1. Lymphocyte Migration © Dr. Colin R.A. Hewitt [email_address]

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

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

4. 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

5. Role of endothelial cells in trafficking and recirculation Endothelial are involved in: Vasomotor tone, vascular permeability, regulation of coagulation, immune modulation and lymphocyte extravasation Molecules expressed by endothelial cells regulate trafficking and recirculation through lymphoid and non-lymphoid tissues 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

6. 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 Tethering and rolling are mediated by SELECTINS and ADDRESSINS Cells normally roll past resting endothelial cells Tethering 4000 microns/sec Rolling 40 microns/sec

7. SELECTINS 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 Selectins & addressins 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

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

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

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

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

12. Arrest Integrins Two chain molecules - that bind to Ig superfamily molecules and extracellular matrix components INTEGRIN Ig FAMILY LIGAND  L  2 (LFA-1) ICAM-1     Integrin   Ig family ligand Activation of lymphocyte increases affinity of integrin (Mn 2+ in vitro)

13. “ Inside out” signalling 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”     Activation of lymphocyte   Remove cytoplasmic tail of integrin  -chain Activation of lymphocyte  

14. “ Outside in” signalling 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” Ligation of lymphocyte integrin by ligand     Activation of lymphocyte

15. Step 4: Migration and diapedesis Firm adhesion causes the leukocyte to flatten and migrate between the endothelial cells ~10 Minutes 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

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

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

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

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

20. 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

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

22. Memory and naïve T cells Naïve 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

23. Integrins facilitate the access of leukocytes to sites of inflammation ICAM-1 VCAM-1 TNF-  Activated vascular endothelium INFLAMMATION Peripheral vascular endothelium Activated effector memory cell with L selectin shed from surface  L  2 (LFA-1)  4  1(VLA-4)

24. 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.

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

26. Discovery of the T cell gut-homing mechanism 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 Murine Lymphoma TK-1 Lymph node HEV Peyer’s patch HEV

27. 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) Cells in the suction blister express CLA - the skin homing receptor E-selectin is the ligand of CLA Apply contact sensitiser Induce delayed-type hypersensitivity Sample T cells by raising a suction blister

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

29. 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

30. Summary L-selectin PNAd (CD34, Gly-CAM)  L  2 (LFA-1) ICAM-1 Contact - Rolling - Arrest - Diapedesis T cells Endothelial cells Naïve cells entering Peripheral Lymph Nodes L-selectin MAdCAM carbohydrate  4  7 MAdCAM-1  L  2 (LFA-1) ICAM-1 Contact - Rolling - Arrest - Diapedesis T cells Endothelial cells Naïve cells entering Peyer’s Patches

31.  4  7 MAdCAM-1  L  2 (LFA-1) ICAM-1 T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis Memory cells homing to Peyer’s Patches CLA E-selectin  4  1 (VLA-4) VCAM-1  L  2 (LFA-1) ICAM-1 T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis Memory cells homing to Skin  4  1 (VLA-4) VCAM-1  L  2 (LFA-1) ICAM-1 T cells Endothelial cells Contact - Rolling - Arrest - Diapedesis Memory cells entering Inflamed tissue