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

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Presentation transcript:

Sphingosine-1-phosphate/sphingosine-1-phosphate receptor 1 signaling is required for migration of naive human T cells from the thymus to the periphery Rachel S. Resop, PhD, Marc Douaisi, PhD, Joshua Craft, BS, Loes C.M. Jachimowski, PhD, Bianca Blom, PhD, Christel H. Uittenbogaart, MD Journal of Allergy and Clinical Immunology Volume 138, Issue 2, Pages 551-557.e8 (August 2016) DOI: 10.1016/j.jaci.2015.12.1339 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Mature human thymocytes migrate to S1P. A, Percentage of immature CD3−CD4−CD8− (double-negative) and CD4+CD8+ (double-positive) and mature CD3hiCD27+ 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 CD3hiCD27+ 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 CD3hiCD27+ thymocytes. For statistical data, please see Table E3. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 S1PR1 and KLF2 mRNA are expressed to the greatest extent within the most mature CD3hiCD69− thymocyte subset. Thymocytes were sorted into 4 populations based on maturation immunophenotype (ie, CD3−CD69−, CD3loCD69+, CD3hiCD69+, and CD3hiCD69−) before real-time PCR. S1PR1, S1PR2, and KLF2 mRNA are shown. For statistical data, please see Table E3. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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+CD3hiCD69− 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. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Expression of S1P-R1 in mature CD3hiCD4+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. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Gating schematic for CD3/CD69 thymocyte populations and profile of S1P-R1+ human thymocytes. Human postnatal thymocytes (age, 0 day to 44 years) were stained with antibodies to CD3, CD27, CD69, and CD45RA and gated as shown on CD27/CD45RA populations, followed by CD3/CD69 populations. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Expression of S1PR3, S1PR4, and S1PR5 mRNA in CD3/CD69 thymocyte populations. Cell sorting was performed as described above to isolate CD3/CD69 populations. One-step RT-quantitative PCR was performed on mRNA isolated from these 4 populations to examine mRNA levels of S1PR3, S1PR4, and S1PR5. ns, Not significant. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Phenotype of S1P-R1+ and total thymocytes. Expression of CD3, CD27, CD69, CD4, CD8, CD62L, and CD45RA on S1P-R1+ thymocytes (5 weeks old; A) compared with the total thymocyte population (B). Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Thymic B and natural killer (NK) cells express S1P-R1. Human postnatal thymocytes were stained for expression of B and NK cell markers to ascertain whether these cells express S1P-R1. A, Expression of CD3, CD27, CD69, CD4, CD8, and CD45RA in total thymocytes (compared with S1P-R1+ thymocytes in Fig 3). B, Gating schematic for thymic B and NK cells. C, Expression of S1P-R1 in thymic B and NK cells relative to the IgG2b isotype control. Journal of Allergy and Clinical Immunology 2016 138, 551-557.e8DOI: (10.1016/j.jaci.2015.12.1339) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions