Cells On Patrol Understanding, quantifying, and Lymphocyte - PowerPoint PPT Presentation

Cells On Patrol Johannes Textor Cells On Patrol Understanding, quantifying, and Lymphocyte migration Intravital imaging modeling lymphocyte migration Motion in numbers Modeling migration Course Immunobiology, May 8th, 2015 Johannes Textor Theoretical Biology & Bioinformatics Universiteit Utrecht 1

Cells On Patrol Learning Objectives Johannes Textor Lymphocyte migration Intravital imaging 1 Understand why and how lymphocytes migrate Motion in numbers 2 Learn about modern intravital imaging Modeling migration 3 Be able to quantify cell migration 4 Appreciate how modeling can contribute to understanding lymphocyte migration 2

Cells On Patrol Lecture Outline Johannes Textor 1 Lymphocyte migration Lymphocyte migration Intravital imaging Motion in numbers 2 Intravital imaging Modeling migration 3 Motion in numbers 4 Modeling migration 3

Cells On Patrol Why cell migration? Johannes Textor Example: T cells in mice vs a previously unseen antigen cells 30,000,000,000 20g 100% lymphocytes 300,000,000 0.2g 1% T cells 150,000,000 0.1g 0.5% Lymphocyte migration 10 − 7 g ag-specific T cells 20-200 0.0000005% Intravital imaging lymph nodes 35 Motion in numbers Modeling migration ∼ 2-3 ag-specific naive T cells per lymph node! T cells migrate because the immune system does not (cannot!) provide complete protection everywhere in the body. Instead, T cells patrol the body by migrating constantly. Il était une fois – la vie Migration is necessary to provide protection everywhere. 4

Cells On Patrol T cell migration takes place on two scales Johannes Textor migration between organs migration within tissue Lymphocyte migration Intravital imaging Motion in numbers Modeling migration • rhythm: hours • rhythm: minutes • studied in the 1960s-70 • studied since 2002 B cells migrate similarly, but more slowly (2-4fold). In this lecture, we’ll focus on T cells to illustrate the basic principle. 5

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, secondary lymhpoid organs (SLOs), and lymph. Lymphocyte migration Intravital imaging Motion in numbers Modeling migration 6

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, secondary lymhpoid organs (SLOs), and lymph. Lymphocyte migration Intravital imaging 1 Circulation in the blood Motion in numbers Modeling migration 6

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, secondary lymhpoid organs (SLOs), and lymph. Lymphocyte migration Intravital imaging 1 Circulation in the blood Motion in numbers Modeling migration 2 Recruitment into SLO 6

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, secondary lymhpoid organs (SLOs), and lymph. Lymphocyte migration Intravital imaging 1 Circulation in the blood Motion in numbers Modeling migration 2 Recruitment into SLO 3 Search for antigen in SLO 6

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, secondary lymhpoid organs (SLOs), and lymph. Lymphocyte migration Intravital imaging 1 Circulation in the blood Motion in numbers Modeling migration 2 Recruitment into SLO 3 Search for antigen in SLO 4 Exit to lymphatic system 6

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, secondary lymhpoid organs (SLOs), and lymph. Lymphocyte migration Intravital imaging 1 Circulation in the blood Motion in numbers Modeling migration 2 Recruitment into SLO 3 Search for antigen in SLO 4 Exit to lymphatic system 5 Re-entry into blood Thoracic duct drains into left subclavian vein 6

Cells On Patrol Circulation routes of Naive T Cells Johannes Textor To find their antigen, naive T Cells circulate between blood, Lymphocyte migration secondary lymhpoid organs Intravital imaging (SLOs), and lymph. Motion in numbers Modeling migration One round takes ≈ 6-30h, depending on the SLO and the cell type (CD4/CD8, Naive/Memory) 6

Cells On Patrol Classic measurements of migration dynamics Johannes Textor Experimental protocol: • Inject labeled naive T cells into subclavian vein Lymphocyte migration • Measure reappearance in Intravital imaging thoracic duct Motion in numbers Modeling migration Westermann, 1994 7

Cells On Patrol How fast do T cells arrive at an infection site? Johannes Textor We can use a simulation to investigate arrival speed at Influenza infection priming in vivo infection sites. cells recruited into response in vivo (%) cells arriving at dLNs in silico (%) 80 9 dLNs Lymphocyte migration 60 6 dLNs Intravital imaging Motion in numbers 40 Modeling migration s N L d 2 20 1 dLN 0 0 1 2 3 4 5 6 7 time p.i. (days) • Depends on number of draining lymph nodes (dLNs) • With few dLNs, these get more blood and swell 8

Cells On Patrol In lymph nodes, T cells meet dendritic cells that present antigen Johannes Textor Normal tissue Inflamed tissue HEV Central� Naive T cell memory T cell Constitutive� Dendritic cell homing Afferent� lymph vessel Lymphocyte migration Lymphoid� organ Intravital imaging Tissue-� Inflammation-� Motion in numbers specific� induced� homing recruitment Modeling migration Postcapillary venule Inflamed venule Antigen Central� memory T cell� (long-lived) � Clonal expansion Activated� T cells� � Effector� Effector T cell� memory T cell� Differentiation (short-lived) (long-lived) � 9

Cells On Patrol Basic structure of lymph nodes Johannes Textor Cortex Paracortex Medulla > 80 % B cells > 80 % T cells Macrophages germinal centers T/DC interactions efferent lymph Lymphocyte migration Intravital imaging Motion in numbers Modeling migration 10

Cells On Patrol Migration of naive T cells through lymph nodes Johannes Textor (1) entry T cells are recruited by surface molecules in high endothelial venules (HEV) in Lymphocyte migration the paracortex. Intravital imaging Motion in numbers Modeling migration Vascular System 2 1 HEV 3 Efferent Lymph Cortex Paracortex Medulla Lymphatic System 11

Cells On Patrol Migration of naive T cells through lymph nodes Johannes Textor (2) search T cells search for antigen in the paracortex. Most of them don’t enter the cortex (B Lymphocyte migration zone) unless activated. Intravital imaging Motion in numbers Modeling migration Vascular System 2 1 HEV 3 Efferent Lymph Cortex Paracortex Medulla Lymphatic System 11

Cells On Patrol Migration of naive T cells through lymph nodes Johannes Textor (3) exit T cells reach the medulla and exit via efferent lymphatic vessels to the lymphatic Lymphocyte migration system. Intravital imaging Motion in numbers Modeling migration Vascular System 2 1 HEV 3 Efferent Lymph Cortex Paracortex Medulla Lymphatic System 11

Cells On Patrol Summary on migration Johannes Textor • T and B cells migrate because it would be too expensive to maintain a sizable number of cells of every specificity in Lymphocyte migration every location. Intravital imaging • T and B cells recirculate between blood, secondary Motion in numbers Modeling migration lymphatic organs (SLOs) and lymph ≈ once per day. • Lymph nodes and other SLOs provide hubs for migrating cells to come together and exchange information. Fun fact to remember A lymph node consists > 90 % of constantly moving cells! 12

Cells On Patrol Intravital imaging – a breakthrough for cell migration research Johannes Textor • Traditional approaches like thoracic duct draining only allowed to indirectly observe cell motion in vivo . Lymphocyte migration • This changed with the advent of two-photon microscopy. Intravital imaging • First immunological applications of two-photon imaging in Motion in numbers 2002 – three back-to-back papers in Science . Modeling migration Terminology intravital “into the living” – any experiment where we observe something within a living being two-photon microscopy – one technique that allows penetrating the skin for intravital imaging 13

Cells On Patrol Fluorescence microscopy Johannes Textor How do we make cells visible in dense tissues? Lymphocyte migration excited state Intravital imaging Motion in numbers Modeling migration Emit low energy photon ground state Excitation of fluorophores Green fluorescent protein 14

Cells On Patrol Two-photon vs confocal microscopy Johannes Textor Basic idea Instead of a single, strong beam shooting through the sample, we have two, weak beams that meet in a single point. Lymphocyte migration Intravital imaging Motion in numbers Infrared laser Scanning mirror (Mode-locked) Modeling migration Dichroic Beam Splitter Green PMT Green Filter Red Filter Red PMT Objective PMT=photomultiplier tube Focal Plane Flurophores 15

Cells On Patrol From stacks to movies Johannes Textor One stack of z -planes is acquired every ∼ 15-30 sec. Lymphocyte migration Intravital imaging Motion in numbers Modeling migration Due to this process, depth resolution ( z ) is much poorer than planar resolution ( x , y ) . 16