Human Naive and Memory T Cells Display Opposite Migratory Responses to Sphingosine-1 Phosphate

This information is current as Annabelle Drouillard, Antoinette Neyra, Anne-Laure of September 29, 2021. Mathieu, Antoine Marçais, Mélanie Wencker, Jacqueline Marvel, Alexandre Belot and Thierry Walzer J Immunol published online 13 December 2017 http://www.jimmunol.org/content/early/2017/12/13/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 13, 2017, doi:10.4049/jimmunol.1701278 The Journal of Immunology

Human Naive and Memory T Cells Display Opposite Migratory Responses to Sphingosine-1 Phosphate

Annabelle Drouillard,*,†,‡,x,{ Antoinette Neyra,*,†,‡,x,{ Anne-Laure Mathieu,*,†,‡,x,{ Antoine Marc¸ais,*,†,‡,x,{ Me´lanie Wencker,*,†,‡,x,{ Jacqueline Marvel,*,†,‡,x,{ Alexandre Belot,*,†,‡,x,{,‖ and Thierry Walzer*,†,‡,x,{

The role of sphingosine-1 phosphate (S1P) in leukocyte trafﬁcking has been well deciphered in mice but remains largely unad- dressed in humans. In this study, we assessed the ex vivo response to S1P of primary human T cell subsets. We found that tonsil but not blood leukocytes were responsive to S1P gradients, suggesting that T cell responsiveness is regulated during their recirculation in vivo. Tonsil naive T cells were readily chemoattracted by S1P in an FTY720-sensitive, S1PR1-dependent manner. Surprisingly, S1P had the opposite effect on effector memory T cells, resident memory T cells, and recently activated T cells, inhibiting their spontaneous or chemokine-induced migration. This inhibition was also more pronounced for CD4 T cells than for CD8 T cell Downloaded from subsets, and was dependent on S1PR2, as shown using the S1PR2 antagonist JTE-013. S1PR1 was progressively downregulated during T cell differentiation whereas S1PR2 expression remained stable. Our results suggest that the ratio between S1PR1 and S1PR2 governs the migratory behavior of T cell subsets. They also challenge previous models of the role of S1P in lymphocyte recirculation and suggest that S1P promotes retention of memory T cell subsets in secondary lymphoid organs, via S1PR2. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/

emory T cell subsets have complementary functions (2). The recently described resident memory T cells (TRM) do not and distinct tissue distributions. Central memory recirculate but are instead resident in both nonlymphoid and lym- M T cells (TCM) defined as CCR7+CD45RA2 in humans phoid organs (3). They are defined as CD69+CD103+/2 in all spe- have superior proliferative capacity and recirculate through sec- cies, irrespective of the expression of other markers, even though ondary lymphoid organs (SLO) via blood and lymph. Effector this definition is known to be imperfect (4). TRM localization, memory T cells (TEM), defined as CCR72CD45RA2CD45RO+ in at sites of pathogen entry, is thought to be essential for recall humans, have important immediate effector functions and recircu- responses. late through the blood and spleen (1). In humans, a high frequency Sphingosine-1 phosphate (S1P) binds to five different G-protein by guest on September 29, 2021 of T cells found in nonreactive lymph nodes (LNs) is of TEM coupled receptors (S1PR1-5). Extracellular S1P is carried in the phenotype, even in infants, suggesting that TEM may recirculate via body by albumin and other lipoproteins. S1P concentration is nonlymphoid tissues to the afferent lymph, as previously proposed maintained at high levels in the blood and lymph and at low levels within tissues (5). S1PR1, S1PR3, and S1PR5 are coupled to Gai and provide attractive cues to lymphocytes. A series of studies † *International Center for Infectiology Research, 69000 Lyon, France; INSERM, found that S1PR1 allows the egress of T and B cells from SLO to U1111, 69000 Lyon, France; ‡E´ cole Normale Supe´rieure de Lyon, 69000 Lyon, France; xUniversite´ Lyon 1, 69000 Lyon, France; {CNRS, UMR5308, 69000 Lyon, the blood and lymph (5). S1PR4 is believed to regulate T cell France; and ‖Service de Ne´phrologie Rhumatologie Dermatologie Pe´diatriques, proliferation and cytokine secretion but not cell migration (6). Hospices Civils de Lyon, Universite´ Claude-Bernard Lyon 1, 69000 Lyon, France S1PR2 is the only S1P receptor coupled to G12/G13 and signals ORCIDs: 0000-0001-7188-7655 (A.D.); 0000-0001-6241-459X (J.M.); 0000-0002- via Rho instead of Rac-like Gai-coupled receptors (7). This 0857-8179 (T.W.). property may explain how S1PR2 mediates chemorepulsion and Received for publication September 5, 2017. Accepted for publication November 7, 2017. antagonizes S1PR1 signaling in mouse osteoclast precursors (8) or germinal center B cells (9), a process important for their proper The laboratory of T.W. was supported by the Agence Nationale de la Recherche (ANR JC SPHINKS to T.W. and ANR JC BaNK to A.M.), the ARC Foundation localization. S1PR2 is also known to be insensitive to the effect of (E´ quipe Labelliseé), and the European Research Council (ERC-Stg 281025) and FTY720, a supra agonist for all other S1P receptors that induces receives institutional grants from INSERM, CNRS, Universite´ Claude Bernard Lyon 1, and E´ cole Normale Supe´rieure de Lyon. their internalization (10, 11). S1P responsiveness is regulated during T cell activation, as A.D., A.N., A.-L.M., A.M., and M.W. performed experiments and analyzed data. J.M., A.B., and M.W. provided intellectual input and reagents. T.W. conceived the shown in mouse studies. Recently activated T cells (act-T cells) ideas and interpreted data; T.W. wrote the paper and edited it with the assistance of upregulate CD69 expression. CD69 binds surface S1PR1 and in- the other authors. duces its internalization, trapping act-T cells in inflamed LNs (12) Address correspondence and reprint requests to Dr. Thierry Walzer, Centre Interna- or in nonlymphoid tissues such as the skin (13). Upon TCR tional de Recherche en Infectiologie, INSERM U1111 – CNRS UMR5308, Univer- site´ Lyon 1, ENS de Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France. stimulation, S1PR1 is also downregulated transcriptionally, which E-mail address: [email protected] may contribute to T cell sequestration in LN. This downregulation The online version of this article contains supplemental material. is, however, transient as S1PR1 levels increase during differenti- Abbreviations used in this article: act-T, activated T; LN, lymph node; SLO, second- ation into effector and memory T cells, which is believed to permit ary lymphoid organ; S1P, sphingosine-1 phosphate; TCM, central memory T cell; egress from SLO by restoring responsiveness to S1P (14, 15). S1P TEM, effector memory T cell; TRM, resident memory T cell. responsiveness is also cyclically modulated during lymphocyte Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 recirculation. In particular, lymphocyte S1PR1 is downregulated

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701278 2 HUMAN T CELL RESPONSE TO S1P in the blood, upregulated in lymphoid organs, and downregulated cells were lysed using Trizol reagent (Invitrogen) and RNA was extracted again in the lymph, in a manner dependent on local S1P con- according to the manufacturer’s instructions. centrations (16) and on the GRK2 kinase (17). Quantitative RT-PCR Few studies have addressed the role of S1P receptors in primary human lymphocytes. It was reported that human thymocytes We used a high-capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, displayed a strong response to S1P in migration assays (18) and CA) to generate cDNA for RT-PCR. PCR was carried out with a SybrGreen- based kit (FastStart Universal SYBR Green Master, Roche, Basel, Swit- that tonsil B cell subsets respond to S1P (19). Moreover, nu- zerland) or SensiFast SYBR No-ROX kit (Bioline) on a StepOne plus merous studies have documented the important lymphopenia instrument (Applied Biosystems) or a LightCycler 480 system (Roche). induced by the treatment with FTY720, a Food and Drug Primers were designed using the Roche software. The following 9 Administration–approved treatment for relapsing multiple scle- primers were used for quantitative PCR: S1PR1 (forward) 5 -AACT- TCGCCCTGCTTGAG-39, S1PR1 (reverse) 59-TCCAGGCTTTTTGTGT- rosis (20–23). FTY720 binds with a higher afﬁnity to S1PR1 and, AGCTT-39,S1PR2(forward)59-CCACTCGGCAATGTACCTGT-39,S1PR2 although the mechanism of FTY720 action is still debated, it is (reverse) 59-ACGCCTGCCAGTAGATCG-39,S1PR3(forward)59-GCAGGC- likely that much of its action comes from its ability to induce AACCTCCTCTCAT-39,S1PR3(reverse)59-GAAAAAGCAGCCAAGTTC- S1PR1 internalization in lymphocytes. In treated patients, CA-39, S1PR4 (forward) 59-AATGGGCTTCCCATGGTC-39, S1PR4 (reverse) 9 9 9 FTY720 induces a quick decrease in peripheral naive cells and 5 -CAGGGTGCTCTCTGCTCCTA-3 , S1PR5 (forward) 5 -GAGTGCAATG- GGCACAATTA-39,S1PR5(reverse)59-GGAACTCCAGGCTTGATCC-39, TCM but it does not affect peripheral TEM and NK cells (20, 21, KLF2 (forward) 59-CATCTGAAGGCGCATCTG-39, KLF2 (reverse) 24), which is attributed to a low S1PR1 expression on these cell 59-CGTGTGCTTTCGGTAGTGG-39, OAZ1 (forward) 59-GGATAAACCC- types and a lower sensitivity to FTY720-induced internalization of AGCGCCAC-39, OAZ1 (reverse) 59-TACAGCAGTGGAGGGAGACC-39.

S1P5 (25). Hence, most of our knowledge of the role of S1P re- Downloaded from Statistics ceptors in lymphocyte trafﬁcking results from the study of loss-of- function mutant mouse models or the study of the impact of Statistical analyses were performed using parametric or nonparametric FTY720 in patients, and the role and regulation of S1P receptors tests (t test or ANOVA) run on the Prism software (GraphPad). Levels of signiﬁcance are expressed as p values (*p , 0.05, **p , 0.01, in human leukocytes remains mostly unexplored. In this study, we ***p , 0.001). revisited this point and measured the response of human memory

T cell subsets to S1P, and assessed the role of the different S1P http://www.jimmunol.org/ receptors in this response. Results Lymphocyte response to S1P is regulated during recirculation Materials and Methods across lymphoid organs Patients and preparation of human lymphocyte suspensions We measured the response of freshly isolated human lymphocyte subsets to S1P using Transwell migration systems. We first All material was used after obtaining informed consent, and the research was conducted in accordance with the Helsinki Declaration. Human pe- tested the response of PBMC obtained from healthy donors, ripheral blood was obtained from healthy donors. Cells were ficollized and taking naive CD4 and CD8 T cells as the prototypic cell types then washed several times in complete medium before resuspension in responsive to S1P. T cell subsets were identified by flow chemotaxis medium. Pediatric tonsils were obtained from patients under- cytometry as shown in Fig. 1A. However, as previously dem- by guest on September 29, 2021 going tonsillectomy and were cut into pieces using a scalpel and then passed onstrated in mice, human blood naive T cells did not respond to through a 70 mM cell strainer to produce a cell suspension. Cells were then ficollized and washed before migration studies. S1P (Supplemental Fig. 1A). A prior incubation in culture medium supplemented (or not) with serum or cytokines did not Chemotaxis assays change this response (data not shown). This unresponsiveness Tonsil lymphocytes or PBMC were suspended in RPMI 1640 supple- was specific to S1P as blood T cells were highly responsive mented with 4 mg/ml fatty acid–free bovine albumin (Sigma, St. Louis, to chemokines such as CXCL12 (Supplemental Fig. 1B). As- MO). The same medium was used to prepare S1P (Sigma) or CXCL12 suming that S1P receptors were desensitized in blood leuko- (R&D Systems, Minneapolis, MN) at the indicated concentrations. Cell migration was analyzed in Transwell chambers (Costar, Cambridge, MA) cytes as a result of high S1P concentrations in this compartment with 5 mm pore-width polycarbonate filters. Pharmacological modulators as described in mice, we measured the response of freshly of S1P receptors FTY720 (Novartis, Basel, Switzerland), JTE-013 isolated tonsil lymphocytes to S1P gradients. As shown in Fig. (Tocris, Bristol, U.K.), SEW2871 (Sigma), and CYM50358 (Tocris) 1B, 1C, tonsil naive T cell chemotaxis was strongly increased were used at the indicated concentrations. PBMC or tonsil cells were when S1P gradients were applied, and compared with the added to the top chambers of the Transwell systems in the presence or absence of the pharmacological inhibitors and incubated at 37˚C for 2 h. control condition without S1P. Maximum migration was ob- The chemoattractants were then added in the lower chamber and the cells tained with 30–60 nM S1P, in the range of previously published were allowed to migrate for 2 h. Transmigrated cells were stained for values for mouse T cells. Higher S1P concentrations inhibited CD3 (UCHT1), CD4 (RPA-T4), CD8 (RPA-T8), CD45RO (UCHL1), this migration, a response typically observed with other G CCR7 (G043H7), CD69 (FN50), CD103 (Ber-act8), CD38 (HIT32), and HLA-DR (LN3) and analyzed/counted by flow cytometry (MACSQuant; protein–coupled receptors. Both naive CD4 and CD8 T cells Miltenyi Biotec, Bergisch Gladbach, Germany). All Abs were purchased migrated in response to S1P, with naive CD8 T cells display- from BD Biosciences (San Jose, CA), eBioscience (San Diego, CA), ing the strongest response. Beckman-Coulter (Miami, FL), or BioLegend (San Diego, CA). The These data show that, like in the mouse (16), human lymphocyte migration index was calculated as the ratio between the number of cells response to S1P fluctuates during recirculation, presumably be- migrating in the S1P (or CXCL12) condition and the number of cells migrating in the control (medium only) condition, as measured by flow cause of receptor internalization in blood lymphocytes. cytometry. The percentage of input was calculated as the ratio between the number of cells migrating in a given condition and the number of S1P inhibit spontaneous migration of memory T cells cells used for the chemotaxis assay. Next, we measured the migration of memory T cell subsets in Cell sorting and RNA preparation response to S1P or CXCL12. Blood memory T cells did not respond to S1P but were strongly reactive to CXCL12 (Supplemental Tonsil lymphocytes were stained for surface markers (similar stainings as for chemotaxis experiments) and sorted into different subsets using a Fig. 1C–F). Surprisingly, when analyzing the response of tonsil FACSAria Cell Sorter (Becton-Dickinson, San Jose, CA). The purity of lymphocytes, S1P did not attract TCM and TEM toward the sorted cell populations was over 98% as checked by flow cytometry. Sorted lower chamber but rather inhibited their spontaneous migration The Journal of Immunology 3

FIGURE 1. Human tonsil T cell subsets display different migratory behaviors in response to S1P. (A–G) Transwell assays of the migration of tonsil T cell subsets assessing movement toward different concentrations of S1P as indicated. T cell subsets were deﬁned as shown in (A). Results in (B)–(G) are presented as migration index or percentage of Downloaded from input for the different conditions and subsets, as detailed in the Materials and Methods section. Lines show biological replicates (n = 9 donors). Asterisks indicate statistical signi- ﬁcance when comparing cell mi- http://www.jimmunol.org/ gration in the control condition (medium) with the conditions with S1P. *p , 0.05, **p , 0.01, ***p , 0.001 (ANOVA test with Dunnett posttest). by guest on September 29, 2021

(Fig. 1D–G), especially for memory CD4 T cells. TCM had an S1PR1 and S1PR2 are respectively involved in naive and intermediate migratory response between naive and TEM, sug- memory T cell response to S1P gesting that the response to S1P was mediated by two different To gain insight into the mechanism underlying the different be- receptors whose expressions were regulated in opposite ways haviors of naive and memory T cell subsets in the presence of during T cell differentiation. When expressing the same migration S1P gradients, we ﬁrst measured the expression of S1P receptors results as a percentage of input, we noticed that tonsil naive T cells by semiquantitative RT-PCR in sorted T cell subsets. As shown in were rather stationary in the absence of chemotactic signals. Re- Fig. 2A and 2B, S1PR1, S1PR2, and S1PR4 were expressed at high ciprocally, memory T cells of both subsets were constitutively levels in T cells, whereas S1PR3 was barely detected and S1PR5 motile, but this migration could be inhibited in a dose-dependent was only expressed at low levels in CD8 TEM. Of note, S1PR1 manner by S1P added in the lower chamber (Fig. 1D–G). Similar results were obtained with LN lymphocytes (data not shown), expression was progressively downregulated upon T cell differen- suggesting that our conclusions on the response of memory T cells tiation into memory cells, whereas S1PR2 and S1PR4 expression to S1P likely apply to those of all SLO. Finally, we also tested the levels remained constitutively expressed. S1PR1 downregulation capacity of tonsil T cells to respond to a physiological source of correlated with KLF2 being strongly downregulated in TEM S1P, i.e., FCS, diluted at different concentrations. As shown in (Fig. 2C). KLF2 is known to induce S1PR1 expression in T cells Supplemental Fig. 2, naive and memory T cells displayed opposite (26). responses to FCS, naive T cells being attracted and memory T cell We then tested the effect of different pharmacological inhibitors migration being inhibited by S1P. of these receptors on the capacity of T cell subsets to respond to Altogether, these results show that T cell response to S1P is S1P. Tonsil naive T cell migration was strongly inhibited by S1PR1 highly regulated during differentiation and that naive and TEM inhibitors FTY720 and SEW2871 (Fig. 2D, 2E) but insensitive to subsets have opposite responses to S1P. These data also suggest that the S1PR4 inhibitor CYM50358. The S1PR2 inhibitor JTE-013 TEM are retained by S1P within SLO whereas naive T cells can exit abrogated the inhibitory effect of S1P on TCM and TEM migra- in response to the same signal. tion (Fig. 2F–I). This inhibitor had, however, no effect on spon- 4 HUMAN T CELL RESPONSE TO S1P

FIGURE 2. S1PR1 and S1PR2 mediate the response to S1P in human naive and memory T cells. (A–C) Quantitative real-time PCR analysis of S1PR (A and B) and KLF2 (C) expression in flow cytometry–sorted T cell subsets from human tonsils. Results show the mean 6 SD of five independent experiments for a total of n =6 donors. *p , 0.05, **p , 0.01, , ***p 0.001 (Mann–Whitney Downloaded from tests). (D–I) Transwell assays of the migration of the indicated tonsil T cell subsets assessing movement toward different concentrations of S1P as indicated. Prior to the addition of S1P in the lower chamber, cells were treated for 2 h in the top http://www.jimmunol.org/ chamber with the indicated pharmacological inhibitors at the indicated concentrations (nanomolars). Results show the mean 6 SD of three independent experiments with n = 3 donors. Asterisks indicate statistical significance when comparing cell migration in the control condition (S1P only) with the conditions with inhibitors. *p , 0.05, by guest on September 29, 2021 **p , 0.01, ***p , 0.001 (ANOVA test with Dunnett posttest).

taneous T cell migration (data not shown). Of note, FTY720 and TRM and recently act-T cells. In mice, TRM are believed to be SEW2871 had a negative impact on memory T cell migration in retained in tissues by default of a response to S1P. Indeed, TRM the presence of S1P whereas JTE-013 treatment tended to increase express very low levels of KLF2 and S1PR1 (27). Moreover, CD69 naive T cell migration to S1P, suggesting that S1PR1 and S1PR2 expression induces S1PR1 internalization, further inhibiting mi- are active in all T cell subsets but that the relative level of each gration toward S1P (12). To test this point in humans, we mea- receptor conditions the migratory behavior in response to S1P. sured the S1P response of human tonsil TRM deﬁned as CD69 Interestingly, when plotting the maximum migration index as a positive T cells coexpressing (or not) CD103. As shown in Fig. 3A function of S1PR1 expression in the various T cell subsets, we and 3B, S1P inhibited spontaneous migration of CD4+ TRM, found a very tight linear correlation between both factors irrespective of their CD103 expression. Again, the S1PR2 antag- (Supplemental Fig. 3), suggesting that S1PR1 expression is the onist JTE-013 abrogated this inhibitory effect (Fig. 3C). For CD8+ limiting rate factor in S1P-induced migration and that the apparent TRM, S1P inhibited spontaneous migration of CD69+CD103+ but repulsion mediated by S1PR2 only occurs when S1PR1 is not CD69+CD1032 cells, and JTE-013 abrogated this effect. expressed at very low levels, i.e., in memory T cells, especially of Upon Ag-mediated activation, T cells are retained within SLOs, the CD4 subset. presumably to sustain their activation and differentiation through serial interactions with Ag presenting cells. Mechanistically, it Spontaneous migration of TRM and act-T cells is inhibited by was previously reported that mouse T cells lose their reactivity S1P in an S1PR2-dependent way during this phase by down regulating S1PR1 expression (14). We As our results suggested that S1P could be an important tissue- addressed this point in humans, exploiting the fact that tonsils retention signal in humans, we next studied the S1P response of contain act-T cells in a variable proportion, classically deﬁned as The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. S1PR2 inhibits spontaneous migration of TRM and act-T cells. Transwell assays of the migration of the indicated tonsil T cell subsets assessing movement toward different concentrations of S1P as indicated. Results are shown as (A) migration index; (B) percentage of input. Lines show biological replicates (n = 6 donors). Asterisks indicate statistical significance when comparing cell migration in the control condition (medium) with the conditions with S1P. *p , 0.05, **p , 0.01, ***p , 0.001 (ANOVA test with Dunnett posttest). (C) Prior to the addition of S1P in the lower chamber, cells were treated for 2 h in the top chamber with the JTE-013 at the indicated concentrations (nanomolars). Results show the mean 6 SD of six independent experiments with n = 6 donors. Asterisks indicate statistical significance when comparing cell migration in the control condition (S1P only) with the conditions with JTE-013. *p , 0.05, **p , 0.01 (Mann–Whitney tests). (D) Quantitative real-time PCR analysis of S1PR expression in the indicated T cell subsets sorted by flow cytometry from human tonsil lymphocyte preparations. n = 3 sorts from three different donors.

CD38+HLA-DR+. As shown in Fig. 3A and 3B, the spontaneous antagonism between S1PR1 and S1PR2 in T cell subsets. As migration of act-CD4+ and act-CD8+ T cells was strongly lymphocytes are constantly exposed to opposing signals inﬂuenc- inhibited by S1P, an effect that could be again curbed by the ing their mobility, we also wanted to test the impact of S1P on the S1PR2 antagonist JTE-013 (Fig. 3C). We also measured the ex- capacity of naive and memory T cells to respond to chemokine pression of S1P receptors in TRM CD69+ and act-T cells. S1PR1 gradients. We therefore measured the migration of tonsil T cells in was found to be highly downregulated in act-T cells compared response to various concentrations of CXCL12, the ligand for with naive T cells and undetectable in TRM whereas the level of CXCR4, a receptor involved in T cell homeostasis (28) and SLO S1PR2 was similar to that of other T cell subsets (Fig. 3D). organization (29), in the presence or absence of S1P given at an optimal concentration (37 nM). As shown in Fig. 4, the addition of S1PR2 engagement inhibits chemokine-induced migration of S1P in the lower chamber slightly increased the migration of naive memory T cell subsets CD4 and CD8 T cells but decreased that of TEM and to a lesser Altogether, our results demonstrate the major role of S1PR2 in extent that of TCM induced by CXCL12. The latter effect was human memory T cell response to S1P and suggest a functional abrogated by JTE-013 in all conditions, demonstrating that S1PR2 6 HUMAN T CELL RESPONSE TO S1P

decreases during T cell differentiation into memory subtypes. This correlated with the migratory behavior of naive versus memory T cells in the presence of S1P; S1PR1 mediated attraction of naive T cells whereas S1PR2 inhibited spontaneous or chemokine-induced memory T cell migration. The affinity of S1P to S1PR2 appears substantially lower than its affinity to S1PR1 (Kd of 27 nM versus Kd of 8 nM for review, see Ref. 30). This suggests that T cells are attracted toward high S1P concentrations as long as S1PR1 expression is sufficient to overcome S1PR2 activity. Our results suggest that TEM, TRM, act-T cells, and to a lesser extent TCM are retained within SLO through S1PR2 signaling. These data challenge the classical view that S1P is a major exit signal for T lymphocytes. S1P-mediated retention of memory and act-T cells within SLO may favor the re-encounter of Ag presented by dendritic cells. These data may also explain why, as recently shown, TEM are very abundant within SLO (2). Importantly, memory T cell subsets were found to display higher spontaneous migration than naive T cells, possibly through con-

stitutive activation of integrins. This intrinsically high mobility Downloaded from may be important for their entry into SLO and to more efﬁciently scan APCs. It has been postulated that egress structures in SLO like LN may be relatively permissive to T lymphocytes, possibly through egress portals (15). In this context, S1PR2 may be important to override the constitutive mobility of memory T cells

and promote their retention within SLO. How memory T cell http://www.jimmunol.org/ subsets reach the blood circulation remains to be determined. S1P-induced S1PR2 desensitization and attraction by other chemotactic signals such as proinflammatory or homeostatic chemokines may allow the egress from SLO. Similar to S1PR1, S1PR2 can be indeed internalized upon stimulation with agonists (31). In zebrafish, the miles apart mutant, S1PR2 R150H alters the migration of cardiac precursor cells to the midline, a phe- nomenon due to constitutive desensitization and internalization of S1PR2 (32). CXCL12 is highly expressed in the medullar region by guest on September 29, 2021 FIGURE 4. S1PR2 inhibits CXCL12-induced migration of memory T of LN (29) and may also contribute to promote entry of memory cell subsets. (A–C) Transwell assays of the migration of the indicated tonsil T cells into lymphatic vessels. T cell subsets assessing movement toward CXCL12 (12 ng/ml) 6 S1P S1PR2 has been previously shown to contribute to accumulation (37 nM) as indicated. Results are shown as migration index. Prior to the of germinal center B cells in the central region of the mouse addition of S1P in the lower chamber, cells were treated (or not) for 2 h in follicle [Wang et al. (6); Green et al. (9)]. Sic et al. (19) also the top chamber with JTE-013 (1000 nM). Results show the mean 6 SD showed that human tonsil germinal center and plasma B cells of four independent experiments with n = 3 donors. Asterisks indicate spontaneous migration was inhibited by S1P. As these cells ex- statistical significance when comparing cell migration in the control con- press high levels of S1PR2, this was likely to be mediated by , , dition (S1P only) with the conditions with JTE-013. *p 0.05, **p 0.01 S1PR2, although this point required formal testing. Likewise, (Mann–Whitney tests). S1PR2 was shown to be critical for mouse follicular helper T cell retention in germinal centers (33) and in the proper localization engagement can oppose chemokine-induced migration in memory of osteoclast precursors in the bone (8). S1PR2 also inhibits Tcellsubsets. migration in many nonhematopoietic cell types, including vascular endothelial and smooth muscle cells as well as tumor cells Discussion (7). Our findings therefore corroborate prior reports suggesting In this study, we demonstrate that human T cell responsiveness to counterbalancing roles of S1PR1 and S1PR2, and suggest that the S1P is regulated at multiple levels. First, as previously demon- antagonism between S1PR1 and S1PR2 is also important to strated in mice (16), S1P receptors are desensitized in blood control the distribution of naive and memory T cells in human. circulating T cells. This is likely the consequence of ligand- Previous studies suggest that S1PR2 usually inhibits S1PR1 induced receptor internalization. All our attempts to restore signaling by activating Rho and inhibiting Rac (34–36). S1PR1 is T cell sensitivity to S1P by preincubation in various conditions also known to signal through Akt (36), an event that has been failed, suggesting that the recycling of S1P receptors on the cell coupled to S1P responsiveness and actin polymerization in hu- surface requires active signals, more complex than the mere man T cells (37). deprivation of S1P. Second, the expression of S1P receptors is The S1PR2 receptor plays important roles in the physiology of highly regulated during human T cell differentiation. S1PR1 several tissues and organs, and thus, a therapeutic application of expression is indeed high in human naive T cells, likely induced S1PR2 antagonists or allosteric modulators will inevitably cause by the transcription factor KLF2 (26) but decreases upon differ- adverse effects if given systemically. However, local targeting of entiation in TCM and even more in TEM, TRM and act-T cells. S1PR2 may alleviate symptoms induced by overt T cell reactions, By contrast, S1PR2 expression remains stable during differenti- in the context of various inflammatory, allergic, or autoimmune ation. Hence the ratio between S1PR1 and S1PR2 progressively syndromes. The Journal of Immunology 7

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0 0 0 CD4 CD4 2,6 CD4 2,6 2,6 S1P (nM)

: Humanblood T cellsubsetsarenotresponsiveto 26 26 26 S1P (nM) S1P (nM) Naive TCM

260 TEM 260 260 2600 2600 2600 0 0 0 CD8 CD8

2,6 CD8 2,6 2,6 26 26 26 260 260 260 2600 2600 2600 D B F Migration index Migration index Migration index 10 15 20 1 2 3 4 5 5 1 2 3 4 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 CD4 12 CD4 12 12 CD4

37 CXCL12 (ng/ml) 37

CXCL12 (ng/ml) 37

CXCL12 (ng/ml) 111 111 111

333 333 333 Naive TCM 1000 TEM 1000 1000 0 0 0 12 12 12 CD8 CD8 37 37 37 CD8 111 111 111 333 333 333 1000 1000 1000 donors. N=4 with experiments four of mean+/-SD the are and index as migration presented are Results 1. figure in shown as defined were subsets cell T indicated. as FCS assessing of subsets concentrations different cell toward movement T blood of migration the of assays Transwell cells toFCS Figure S2:

Migration index Migration index 1 1 1 0 5 0 5 8 0 2 4 6 Opposite migratory responses of tonsil naïve and memory T 0 CD4 T cells

0 CD8 T cells 0 0,01 0,01 0,1 0,1 1 1 10 10 100 100 0 0 FCS FCS 0,01 0,01 0,1 0,1

1 ( ( % % 10 10 ) ) 100 100 0 0 0,01 0,01 TEM TCM Naive 0,1 0,1 TEM TCM Naive 1 1 10 10 100 100 R2=0.94 Migration index

S1PR1 relative level

Figure S3: The migration induced by S1P is highly correlated with the level of S1PR1 mRNA expression in tonsil T cell subsets Graph showing the migration index of T cell subsets at 37nM S1P in relation with the S1PR1 mRNA relative expression as measured by Q-PCR.