Neural Crest–Derived Pericytes Promote Egress of Mature Thymocytes at the Corticomedullary Junction

Marcus A. Zachariah and Jason G. Cyster

JC@ZIB, 14.02.2011Bahram Kasmapour

Research Article

Background

?

Spleen, lymph nodes, peyer’s patches, …

• Lymphocyte circulation: – constant circulation & survey of secondary lymphoid

organs– Entering lymphoid organs: selectins, chemokine receptors

and integrins– Leaving lymphoid organs:

• Myriocin discovered in screening for imm. Supressor drugs FTY720

• FTY720 is similar to lipid Sphingosine phosphorylation Potent agonist for lysophospholipid S1P receptor(s) : S1P1

• S1P1: a G-protein coupled receptor

• “Signaling sphingolipid”• Can block egress

Background• Egress based on S1P concentration difference: blood vs. lymph (6-fold Δ)• S1P not produced only by erythrocytes in plasma… • Lymph source of S1P lymphatic endothelial cells (LEC)?• Mature lymphocytes express S1P1: through Kruppel-like factor (KLP2)

• Egress route? blood or lymph? – Transmigration through corticomedullary junction? Thymic lymphatics?

• Model of egress:

Schwab et al., Nature Immunology, 2007

S1P1 is sufficient to mediate egress (of immature T-cells)

It responds to S1P as chemotaxis signal

A&D: transgenic lines carrying S1P1 transgeneDP: double positive (CD4+/CD8+) TH or TC

SP: single positive (CD4) TH

High S1P1 expression level

S1P1 is sufficient to mediate egress (of immature T-cells)

DP ↑ presence in spleen↑ in blood & spleen       ↓ in thymus

S1P1 is sufficient to mediate egress (of immature T-cells)

• Premature egress?• Intercrossed A line with RAG-GFP reporter mice• RAG-1+ (GFP+) during maturation, decay over few days time

• S1P1 only KLF2 target?• Bred A line with KLF2f/f-CD4Cre mice (conditional KO)• Selective deletion of KLF2 in DP stage

↑ recent Thymic immigrants (immature)  present in periphery

No difference (transgene)

Mature SP T-cells

S1P1 is the essential KLF2 target gene needed for egress

Perivascular accumulation of S1P1 transgenic T cells

S1P1 dependent perivascular accumulation, disrupted by FTY720 More S1P and/or higher sensitivity of A line

Laminin & CK5+: epithelial & endothelial basement membraneERTR7+/PDGFRβ+: pericytes (special blood vessel ensheathing support cells)

Bone marrow

Intravascular labeling reveals emigrating thymocytes

• Intravenous injection with α-CD4-PE thymus isolated shortly thereafter

Small but reproducible thymic DP population detected (not from blood)

CD-PE+ in blood vessels of cortex and medulla

Intravascular labeling reveals emigrating thymocytes

FTY720 blocks egress (?)

Recent migration, not blood circulating thymocytes

DC69

CD69 regulates timing of egress (could help with full selection and maturation)

Thymocytes emigrate by blood vessels at the corticomedullary junction (CMJ)

~70 thymocytes per section and 2500 per thymus  estimated 1% of total 

or 1E6 per day egress to blood

In vivo labled CD4 SP in thymus, CD31 for vascular endotheliumCD8 for cortical regions

Thymocyte imaged during egress to blood

Thymocyte within 50µm of CMJ

Neural crest-derived pericytes promote thymocyte egress

Pericytes: diverse population of cells closely ensheating blood vesselsThymic prevascular cells neural crest origin* encountered before endothelium T-cell acumulation... Pericytes producing S1P?

N.crest ΔSphk mice ΔSphk

S1P has (also) a pericyte origin S1P1 up-regulation

Less DC4+ in blood (?) Less recent migrants in PLN

Neural crest-derived pericytes promote thymocyte egress

Accumulation of mature SP ThymocytesS1P needed for CD69 down-regulation 

during maturation

Normal pericyte distribution

Lymphatic S1P is not required for thymic egress

No increase in mature SP thymocytes  in thymus, no major role for lymphatic v.

Thy LEC ΔSphk

GP38+CD31+ : lymphatic endothelial cells (LEC)GP38-CD31+: blood vessel endothelial cells (BEC)PDGFRβ+CD31-: pericytes

Abalation (Cre-Rosa26eYFP) of Sph kinsae activity in lymphatic endothelium does not inhibit thymic egress

dige

sted

thym

us

Not much LEC in thymus. In LN all LEC are ΔSphk

Take home messages…

• Egress of thymocytes is Sphingosine-1-P dependent• S1P receptor, S1P1 is expressed on cell surface• S1P1 is only target for KLF2 needed for mature SP egress• FTY720 a potent agonist for Lysophospid receptors is derived

from Myriocin (fungal )• S1P for egress is provided by the cooperation of pericytes/b.v.

endothelial and plasma• Egress takes place at corticomedullary junction b. vessels • No major role for Lymphatic vessles in egress• Blocking egress could be used for “safer” immunosuppression

for organ transplantation

Thank you for listening!