluorescence imaging setup, taking care that the laser source is blocked before looking into the eyepiece

1 In live view, scan the tissue to find imaging regions of interest For example, when determining accumulation and migration

of thymocytes within cortical and medullary thymic regions, the imaging field should ideally encompass a complete medul-lary patch and an intact cortical expanse extending from the cortical medullary junction to the capsule Various focal depths should be explored to find a suitable imaging volume, as med-ullary tissue tends to be less rigid than cortical tissue and thus can have less integrity closer to the tissue surface Alternately,

if observing cell-cell contacts, the field of view should be focused onto the thymocytes and/or stromal cells at suffi-ciently high magnification to resolve such contacts Other imaging parameters to be set include:

2 Excitation intensity—Pockels cell voltage for attenuating

exci-tation laser intensity should be set so that the image is bright, while bearing in mind that high illumination comes at the cost

of photobleaching during extended imaging times

3 Excitation wavelength—For CellTracker dyes, GFP and its

wavelengths are also ideal because they cause less tissue age; however the optimal excitation wavelength must be deter-mined based on the specs of individual lasers with respect to power emitted at each wavelength, and the combination of fluorophores to be excited by a single wavelength in a given experiment In order to image intracellular calcium concentra-tions using Indo1AM, the laser must be tuned to a shorter wavelength For our experiments observing both Indo1AM and GFP, we use simultaneous excitation by two laser sources

dam-at 730 nm and 840 nm, respectively

4 Focal plane interval distance (z-step)—The top of the imaging

volume should be set so that it captures thymocytes migrating within the tissue, rather than unincorporated cells floating on top of the slice For this reason, we set the top imaging plane

≥20 μm below the cut surface of the slice Given an average

smaller step sizes can be used in order to obtain greater lateral resolution, this will increase the number of focal planes to be acquired for a given tissue volume and thus increase laser expo-sure and tissue damage We generally acquire nine layers at 5

μm steps in order to observe a tissue volume depth of 40 μm

difficult due to attenuation of fluorescence through the tissue slice, with image resolution dependent on the efficiency of the fluorophore and the density of structures labeled

5 Frame and/or line averaging—The use of signal averaging will

improve the signal-to-noise ratio (SNR) and provide a clearer image However, the trade-offs include increased laser expo-sure resulting in photobleaching as well as increased computa-tional load during data acquisition

6 PMT voltage and preamp—Increased PMT voltage can be

employed in order to minimize the amount of laser intensity that must be applied in order to obtain a bright image signal; however high gain will increase noise For improved SNR, pre-amp gain and offset can be adjusted in order to cover the whole

7 Pixel dwell time—Similar to signal averaging, increased dwell

times will result in increased signal and improve image quality, with the caveat that long dwell times will increase laser expo-sure, inducing photobleaching and tissue damage

8 Time interval duration—Duration between time points must

be selected so that cellular movements are adequately sampled, while not oversampling and thus unnecessarily exposing the sample to laser illumination For thymocyte migration within the thymic slice, we generally select intervals of 15–20 s, for total imaging durations of 15–30 min

The data acquisition file type should be considered since this affects their readability by the analysis program Proprietary file types may require file conversion programs, though many dedicated image analysis programs can now read most file types and associated metadata employed by microscope vendors

Motility parameters enable quantitative comparisons among imental conditions Calculation of many parameters is automatic in the data analysis program Imaris Bitplane once thymocytes have been rendered as Spot or Surface objects Otherwise, these param-eters can be calculated manually by plotting thymocyte positions as

exper-a function of time Useful metrics for compexper-aring motility under different experimental conditions can include:

1 Displacement—the absolute distance traveled between given

time points; plotting displacement as a function of the square

3.5 Image Analysis

3.5.1 Import the Image

Data Files to the Analysis

root of time can reveal whether the cell tends to travel in a random walk (slope ~ 1) or in a confined path, in which the plotted curve quickly reaches a plateau

2 Path straightness—displacement divided by the length of the

path traveled by the cell Path straightness reveals whether the cell travels in a straight line (straightness of 1) or in a more tortuous manner (decreasing straightness value approaching 0)

3 Velocity—path length divided by the time duration sampled

Mean cell velocities can be impacted by such factors as cellular interactions, chemokine gradients, and activation state

In order to quantify cellular interactions, the shortest distance between the surfaces of each thymocyte and stromal cell must be computed for every time point; for this purpose we have utilized the ImarisXT package of Imaris:

1 Mask the fluorescence channel marking the stromal cells (most often, stationary cells), using Surfaces

2 Convert the datatype to 32-bit floating

3 With the stromal cell Surfaces object active, generate a new channel using Imaris XT Distance Transformation Specify dis-tances outside the object In the newly created channel, the intensity denotes distance away from the mask

4 Once rendered as an Imaris object, each thymocyte will now have channel intensities which represent its distance to the nearest stromal cell at any given time point

5 In order to call a time point of interaction for the thymocyte, determine a threshold distance; based on visual inspection of

threshold for a thymocyte:stromal cell interaction

6 The duration of individual contact events was measured based

on consecutive interaction time frames Thus parameters such

as frequency of contact, mean contact duration (dwell time), and percent of time contacting stromal cells can be quantified

1 We employ the ratiometric calcium indicator Indo1AM in order to visualize changes in intracellular calcium concentra-tions as a proxy for TCR signaling during thymocyte selection

sig-nal, the total fluorescence intensity of the cell volume from the

400 nm emission channel is divided by that of the 480 nm emission channel

2 By plotting individual cell calcium ratios, periods of elevated or baseline intracellular calcium can be described; TCR activation

3.5.3 Quantification

of Thymocyte: Stromal Cell

Interactions

3.5.4 Calcium Imaging

as a Proxy for Activation

Jessica N Lancaster and Lauren I.R Ehrlich

events can be determined based on relative fluxes in the cium signal, followed by signal decay The fluorescence ratio values that indicate peak flux and elevated intracellular calcium signals are specific to the experimental conditions, and can be determined by calibration experiments

3 To calibrate, an ionophore, such as ionomycin, or the cognate antigen of the TCR transgenic thymocytes is introduced into the perfusion medium to induce TCR activation The addition

of the activating reagent will result in rapid calcium flux within all the thymocytes in the sample

4 Since the time of reagent addition and thus TCR activation is known, we can obtain relative fluorescence ratio values for resting (before addition of reagent) and activated states

5 After any calibration the perfusion line must be flushed and the specimen holder cleaned completely, as any residual iono-phore/antigen will contaminate subsequent imaging

4 Notes

1 Any excess fluorescent dye will interfere with imaging by ing fluorescent debris on the thymic slice It is essential that the dye is thoroughly mixed into the medium during preparation, which we accomplish by vigorous vortexing of the dye after adding to media During the wash steps after cell staining, it is also critical that any unbound dye is removed Towards this end, it is helpful to centrifuge tubes in a fixed angle centrifuge rotor and aspirate the non-cell debris, which distributes along the wall of the tube, away from the cell pellet in the tube

2 Removing all connective tissue from the thymus is essential to generating high quality thymic slices for imaging Large sec-tions can be cut away with scissors, while smaller pieces can

be carefully peeled off using angled forceps Complete removal

of connective tissue is facilitated by dissection under a stereomicroscope

3 Along with using a completely clean thymic lobe, the manner

in which the lobe is set into the agarose is critical to obtaining good thymic slices Upon introducing the lobe into the cool-ing agarose, the boundaries of the lobe must completely con-tact the agarose, without any air pockets, to ensure that the subsequent slicing step cuts through the lobe rather than crushing the tissue Before the agarose has set, a spatula can be used to agitate the agarose, directing it towards the lobe

4 Several difficulties may arise during sectioning that can result

in inadequate thymic slices If connective tissue remaining on the thymic slice attaches to the blade, the blade can pull the

Multi-photon Microscopy of Thymocyte Migration

tissue out of the block If there is a gap between the lobe and the agarose, the blade can crush the tissue For these issues, slices may be salvaged by pausing the blade and trimming any uncut tissue away with fine spring scissors before resuming It

is also advised to prepare extra tissue blocks to ensure sufficient thymic slices can be obtained for imaging In addition, large thymuses, for example from mice 6 weeks of age, may prove difficult to slice, as their large cross-sectional area may collapse after sectioning We have found it easiest to section from mice

3 to 4 weeks of age However, ages can be adjusted per mental needs While keeping the cut slices on ice will maintain tissue integrity, tissue structures look best shortly after section-ing; therefore sectioning should be timed to minimize the time the slices sit before imaging

5 Practically speaking, there should be a balanced number of

the bit-depth of the image) intensity Thus, the greatest range and variation of intervening graytones can be acquired in the image, providing improved image resolution Adjust PMT preamp gain and offset while observing the live image with a pixel intensity lookup table (LUT), with the goal of having sparse zero intensity pixels and very few regions of saturated pixel intensity

6 Drawbacks to Indo1AM include a blue-shifted excitation peak wavelength compared to most fluorophores, scattering of emission fluorescence resulting in noisy signals from single cells, and changes in calcium sensitivity over time as the dye is compartmentalized, making its use practical only within the first 6 h after loading In addition, the capacity to observe Indo1AM and GFP simultaneously has necessitated the use of two excitation lasers, which increases exposure of the tissue to phototoxicity However, Indo1AM remains a good tool for calcium detection due to its ease of loading and brightness

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Multi-photon Microscopy of Thymocyte Migration

George Edward Rainger and Helen M Mcgettrick (eds.), T-Cell Trafficking: Methods and Protocols, Methods in Molecular Biology,

vol 1591, DOI 10.1007/978-1-4939-6931-9_3, © Springer Science+Business Media LLC 2017

Chapter 3

Visualizing and Tracking T Cell Motility In Vivo

Robert A Benson, James M Brewer, and Paul Garside

Abstract

Advanced cellular tracking and imaging techniques allow the dynamic nature of immune responses to be studied in detail and in a physiological context Here we describe two methods applying multiphoton laser scanning microscopy to the visualization and tracking of fluorescently labeled CD4 + T cells and dendritic cells (DCs) within the complex lymph node (LN) environment Ex vivo imaging of LNs allows the study

of cell populations without the need for skilled surgical techniques while providing comparable data While more technically demanding, intravital imaging of the popliteal LN allows aspects of T cell/DC responses

to be studied in the context of an intact lymph and blood supply We also describe methods to aid the acquisition of time series data suitable for cellular tracking, providing a quantitative approach to real-time analysis of DC and T cell LN responses.

Key words T cell, Dendritic cell, Adoptive transfer, In vivo, Intravital imaging, Multiphoton,

Migration

1 Introduction

The application of multiphoton laser scanning microscopy (MPLSM) to the study of dynamic immune cell responses allows the visualization and quantification of behaviors underpinning both successful and aberrant immune responses Of note is the application of MPLSM to visualize cellular responses in secondary lymphoid tissues Such imaging studies have provided insight into

5–9], and subsequent impact on clonal expansion [10], tion [11, 12], and exit [13, 14] to peripheral tissues to execute effector functions

differentia-The use of MPLSM offers several advantages over more ventional single-photon imaging approaches Firstly, fluorophore excitation is achieved using long wavelength, femtosecond-pulsed laser light that has low average power Secondly, excitation usually requires infrared light, reducing light scattering and absorption by

the tissues, facilitating deeper imaging The inherently low energy

exci-tation requires near simultaneous absorption of two or more tons of light by the fluorophore, out of focus light is eliminated as excitation only occurs at the focal point of the lens where photon density is highest Thus there is no wasted illumination out of the plane of focus and consequently no requirement for a pinhole as in confocal laser scanning systems These attributes make MPLSM extremely applicable to studying dynamic cellular responses in the environmentally complex and three-dimensional context of intact tissues

pho-Here we present two methods allowing visualization of T cell behavior and interaction with antigen presenting cells Imaging of explanted tissues provides a robust, reproducible, and accessible method for imaging dynamic cellular behavior in secondary lym-

to produce results comparable to those achieved using intravital

training However intravital imaging allows additional factors such

under more physiological settings where lymph and blood vessels remain intact

2 Materials

1 C57BL/6 mice or appropriate fluorescent reporter strain, e.g.,

institute specific approval for the relevant animal work

10% fetal calf serum (FCS)

3 Conditioned culture supernatant from X63 myeloma cells transfected with mouse GM-CSF cDNA or recombinant GM- CSF [21]

4 Hemocytometer and trypan blue

protein (Worthington Biochemical, Lakewood, NJ, USA)

6 LPS (Escherichia coli 055:B5, Sigma-Aldrich).

1 Cell tracker dye (ThermoFisher Scientific, Loughborough, UK)

(a) Preparation of stock CellTracker™ red CMPTX: Warm product to room temperature and dissolve in high quality

with Exogenous Dye

and Adoptive Transfer

Robert A Benson et al.

(b) Preparation of stock CellTracker™ green CMFDA: Warm product to room temperature and dissolve in high quality DMSO to a final concentration of 10 mM Resuspend 500

μg of lyophilized CMFDA (mwt 464.9) in 90 μL of DMSO This gives a 10 mM stock Vortex to dissolve

2 Phosphate buffered saline (PBS)

5 RPMI-1640

6 RMPI-1640 plus 10% FCS

7 Hemocytometer and trypan blue

8 Warmed air box preheated to 37 °C

9 1 mL syringe and 27 G needle

1 Appropriate country/institute approval to carry out the vant animal work

2 Appropriate reporter mice T cell receptor (TCR) transgenic

3 PBS

4 Mouse dissection board and dissecting tools

5 CD4 T cell isolation kit, e.g., from Miltenyi Biotec or Stemcell Technologies

6 Buffer to prepare single cell suspension of TCR transgenic cells, e.g., HBSS or PBS containing 2% FCS

7 Hemocytometer and trypan blue

8 Warmed air box preheated to 37 °C

9 1 mL syringe and 27 G needle to inject the TCR Tg cells intravenously

3 Peristaltic pump (Harvard apparatus)

4 Inline heater (Harvard Apparatus)

5 Homeothermic monitor (Harvard Apparatus)

6 Oxygen concentrator (Vet Tech Solutions, Cheshire, UK)

7 Vacuum unit (Dymax 5, Charles Austin) and conical flask

8 Plastic tubing (Harvard Apparatus)

9 Veterinary grade superglue, e.g., Vetbond tissue adhesive (3M Direct)

10 CS-12R coverslips (Harvard Apparatus)

11 High vacuum silicone grease

12 Dissection tools

Fig 1 Tissue bath for imaging of excised lymphoid tissues Heat from two power

film resistors is distributed via a metal base plate throughout the tissue bath A perspex chamber is placed on top of the base plate and sealed using silicone vacuum grease Two metal side plates positioned over the lip of the perspex component hold the chamber in position, tightened by metal screws Perfusion input and exhaust ports are positioned at opposite ends of the chamber with a plastic baffle inserted to counter flow effects from the input port A strong direct flow can contribute to tissue movement artifacts during imaging Temperature probes can be inserted directly into the chamber and/or positioned in a channel

in the side of the metal plate to feedback temperature for auto-adjust of inline heater and power film resistors via the homeothermic monitor Lymph nodes adhered to a small plastic coverslip can be positioned in the center of the chamber

Robert A Benson et al.

2 Prewarmed PBS (37 °C)

3 Heat mat and homeothermic monitor (Harvard Apparatus)

4 Inhalation anesthesia (Isoflurane, mask, scavenger, oxygen concentrator/cylinder) (Vet Tech Solutions)

5 Ocular lubricant (eye cream)

6 Depilatory cream, e.g., Nair, Veet, etc Such creams have been proven effective and nontoxic for use on animals under

7 Zink oxide waterproof tape, e.g., Leukoplast

2.5 Imaging

of Lymph Node In Situ

Fig 2 Imaging chamber for in situ visualization of popliteal lymph nodes Using

a hand drill, cut a 100 mm plastic petri dish lid into a crescent shape Attach this

to the lip edge of a 100 mm glass petri dish using epoxy resin The glass petri dish will act as a submersion chamber for the hind legs of the mouse while the plastic lid acts to support the upper torso Attach two domed microcentrifuge caps to the center of the glass dish These will act as fixing points for the leg and

skin flaps For additional detailed images of the chamber please see Liou et al

[23] The nose cone for provision of inhaled anesthesia can be mounted to the plastic upper portion of the rig using adhesive tape A temperature regulated heating mat can be sited below the chamber to aid warming Temperature feed-back can be provided by either placing a probe in the bath below the animal or where available use a rectal probe to allow monitoring of core body temperature

In vivo Imaging of T Cells