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 Highly professional antigen presenting cells  DC reside in the peripheral tissues  Upon exposure to antigen they migrate to the lymphoid organs via the lymphatics  In the lymphoid tissue (e.g. lymph nodes) they activate naïve T cells

 This is an active process  Involves chemokines  adhesion molecules  Inflammatory molecules  The dendritic cell must undergo morphological changes in order to pass through the endothelial junctions  Non-muscle myosin II is known to propel DCs forward and pass through narrow gaps.

Initially identified as axonal guidance cues that determine the direction and migration of neurons. Their discovery was in regards to axon guidance in the limb buds of grasshoppers in 1992 subsequently found to be involved in many other systems. such as heart development Vascular growth Tumour progression Immune responses Bone development

 In the nervous system semaphorins regulate cell motility and morphology through interactions with receptors of the plexin family  The plexin family includes plexin-A1  This is the main receptor for class III and class IV semaphorins  In the immune system plexin-A1 is expressed specifically in dendritic cells  Plexin-A1 deficient mice have severe defects in antigen-specific T cell responses

 Plexin-A1 has a crucial role in DC trafficking  In particular, entry into the lymphatics  Sem3A produced in the lymphatics is a ligand for plexin-A1  Sema3A acts on the rear side of DCs where plexin-A1 is also localised  Sem3A induced phosphorylation of the myosin light chain resulting in the ability of DCs to squash through narrow gaps.

 CFSE labelled activated OT-II T cells were introduced into WT and Plxna1 -/- mice  These OT-II T cells specifically recognise and respond to ova peptide  Then ova peptide administered in freunds adjuvant subcutaneously into the footpads. T cells were isolated from the draining LNs They found that antigen specific T cell proliferative response was much lower in Plxna1-/- mice then in WT.

 Ova-pulsed DCs from WT and Plxna1-/- mice labelled with CMTMR (orange) were injected into footpads and analysed for their ability to move to the popliteal lymph nodes. OT-II CD4 T cells were labelled green and injected IV.  Yes!!  In WT mice DC traffic into the T cell area of LNs within 24hrs  In Plxna1-/- mice only a few DCs made it into the T cell area of the LN  Thus Plxna1 is required for the migratory ability of DCs.

 CFSE labelled DCs injected into footpads of WT and Plxna1-/- mice and their arrival in draining LNs was determined (1C)  Applied FITC epicutaneously to the shoulder skin of WT and Plxna1-/- mice and looked for FITC+ DCs in draining Brachial LNs (1D).  Migrating DCs in 1C calculated as follows:  % input cells=total cells x (%CFSE + cells/input cells).  ID shows that actual number of endogenous DCs isolated from the brachial LN 24 and 48hrs following epicutaneous administartion of FITC-isomer to the shoulder skin

 U ptake of FITC-dextran at 37C for 30 mins was the same in WT and KO mice  Plexin-A1 not required for antigen uptake by DCs  Cells were cultured with FITC-dextran at 37C for 30 minutes. Cells were analysed for presence of FITC+ cells

 Analysis of chemotaxis of WT and KO DCs towards a gradient of CCL19,CCL21 or CXCL12 in transwell system was unaltered in the KO mice  As was their ability to sense a directional gradient : see movie 1

CFSE labelled wildtype or Plexin-A1-/- DCs (green) were injected intradermally into the ears of oxazalone-treated mice. lymphatic endothelial cells were stained with anti-LYVE-1 (red) DCs from Plxna-1-/- mice were retained along the endothelium lining the lymphatics located in the dermis DCs from wildtype mice were not retained

Does Plexin-1A affect the initial contact between DC and endothelial cell? using time-lapse imaging DCs were investigated for their ability to move across a lymphatic endothelial cell monolayer Yellow dotted lines indicate junctions of endothelial cells. White arrows indicate DCs in contact with lymphatic endothelial cells Red arrows indicate the transmigration process of wildtype DCs Show movie 2

And further confirmation…… CFSE labelled wildtype or Plxna-1-/- DCs (green) were added to monolayers of endothelial lymphatic cells which had been stained with F-actin marker phalloidin (red) Migration of DCs monitored by z-stack imaging (confocal microscopy) wildtype DCs observed from the top to the bottom of the stack In contrast Plxna1-/- DCs were able to make contact with the endothelial cells but could not trnasmigrate across them Show movie 3

The role of the ligand for Plxna-1  Plexin-A1 is a receptor for semaphorin 3A and semaphorins 6C and 6D.  All are expressed in lymphatic endothelial cells  Plexin-A1 binds to Sem3A in association with NRP-1  Sem3A is soluble  Sem6C and Sem6D are both transmembrane molecules

Which is the most important of the three semaphorins for transmigration  Wildtype DCs were transferred into Sem3A -/-, Sem6C -/- and Sem 6D -/- mice.  Presence of DCs in draining lymph nodes estimated DCs from Sem3A -/- mice only, showed impaired migration

 Sem3A has been identified as a chemorepellent factor that guides the direction of neurons.  This data shows that it promotes trafficking.  the authors hypothesise that cell polarity generated by chemokines during DC migration may be influenced by Sem3A  They test this using chemotaxis assays

 Sem3A was added to the upper or lower chamber of a Transwell system in the presence or absence of chemokines  In the absence of chemokines Sem3A did not show an effect on DC migration  In the presence of chemokines Sem3A enhanced DC migration when added to the upper chamber only  In this set-up Sem3A is acting on the rear of the cells

 Authors used confocal time-lapse video microscopy  Plexin-A1transfected DCs (green) were treated with LPS and suspended in type 1 collagen.  Gels were then placed in a Zigmond chamber with a chemokine gradient applied to them  DC locomotion was examined at 1min intervals  Plexin-A1 found to be localised in the trailing edge of migrating DCs  Play Movie 5

 Myosin II is known to be involved in DC trafficking  It is phosphorylated by MLC and the Rho kinase : ROCK  Localisation of these molecules resembles that of Plexin-A1  Myosin II is believed to be required for squeezing of the cell body and induction of actinomycin contraction when cells pass through narrow gaps or constricted areas.

 Confocal Z stack imaging was performed on DCs on fibronectin-coated coverslips treated with human IgG or recombinant Sem3A fusion protein  Stained with antibody to phosphorylated MLC (pMLC)  Phosphorylation of MLC enhanced in the presence of Sem3A  This affect not observed in Plxn-A1 -/- mice thus Sem3a promotes MLC phosphorylation via its interaction Plexin-A1  Phosphorylation of the myosin light chain promotes actinomycin contraction.

 Semaphorins are immunomodulatory molecules  Here the authors demonstrate that Semaphorin mediated signals are crucial for DC trafficking  In particular in their entrance into the lymphatics  Sem3A promotes actinomycin contraction at the trailing edge of migrating DCs so that they may pass through narrow gaps.