Lymphocyte Migration ©Dr. Colin R.A. Hewitt crah1@le.ac.uk

Differences between armed, effector T cells and naïve T cells - Naïve CD4 cells Associates with TcR and CD4 - phosphatase activity reduces threshold of T cell signalling CD45RA CD45RO Differential splicing of CD45 mRNA in naïve & armed T cells CD2 LFA-1 CD44 Adhesion molecules Homing to lymph node L-selectin VLA-4 Homing to inflamed vascular endothelium Naïve Activated + + + + + - - - ++ ++ ++ - + ++

Patterns of lymphocyte trafficking Naïve T cells Thymus Lymph node HEV Naïve T cell Bone Marrow

High endothelial venules Post capillary venules in 2º lymphoid tissue HIGH ENDOTHELIAL VENULES. Specialised to allow lymphocytes and nothing else into the lymph node HEV Post capillary venules in other tissues are lined by simple squamous epithelium

Role of endothelial cells in trafficking and recirculation Endothelial are involved in: Vasomotor tone, vascular permeability, regulation of coagulation, immune modulation and lymphocyte extravasation High endothelial venules Constitutively present in secondary lymphoid tissue Need to allow egress of naïve cells from the circulation Post-capillary venules Present in non-lymphoid tissues Molecules expressed by endothelial cells regulate trafficking and recirculation through lymphoid and non-lymphoid tissues

The multi-step paradigm of leukocyte migration: Step 1: Tethering & rolling Cytokine activated endothelial cells express adhesion molecules Leukcocytes ‘marginate’ from the peripheral pool to the marginal pool Cells normally roll past resting endothelial cells Tethering 4000 microns/sec Rolling 40 microns/sec Tethering and rolling are mediated by SELECTINS and ADDRESSINS

Selectins & addressins Leucocytes inc. Naive T cells: L SELECTIN Endothelial cells: P SELECTIN & E SELECTIN P selectin: Weibel-Palade bodies. E selectin: TNF & IL-1 induced A common core with different extracellular C type lectin domains that bind carbohydrates in a Ca2+ dependent manner. Each selectin binds to specific carbohydrates and is able to transduces signals into the cell VASCULAR ADDRESSINS On high endothelial venules in lymphoid tissue: Carbohydrates that “decorate” CD34 and GlyCAM-1 Sialyl LewisX molecules Peripheral Node addressins (PNAd) Mucosal endothelium: MAdCAM-1 Guides lymphocyte entry into lymphoid tissues

Steps 2 & 3: Activation & arrest Cytokines from epithelium activate expression of Intracellular adhesion molecules (ICAMs) Rolling Neutrophil is activated by chemokines Selectin is shed Cell activation changes integrin to high affinity format INTEGRIN (adhesion molecule) has low affinity for ICAM

Activation 1-3 seconds G-protein-linked seven transmembrane spanning receptors For granulocyte activation: Chemokines Platelet activating factor C5a In T cells: ??

Activation Inhibit G protein with pertussis toxin Occupancy of large numbers of surface receptors Rolling phenotype only - no stable adhesion Ligand of lymphocyte toxin-sensitive receptor not yet identified

Steps 2 & 3: Activation & arrest Cytokines from epithelium activate expression of Intracellular adhesion molecules (ICAMs) Rolling Neutrophil is activated by chemokines Selectin is shed Cell activation changes integrin to high affinity format INTEGRIN (adhesion molecule) has low affinity for ICAM

Arrest       Integrin Activation of lymphocyte INTEGRIN Ig FAMILY LIGAND aLb2 (LFA-1) ICAM-1       Integrin Activation of lymphocyte increases affinity of integrin (Mn2+ in vitro) Ig family ligand Integrins Two chain molecules - that bind to Ig superfamily molecules and extracellular matrix components

“Inside out” signalling   Activation of lymphocyte     Remove cytoplasmic tail of integrin a-chain Activation of lymphocyte   Activation of the extracellular high affinity integrin binding site is dependent upon activation of the lymphocyte, & the cytoplasmic domain of the integrin i.e. signals from “inside” the cell have an effect “outside”

“Outside in” signalling Ligation of lymphocyte integrin by ligand     Activation of lymphocyte High affinity interaction of integrins with their ligands may alter the behaviour of the cell i.e. signals from “outside” the cell have an effect “inside”

Step 4: Migration and diapedesis Leukocyte migrates towards site of infection by detecting and following a gradient of chemokine. Step 4: Migration and diapedesis Firm adhesion causes the leukocyte to flatten and migrate between the endothelial cells Leukocytes migrate readily to the chemokine RANTES made by epithelilal cells that have encountered microorganisms Arrest is reversible if diapeisis does not occur ~10 Minutes

Diapedesis Metalloproteases digest the basement membrane PECAM expressed at intercellular junctions of endothelial cells and on the lymphocyte Metalloproteases digest the basement membrane

Migration Signals similar to those important in step 2 are involved i.e. chemokines Simultaneous occupancy of large numbers of surface receptors - the cell will stay still. Extracellular matrix provides traction for moving cells Chemotactic gradient Differential receptor occupancy between the trailing and leading edges of the cells. Operates at low levels of receptor expression

Recirculation Non-lymphoid cells Naïve lymphoid cells Pass through the blood vessels in the lymph node and continue arterio-venous circulation HEV Naïve lymphoid cells Adhere to and squeeze between High Endothelial Venules (HEV), then percolate through the lymph node and exit via the efferent lymphatic vessel HEV

Inflammation Normal oesophagus Normal palatine tonsils Normal skin Candida infection Streptococcal infection Staphylococcal infection

Role of endothelial cells in trafficking and recirculation High endothelial venules Constitutively present in secondary lymphoid tissue Need to allow egress of naïve cells from the circulation Post-capillary venules Present in non-lymphoid tissues Injury and inflammation alters morphology to resemble HEV Need to allow egress of memory cells to sites of infection

Inflammation or injury induces changes in endothelial cells Injury or irritation generates thrombin histamine, Leukotrienes etc t = seconds Weibel-Palade bodies with pre-formed adhesion molecules Adhesion molecule expression IkB phosphorylated & degraded. NF-kB translocates to nucleus t = hours Adhesion molecule expression TNF & IL-1 released due to inflammation in tissue

Memory and naïve T cells Activated + - L-selectin VLA-4 CD45RA CD45RO CD2 LFA-1 CD44 ++ Associates with TcR and CD4 - phosphatase activity reduces threshold of T cell signalling Naïve cells need to access lymphoid tissue to become stimulated Memory cells need to access sites of inflammation

Integrins facilitate the access of leukocytes to sites of inflammation Peripheral vascular endothelium Activated effector memory cell with L selectin shed from surface aLb2 (LFA-1) a4b1(VLA-4) ICAM-1 VCAM-1 TNF-a Activated vascular endothelium INFLAMMATION

Trafficking, homing and adhesion Trafficking: Non-random movement of cells from tissues, blood or lymph. Includes migration to and from sites of lymphocyte maturation as well as homing. Adhesion: Binding of cells to other cells or extracellular matrix Homing: Tendency of lymphocytes activated in a particular region of the body to preferentially return to the same region Includes localisation of cells in distinct regions of lymphoid tissue.

Evidence that lymphocytes exhibit specialised trafficking patterns 3H-labelled lymphocytes from mesenteric lymph nodes Remove tissues, section and autoradiograph A Section through A 3H-labelled lymphocytes from skin

Discovery of the T cell gut-homing mechanism Murine Lymphoma TK-1 Lymph node HEV Peyer’s patch Inhibition of binding using a panel of monoclonal antibodies identified the lymphocyte molecule that mediated binding to Peyer’s Patch HEV: the integrin a4b7. A similar approach was used to identify the endothelial ligand of a4b7: the mucosal addressin: MAdCAM-1

Skin-homing T cells Cutaneous T cell lymphomas Extensive infiltration of epidermis with T cells Cells home to the skin and express the cutaneous lymphocyte associated antigen (CLA) Apply contact sensitiser Induce delayed-type hypersensitivity Sample T cells by raising a suction blister Cells in the suction blister express CLA - the skin homing receptor E-selectin is the ligand of CLA

Why is lymphocyte homing necessary? Tendency of lymphocytes activated in a particular region of the body to preferentially return to the same region. Anti-rotavirus T cells will never be needed in the skin Gut pathogen e.g. rotavirus Gut Anti-rotavirus T cells will be needed in the gut Anti-rotavirus T cells activated Response resolves, lymphocytes non-randomly redistributed

Quantitative aspects of lymphocyte migration Traffic between lymphoid/non-lymphoid tissues involves~ 5 x 1011 cells per day Only ~2% (1 x 1010) of these cells are in the blood at any one time Lymphocytes only stay in the blood for ~30 minutes Circulating blood pool of lymphocytes is exchanged 48 times a day However…… Less than 10% of blood lymphocytes migrate into lymph nodes, tonsils & Peyer’s patches. ~90% of lymphocytes leave the blood to enter organs such as the liver, lung spleen and bone marrow. Traffic is 5 times faster than traffic through lymphoid tissue

Summary Naïve cells entering Peripheral Lymph Nodes L-selectin PNAd (CD34, Gly-CAM) aLb2 (LFA-1) ICAM-1 Contact - Rolling - Arrest - Diapedesis T cells Endothelial cells Naïve cells entering Peripheral Lymph Nodes L-selectin MAdCAM carbohydrate a4b7 MAdCAM-1 aLb2 (LFA-1) ICAM-1 Contact - Rolling - Arrest - Diapedesis T cells Endothelial cells Naïve cells entering Peyer’s Patches

Memory cells entering Inflamed tissue a4b1 (VLA-4) VCAM-1 aLb2 (LFA-1) ICAM-1 T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis Memory cells entering Inflamed tissue a4b7 MAdCAM-1 aLb2 (LFA-1) ICAM-1 T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis Memory cells homing to Peyer’s Patches CLA E-selectin a4b1 (VLA-4) VCAM-1 aLb2 (LFA-1) ICAM-1 T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis Memory cells homing to Skin