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Lymphocyte Migration ©Dr. Colin R.A. Hewitt

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 Naïve + Activated Homing to lymph node + ++ + ++ + ++ Adhesion molecules + + ++ Differential splicing of CD45 mRNA in naïve & armed T cells Homing to inflamed vascular endothelium

Patterns of lymphocyte trafficking Naïve T cells

Thymus Bone Marrow Naïve T cell Lymph node HEV

High endothelial venules

Post capillary venules in 2 º 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

SELECTINS

Leucocytes inc. Naive T cells: Endothelial cells:

L SELECTIN 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 Lewis X 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 Selectin is shed is activated by INTEGRIN chemokines (adhesion molecule) has low affinity for ICAM Cell activation changes integrin to high affinity format

1-3 seconds

Activation

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

Arrest

INTEGRIN

 L  2 (LFA-1)

Ig FAMILY LIGAND

ICAM-1  Integrin  Activation of lymphocyte increases affinity of integrin

(Mn 2+ 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  -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

Firm adhesion causes the leukocyte to flatten and migrate between the endothelial cells Leukocyte migrates towards site of infection by detecting and following a gradient of chemokine.

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

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 Extracellular matrix provides traction for moving cells Chemotactic gradient Simultaneous occupancy of large numbers of surface receptors the cell will stay still.

Differential receptor occupancy between the trailing and leading edges of the cells.

Operates at low levels of receptor expression

HEV HEV

Recirculation

Non-lymphoid cells Pass through the blood vessels in the lymph node and continue arterio venous circulation 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

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

t = seconds Weibel-Palade bodies with pre-formed adhesion molecules Injury or irritation generates thrombin histamine, Leukotrienes etc Adhesion molecule expression t = hours TNF & IL-1 released due to inflammation in tissue I k B phosphorylated & degraded.

NF k B translocates to nucleus Adhesion molecule expression

Memory and naïve T cells

Associates with TcR and CD4 - phosphatase activity reduces threshold of T cell signalling Naïve Activated + + ++ + ++ + ++ + + ++ 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

Activated effector memory cell with L selectin shed from surface  L  2 (LFA-1)  4  1(VLA-4) Peripheral vascular endothelium INFLAMMATION ICAM-1 VCAM-1 Activated vascular endothelium TNF 

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: Homing:

Binding of cells to other cells or extracellular matrix 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

3 H-labelled lymphocytes from mesenteric lymph nodes 3 H-labelled lymphocytes from skin Remove tissues, section and autoradiograph A Section through A

Discovery of the T cell gut-homing mechanism

Murine Lymphoma TK-1 Lymph node HEV Peyer’s patch HEV Inhibition of binding using a panel of monoclonal antibodies identified the lymphocyte molecule that mediated binding to Peyer’s Patch HEV: the integrin  4  7.

A similar approach was used to identify the endothelial ligand of  4  7: 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.

Gut pathogen e.g. rotavirus Anti-rotavirus T cells will never be needed in the skin Gut Anti-rotavirus T cells activated Response resolves, lymphocytes non randomly redistributed Anti-rotavirus T cells will be needed in the gut

Quantitative aspects of lymphocyte migration

Traffic between lymphoid/non-lymphoid tissues involves~ 5 x 10 11 cells per day Only ~2% (1 x 10 10 ) 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