1. Lymphocyte Migration
©Dr. Colin R.A. Hewitt

2. 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 + + + + + - - - ++ ++ ++ - + ++

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

4. 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

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
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

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

7. 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

8. 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

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

10. 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

11. 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

12. 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

13. “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”

14. “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”

15. 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

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

17. 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

18. 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

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
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

22. 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

23. 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

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

26. 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

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)
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

28. 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

29. 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

30. 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

31. 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