Download S1P1 is sufficient to mediate egress (of immature T

Neural Crest–Derived Pericytes
Promote Egress of Mature Thymocytes at
the Corticomedullary Junction
Marcus A. Zachariah and
Jason G. Cyster
JC@ZIB, 14.02.2011
Bahram Kasmapour
Research Article
Background
• Lymphocyte circulation:
– constant circulation & survey of secondary lymphoid
organs
– Entering lymphoid organs: selectins, chemokine receptors
and integrins
Spleen, lymph nodes,
– Leaving lymphoid organs:
peyer’s patches, …
• 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)
A&D: transgenic lines carrying S1P1 transgene
DP: double positive (CD4+/CD8+)  TH or TC
SP: single positive (CD4) TH
High S1P1 expression level
It responds to S1P as chemotaxis signal
S1P1 is sufficient to mediate egress (of immature T-cells)
↑ in blood & spleen
DP ↑ presence in 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
Mature SP T-cells
↑ recent Thymic immigrants (immature)
present in periphery
S1P1 is the essential KLF2 target gene needed for egress
No difference
(transgene)
Perivascular accumulation of S1P1 transgenic T cells
Laminin & CK5+: epithelial & endothelial basement membrane
ERTR7+/PDGFRβ+: pericytes (special blood vessel ensheathing support cells)
Bone marrow
S1P1 dependent perivascular accumulation, disrupted by FTY720
 More S1P and/or higher sensitivity of A line
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 (?)
DC69
Recent migration, not blood circulating thymocytes
CD69 regulates timing of
egress (could help with full
selection and maturation)
Thymocytes emigrate by blood vessels at
the corticomedullary junction (CMJ)
In vivo labled CD4 SP in thymus, CD31 for vascular endothelium
CD8 for cortical regions
Thymocyte with
in 50µm of CMJ
~70 thymocytes per section and 2500
per thymus  estimated 1% of total
or 1E6 per day egress to blood
Thymocyte imaged during egress to blood
Neural crest-derived pericytes promote thymocyte egress
Pericytes: diverse population of cells closely ensheating blood vessels
Thymic prevascular cells  neural crest origin*  encountered before endothelium
T-cell acumulation... Pericytes producing S1P?
N.crest ΔSphk
S1P has (also) a pericyte origin
Less DC4+ in blood (?)
mice ΔSphk
S1P1 up-regulation
Less recent migrants in PLN
Neural crest-derived pericytes promote thymocyte egress
Accumulation of mature SP Thymocytes
S1P needed for CD69 down-regulation
during maturation
Normal pericyte distribution
Lymphatic S1P is not required for thymic egress
digested thymus
Abalation (Cre-Rosa26eYFP) of Sph kinsae activity in lymphatic endothelium does not inhibit thymic egress
GP38+CD31+ : lymphatic endothelial cells (LEC)
GP38-CD31+: blood vessel endothelial cells (BEC)
PDGFRβ+CD31-: pericytes
Not much LEC in thymus. In LN all LEC are ΔSphk
Thy LEC ΔSphk
No increase in mature SP
thymocytes in thymus, no
major role for lymphatic v.
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!