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Marcus A. Zachariah and  Jason G. Cyster JC@ZIB, 14.02.2011 Bahram Kasmapour

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:

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Marcus A. Zachariah and  Jason G. Cyster JC@ZIB, 14.02.2011 Bahram Kasmapour

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  1. 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

  2. Background • 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 Spleen, lymph nodes, peyer’s patches, … ?

  3. 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

  4. 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 high High S1P1 expression level It responds to S1P as chemotaxis signal

  5. S1P1 is sufficient to mediate egress (of immature T-cells) ↑ in blood & spleen ↓ in thymus DP ↑ presence in spleen

  6. 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 No difference (transgene) S1P1 is the essential KLF2 target gene needed for egress

  7. 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

  8. 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

  9. 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)

  10. 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

  11. N.crest ΔSphk mice ΔSphk 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? S1P has (also) a pericyte origin S1P1 up-regulation Less DC4+ in blood (?) Less recent migrants in PLN

  12. Neural crest-derived pericytes promote thymocyte egress Accumulation of mature SP Thymocytes S1P needed for CD69 down-regulation during maturation Normal pericyte distribution

  13. Lymphatic S1P is not required for thymic egress 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 Thy LEC ΔSphk digested thymus No increase in mature SP thymocytes in thymus, no major role for lymphatic v. Not much LEC in thymus. In LN all LEC are ΔSphk

  14. 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!

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