Neural Crest–Derived Pericytes Promote Egress of Mature Thymocytes at the Corticomedullary Junction Marcus A. Zachariah and Jason G. Cyster Bahram 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) : S1P 1 S1P 1 : 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 S1P 1 : 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 transgene DP: double positive (CD4+/CD8+)  T H or T C SP: single positive (CD4) T H high 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 KLF2 f/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 membrane ERTR7+/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 endothelium CD8 for cortical regions Thymocyte imaged during egress to blood Thymocyte with in 50µm of CMJ

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 ΔSphkmice ΔSphk S1P has (also) a pericyte originS1P1 up-regulation Less DC4+ in blood (?)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 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 digested thymus 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!