Sphingosine-1-phosphate/sphingosine-1-phosphate receptor 1 signaling is required for migration of naive human T cells from the thymus to the periphery

Abstract

Background: The mechanisms that govern the egress of mature thymocytes from the human thymus to the periphery remain understudied yet are of utmost importance to the field of basic immunology, as well as T-cell reconstitution in various immunodeficiencies. We examined the expression and function of sphingosine-1-phosphate (S1P) receptors in human thymocyte egress.

Objectives: We aimed to determine whether S1P receptors (S1P-Rs) play a role in mature human thymocyte egress and to identify the thymocyte population or populations that express S1P-Rs and respond to S1P by migrating across a concentration gradient.

Methods: Human thymocytes were exposed to S1P in Transwell plate migration assays coupled to flow cytometry to evaluate the response to S1P of thymocytes at different stages of maturation. Constitutive S1P-R expression was quantified by means of real-time PCR in sorted thymocyte subsets and flow cytometry. S1P-R1 and Kruppel-like factor 2 expression were monitored after S1P exposure by using flow cytometry and quantitative PCR.

Results: S1P-R1 was the prevalent S1P receptor on mature human thymocytes (CD3(hi)CD27(+)CD69(-)), the population that also demonstrated the greatest response to S1P in migration assays. Pretreatment with FTY720, an S1P-R1 nonselective modulator significantly reduced migration and suggested a role for S1P-R2 in retaining thymocytes in the tissue. Lastly, surface S1P-R1 expression, as well S1PR1 and Kruppel-like factor 2 (KLF2) transcripts, were significantly decreased in mature thymocytes on exposure to S1P.

Conclusion: Mature human thymocytes rely on S1P-R1 to migrate toward S1P. Taken in the context of murine work demonstrating that S1P is required for thymocyte egress to the periphery, our data highlight a new key chemokine for human thymocyte egress.

Keywords: S1P-R1; S1P-R2; Sphingosine-1-phosphate; T-cell reconstitution; human immunology; migration; thymic egress; thymus.

FIG 1

Mature human thymocytes migrate to S1P. A, Percentage of immature CD3⁻CD4⁻CD8⁻ (double-negative) and CD4⁺CD8⁺ (double-positive) and mature CD3^hiCD27⁺ thymocyte subsets expressing CD4 or CD8 that migrate to 100 nmol/L S1P in a Transwell migration assay (total thymocytes in the upper chamber). B, Migration of mature CD3^hiCD27⁺ thymocyte populations identified by using antibodies to CD69 and CD62L. C, Effect of FTY720, an S1P-R1, S1P-R3, S1P-R4, and S1P-R5 modulator, on migration of mature CD3^hiCD27⁺ thymocytes. For statistical data, please see Table E3.

FIG 2

S1PR1 and KLF2 mRNA are expressed to the greatest extent within the most mature CD3^hiCD69⁻ thymocyte subset. Thymocytes were sorted into 4 populations based on maturation immunophenotype (ie, CD3⁻CD69⁻, CD3^loCD69⁺, CD3^hiCD69⁺, and CD3^hiCD69⁻) before real-time PCR. S1PR1, S1PR2, and KLF2 mRNA are shown. For statistical data, please see Table E3.

FIG 3

S1P-R1 protein is expressed to the greatest extent on the most mature thymocyte subset. Human postnatal thymocytes were stained with surface antibodies (see Table E1). A, S1P-R1 profile of 1 representative postnatal (2-year-old) thymus gated as described above. B, Summary of S1P-R1 expression on cells from 14 postnatal thymi (median age, 9 months) showing comparison of S1P-R1 expression on CD27⁺CD3^hiCD69⁻ thymocytes to less mature populations. C, Expression of S1P-R1 on fetal thymocyte populations (median age, 16.5 weeks of gestation). For statistical data, please see Table E3.

FIG 4

Expression of S1P-R1 in mature CD3^hiCD4⁺CD69⁻ thymocytes after stimulation with S1P. Postnatal thymocytes were exposed to 100 nmol/L S1P in serum-free medium for 30 minutes or untreated and immediately stained for S1P-R1 and other surface markers. A, Change in surface expression of S1P-R1 between thymocytes exposed to S1P (black) versus medium (mock, light gray). B, Fold change in S1P-R1 expression after 30 minutes of S1P exposure of postnatal (PN) and fetal (FT) thymocytes. For statistical data, please see Table E3.

FIG 5

S1PR1 and KLF2 mRNA expression decrease after exposure of fetal thymocytes to S1P. Thymocytes from human fetal thymus/liver implants in immunodeficient mice were exposed to 100 nmol/L S1P or medium alone for 30 minutes and subsequently analyzed for S1PR1 and KLF2 mRNA expression. Fold change of S1PR1, KLF2, and IL7R mRNA after S1P exposure is depicted. For statistical data, please see Table E3.