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

Marvel, Alexandre Belot and Thierry WalzerMathieu, Antoine Marçais, Mélanie Wencker, Jacqueline Annabelle Drouillard, Antoinette Neyra, Anne-Laure

ol.1701278http://www.jimmunol.org/content/early/2017/12/13/jimmun

published online 13 December 2017J Immunol 

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The Journal of Immunology

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

Annabelle Drouillard,*,†,‡,x,{ Antoinette Neyra,*,†,‡,x,{ Anne-Laure Mathieu,*,†,‡,x,{

Antoine Marcais,*,†,‡,x,{ Melanie 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

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.

Memory T cell subsets have complementary functionsand distinct tissue distributions. Central memoryT cells (TCM) defined as CCR7+CD45RA2 in humans

have superior proliferative capacity and recirculate through sec-ondary lymphoid organs (SLO) via blood and lymph. Effectormemory T cells (TEM), defined as CCR72CD45RA2CD45RO+ inhumans, have important immediate effector functions and recircu-late through the blood and spleen (1). In humans, a high frequencyof T cells found in nonreactive lymph nodes (LNs) is of TEMphenotype, even in infants, suggesting that TEM may recirculate vianonlymphoid tissues to the afferent lymph, as previously proposed

(2). The recently described resident memory T cells (TRM) do notrecirculate but are instead resident in both nonlymphoid and lym-phoid organs (3). They are defined as CD69+CD103+/2 in all spe-cies, irrespective of the expression of other markers, even thoughthis definition is known to be imperfect (4). TRM localization,at sites of pathogen entry, is thought to be essential for recallresponses.Sphingosine-1 phosphate (S1P) binds to five different G-protein

coupled receptors (S1PR1-5). Extracellular S1P is carried in thebody by albumin and other lipoproteins. S1P concentration ismaintained at high levels in the blood and lymph and at low levelswithin tissues (5). S1PR1, S1PR3, and S1PR5 are coupled to Gaiand provide attractive cues to lymphocytes. A series of studiesfound that S1PR1 allows the egress of T and B cells from SLO tothe blood and lymph (5). S1PR4 is believed to regulate T cellproliferation and cytokine secretion but not cell migration (6).S1PR2 is the only S1P receptor coupled to G12/G13 and signalsvia Rho instead of Rac-like Gai-coupled receptors (7). Thisproperty may explain how S1PR2 mediates chemorepulsion andantagonizes S1PR1 signaling in mouse osteoclast precursors (8) orgerminal center B cells (9), a process important for their properlocalization. S1PR2 is also known to be insensitive to the effect ofFTY720, a supra agonist for all other S1P receptors that inducestheir internalization (10, 11).S1P responsiveness is regulated during T cell activation, as

shown in mouse studies. Recently activated T cells (act-T cells)upregulate CD69 expression. CD69 binds surface S1PR1 and in-duces its internalization, trapping act-T cells in inflamed LNs (12)or in nonlymphoid tissues such as the skin (13). Upon TCRstimulation, S1PR1 is also downregulated transcriptionally, whichmay contribute to T cell sequestration in LN. This downregulationis, however, transient as S1PR1 levels increase during differenti-ation into effector and memory T cells, which is believed to permitegress from SLO by restoring responsiveness to S1P (14, 15). S1Presponsiveness is also cyclically modulated during lymphocyterecirculation. In particular, lymphocyte S1PR1 is downregulated

*International Center for Infectiology Research, 69000 Lyon, France; †INSERM,U1111, 69000 Lyon, France; ‡Ecole Normale Superieure de Lyon, 69000 Lyon,France; xUniversite Lyon 1, 69000 Lyon, France; {CNRS, UMR5308, 69000 Lyon,France; and ‖Service de Nephrologie Rhumatologie Dermatologie Pediatriques,Hospices Civils de Lyon, Universite Claude-Bernard Lyon 1, 69000 Lyon, France

ORCIDs: 0000-0001-7188-7655 (A.D.); 0000-0001-6241-459X (J.M.); 0000-0002-0857-8179 (T.W.).

Received for publication September 5, 2017. Accepted for publication November 7,2017.

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(Equipe Labellisee), and the European Research Council (ERC-Stg 281025) andreceives institutional grants from INSERM, CNRS, Universite Claude Bernard Lyon1, and Ecole Normale Superieure de Lyon.

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 theideas and interpreted data; T.W. wrote the paper and edited it with the assistance ofthe other authors.

Address correspondence and reprint requests to Dr. Thierry Walzer, Centre Interna-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.E-mail address: Thierry.walzer@inserm.fr

The online version of this article contains supplemental material.

Abbreviations used in this article: act-T, activated T; LN, lymph node; SLO, second-ary lymphoid organ; S1P, sphingosine-1 phosphate; TCM, central memory T cell;TEM, effector memory T cell; TRM, resident memory T cell.

Copyright� 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00

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in the blood, upregulated in lymphoid organs, and downregulatedagain in the lymph, in a manner dependent on local S1P con-centrations (16) and on the GRK2 kinase (17).Few studies have addressed the role of S1P receptors in primary

human lymphocytes. It was reported that human thymocytesdisplayed a strong response to S1P in migration assays (18) andthat tonsil B cell subsets respond to S1P (19). Moreover, nu-merous studies have documented the important lymphopeniainduced by the treatment with FTY720, a Food and DrugAdministration–approved treatment for relapsing multiple scle-rosis (20–23). FTY720 binds with a higher affinity to S1PR1 and,although the mechanism of FTY720 action is still debated, it islikely that much of its action comes from its ability to induceS1PR1 internalization in lymphocytes. In treated patients,FTY720 induces a quick decrease in peripheral naive cells andTCM but it does not affect peripheral TEM and NK cells (20, 21,24), which is attributed to a low S1PR1 expression on these celltypes and a lower sensitivity to FTY720-induced internalization ofS1P5 (25). Hence, most of our knowledge of the role of S1P re-ceptors in lymphocyte trafficking results from the study of loss-of-function mutant mouse models or the study of the impact ofFTY720 in patients, and the role and regulation of S1P receptorsin human leukocytes remains mostly unexplored. In this study, werevisited this point and measured the response of human memoryT cell subsets to S1P, and assessed the role of the different S1Preceptors in this response.

Materials and MethodsPatients and preparation of human lymphocyte suspensions

All material was used after obtaining informed consent, and the researchwas conducted in accordance with the Helsinki Declaration. Human pe-ripheral blood was obtained from healthy donors. Cells were ficollized andthen washed several times in complete medium before resuspension inchemotaxis medium. Pediatric tonsils were obtained from patients under-going tonsillectomy and were cut into pieces using a scalpel and then passedthrough a 70 mM cell strainer to produce a cell suspension. Cells were thenficollized and washed before migration studies.

Chemotaxis assays

Tonsil lymphocytes or PBMC were suspended in RPMI 1640 supple-mented with 4 mg/ml fatty acid–free bovine albumin (Sigma, St. Louis,MO). The same medium was used to prepare S1P (Sigma) or CXCL12(R&D Systems, Minneapolis, MN) at the indicated concentrations. Cellmigration was analyzed in Transwell chambers (Costar, Cambridge, MA)with 5 mm pore-width polycarbonate filters. Pharmacological modulatorsof S1P receptors FTY720 (Novartis, Basel, Switzerland), JTE-013(Tocris, Bristol, U.K.), SEW2871 (Sigma), and CYM50358 (Tocris)were used at the indicated concentrations. PBMC or tonsil cells wereadded to the top chambers of the Transwell systems in the presence orabsence of the pharmacological inhibitors and incubated at 37˚C for 2 h.The chemoattractants were then added in the lower chamber and the cellswere allowed to migrate for 2 h. Transmigrated cells were stained forCD3 (UCHT1), CD4 (RPA-T4), CD8 (RPA-T8), CD45RO (UCHL1),CCR7 (G043H7), CD69 (FN50), CD103 (Ber-act8), CD38 (HIT32), andHLA-DR (LN3) and analyzed/counted by flow cytometry (MACSQuant;Miltenyi Biotec, Bergisch Gladbach, Germany). All Abs were purchasedfrom BD Biosciences (San Jose, CA), eBioscience (San Diego, CA),Beckman-Coulter (Miami, FL), or BioLegend (San Diego, CA). Themigration index was calculated as the ratio between the number of cellsmigrating in the S1P (or CXCL12) condition and the number of cellsmigrating in the control (medium only) condition, as measured by flowcytometry. The percentage of input was calculated as the ratio betweenthe number of cells migrating in a given condition and the number ofcells used for the chemotaxis assay.

Cell sorting and RNA preparation

Tonsil lymphocytes were stained for surface markers (similar stainings asfor chemotaxis experiments) and sorted into different subsets using aFACSAria Cell Sorter (Becton-Dickinson, San Jose, CA). The purity ofsorted cell populations was over 98% as checked by flow cytometry. Sorted

cells were lysed using Trizol reagent (Invitrogen) and RNA was extractedaccording to the manufacturer’s instructions.

Quantitative RT-PCR

We used a high-capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad,CA) to generate cDNA for RT-PCR. PCRwas carried out with a SybrGreen-based kit (FastStart Universal SYBR Green Master, Roche, Basel, Swit-zerland) or SensiFast SYBR No-ROX kit (Bioline) on a StepOne plusinstrument (Applied Biosystems) or a LightCycler 480 system (Roche).Primers were designed using the Roche software. The followingprimers were used for quantitative PCR: S1PR1 (forward) 59-AACT-TCGCCCTGCTTGAG-39, S1PR1 (reverse) 59-TCCAGGCTTTTTGTGT-AGCTT-39, S1PR2 (forward) 59-CCACTCGGCAATGTACCTGT-39, S1PR2(reverse) 59-ACGCCTGCCAGTAGATCG-39, S1PR3 (forward) 59-GCAGGC-AACCTCCTCTCAT-39, S1PR3 (reverse) 59-GAAAAAGCAGCCAAGTTC-CA-39, S1PR4 (forward) 59-AATGGGCTTCCCATGGTC-39, S1PR4 (reverse)59-CAGGGTGCTCTCTGCTCCTA-39, S1PR5 (forward) 59-GAGTGCAATG-GGCACAATTA-39, S1PR5 (reverse) 59-GGAACTCCAGGCTTGATCC-39,KLF2 (forward) 59-CATCTGAAGGCGCATCTG-39, KLF2 (reverse)59-CGTGTGCTTTCGGTAGTGG-39, OAZ1 (forward) 59-GGATAAACCC-AGCGCCAC-39, OAZ1 (reverse) 59-TACAGCAGTGGAGGGAGACC-39.

Statistics

Statistical analyses were performed using parametric or nonparametrictests (t test or ANOVA) run on the Prism software (GraphPad). Levelsof significance are expressed as p values (*p , 0.05, **p , 0.01,***p , 0.001).

ResultsLymphocyte response to S1P is regulated during recirculationacross lymphoid organs

Wemeasured the response of freshly isolated human lymphocytesubsets to S1P using Transwell migration systems. We firsttested the response of PBMC obtained from healthy donors,taking naive CD4 and CD8 T cells as the prototypic cell typesresponsive to S1P. T cell subsets were identified by flowcytometry as shown in Fig. 1A. However, as previously dem-onstrated in mice, human blood naive T cells did not respond toS1P (Supplemental Fig. 1A). A prior incubation in culturemedium supplemented (or not) with serum or cytokines did notchange this response (data not shown). This unresponsivenesswas specific to S1P as blood T cells were highly responsiveto chemokines such as CXCL12 (Supplemental Fig. 1B). As-suming that S1P receptors were desensitized in blood leuko-cytes as a result of high S1P concentrations in this compartmentas described in mice, we measured the response of freshlyisolated tonsil lymphocytes to S1P gradients. As shown in Fig.1B, 1C, tonsil naive T cell chemotaxis was strongly increasedwhen S1P gradients were applied, and compared with thecontrol condition without S1P. Maximum migration was ob-tained with 30–60 nM S1P, in the range of previously publishedvalues for mouse T cells. Higher S1P concentrations inhibitedthis migration, a response typically observed with other Gprotein–coupled receptors. Both naive CD4 and CD8 T cellsmigrated in response to S1P, with naive CD8 T cells display-ing the strongest response.These data show that, like in the mouse (16), human lymphocyte

response to S1P fluctuates during recirculation, presumably be-cause of receptor internalization in blood lymphocytes.

S1P inhibit spontaneous migration of memory T cells

Next, we measured the migration of memory T cell subsets inresponse to S1P or CXCL12. Blood memory T cells did not re-spond to S1P but were strongly reactive to CXCL12 (SupplementalFig. 1C–F). Surprisingly, when analyzing the response of tonsillymphocytes, S1P did not attract TCM and TEM toward thelower chamber but rather inhibited their spontaneous migration

2 HUMAN T CELL RESPONSE TO S1P

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(Fig. 1D–G), especially for memory CD4 T cells. TCM had anintermediate migratory response between naive and TEM, sug-gesting that the response to S1P was mediated by two differentreceptors whose expressions were regulated in opposite waysduring T cell differentiation. When expressing the same migrationresults as a percentage of input, we noticed that tonsil naive T cellswere rather stationary in the absence of chemotactic signals. Re-ciprocally, memory T cells of both subsets were constitutivelymotile, but this migration could be inhibited in a dose-dependentmanner by S1P added in the lower chamber (Fig. 1D–G). Similarresults were obtained with LN lymphocytes (data not shown),suggesting that our conclusions on the response of memory T cellsto S1P likely apply to those of all SLO. Finally, we also tested thecapacity of tonsil T cells to respond to a physiological source ofS1P, i.e., FCS, diluted at different concentrations. As shown inSupplemental Fig. 2, naive and memory T cells displayed oppositeresponses to FCS, naive T cells being attracted and memory T cellmigration being inhibited by S1P.Altogether, these results show that T cell response to S1P is

highly regulated during differentiation and that naive and TEMsubsets have opposite responses to S1P. These data also suggest thatTEM are retained by S1P within SLOwhereas naive T cells can exitin response to the same signal.

S1PR1 and S1PR2 are respectively involved in naive andmemory T cell response to S1P

To gain insight into the mechanism underlying the different be-haviors of naive and memory T cell subsets in the presence of

S1P gradients, we first measured the expression of S1P receptors

by semiquantitative RT-PCR in sorted T cell subsets. As shown in

Fig. 2A and 2B, S1PR1, S1PR2, and S1PR4 were expressed at high

levels in T cells, whereas S1PR3 was barely detected and S1PR5

was only expressed at low levels in CD8 TEM. Of note, S1PR1

expression was progressively downregulated upon T cell differen-

tiation into memory cells, whereas S1PR2 and S1PR4 expression

levels remained constitutively expressed. S1PR1 downregulation

correlated with KLF2 being strongly downregulated in TEM

(Fig. 2C). KLF2 is known to induce S1PR1 expression in T cells

(26).We then tested the effect of different pharmacological inhibitors

of these receptors on the capacity of T cell subsets to respond to

S1P. Tonsil naive T cell migration was strongly inhibited by S1PR1

inhibitors FTY720 and SEW2871 (Fig. 2D, 2E) but insensitive to

the S1PR4 inhibitor CYM50358. The S1PR2 inhibitor JTE-013

abrogated the inhibitory effect of S1P on TCM and TEM migra-

tion (Fig. 2F–I). This inhibitor had, however, no effect on spon-

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 concen-

trations 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

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-

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).

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taneous T cell migration (data not shown). Of note, FTY720 andSEW2871 had a negative impact on memory T cell migration inthe presence of S1P whereas JTE-013 treatment tended to increasenaive T cell migration to S1P, suggesting that S1PR1 and S1PR2are active in all T cell subsets but that the relative level of eachreceptor conditions the migratory behavior in response to S1P.Interestingly, when plotting the maximum migration index as afunction of S1PR1 expression in the various T cell subsets, wefound a very tight linear correlation between both factors(Supplemental Fig. 3), suggesting that S1PR1 expression is thelimiting rate factor in S1P-induced migration and that the apparentrepulsion mediated by S1PR2 only occurs when S1PR1 isexpressed at very low levels, i.e., in memory T cells, especially ofthe CD4 subset.

Spontaneous migration of TRM and act-T cells is inhibited byS1P in an S1PR2-dependent way

As our results suggested that S1P could be an important tissue-retention signal in humans, we next studied the S1P response of

TRM and recently act-T cells. In mice, TRM are believed to beretained in tissues by default of a response to S1P. Indeed, TRMexpress very low levels of KLF2 and S1PR1 (27). Moreover, CD69expression induces S1PR1 internalization, further inhibiting mi-gration toward S1P (12). To test this point in humans, we mea-sured the S1P response of human tonsil TRM defined as CD69positive T cells coexpressing (or not) CD103. As shown in Fig. 3Aand 3B, S1P inhibited spontaneous migration of CD4+ TRM,irrespective of their CD103 expression. Again, the S1PR2 antag-onist JTE-013 abrogated this inhibitory effect (Fig. 3C). For CD8+

TRM, S1P inhibited spontaneous migration of CD69+CD103+ butnot CD69+CD1032 cells, and JTE-013 abrogated this effect.Upon Ag-mediated activation, T cells are retained within SLOs,

presumably to sustain their activation and differentiation throughserial interactions with Ag presenting cells. Mechanistically, itwas previously reported that mouse T cells lose their reactivityduring this phase by down regulating S1PR1 expression (14). Weaddressed this point in humans, exploiting the fact that tonsilscontain act-T cells in a variable proportion, classically defined as

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

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 addi-

tion of S1P in the lower chamber,

cells were treated for 2 h in the top

chamber with the indicated phar-

macological inhibitors at the indi-

cated concentrations (nanomolars).

Results show the mean 6 SD of

three independent experiments with

n = 3 donors. Asterisks indicate

statistical significance when com-

paring cell migration in the control

condition (S1P only) with the con-

ditions with inhibitors. *p , 0.05,

**p , 0.01, ***p , 0.001 (ANOVA

test with Dunnett posttest).

4 HUMAN T CELL RESPONSE TO S1P

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CD38+HLA-DR+. As shown in Fig. 3A and 3B, the spontaneousmigration of act-CD4+ and act-CD8+ T cells was stronglyinhibited by S1P, an effect that could be again curbed by theS1PR2 antagonist JTE-013 (Fig. 3C). We also measured the ex-pression of S1P receptors in TRM CD69+ and act-T cells. S1PR1was found to be highly downregulated in act-T cells comparedwith naive T cells and undetectable in TRM whereas the level ofS1PR2 was similar to that of other T cell subsets (Fig. 3D).

S1PR2 engagement inhibits chemokine-induced migration ofmemory T cell subsets

Altogether, our results demonstrate the major role of S1PR2 inhuman memory T cell response to S1P and suggest a functional

antagonism between S1PR1 and S1PR2 in T cell subsets. Aslymphocytes are constantly exposed to opposing signals influenc-ing their mobility, we also wanted to test the impact of S1P on thecapacity of naive and memory T cells to respond to chemokinegradients. We therefore measured the migration of tonsil T cells inresponse to various concentrations of CXCL12, the ligand forCXCR4, a receptor involved in T cell homeostasis (28) and SLOorganization (29), in the presence or absence of S1P given at anoptimal concentration (37 nM). As shown in Fig. 4, the addition ofS1P in the lower chamber slightly increased the migration of naiveCD4 and CD8 T cells but decreased that of TEM and to a lesserextent that of TCM induced by CXCL12. The latter effect wasabrogated by JTE-013 in all conditions, demonstrating that S1PR2

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.

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engagement can oppose chemokine-induced migration in memoryT cell subsets.

DiscussionIn this study, we demonstrate that human T cell responsiveness toS1P is regulated at multiple levels. First, as previously demon-strated in mice (16), S1P receptors are desensitized in bloodcirculating T cells. This is likely the consequence of ligand-induced receptor internalization. All our attempts to restoreT cell sensitivity to S1P by preincubation in various conditionsfailed, suggesting that the recycling of S1P receptors on the cellsurface requires active signals, more complex than the meredeprivation of S1P. Second, the expression of S1P receptors ishighly regulated during human T cell differentiation. S1PR1expression is indeed high in human naive T cells, likely inducedby the transcription factor KLF2 (26) but decreases upon differ-entiation in TCM and even more in TEM, TRM and act-T cells.By contrast, S1PR2 expression remains stable during differenti-ation. Hence the ratio between S1PR1 and S1PR2 progressively

decreases during T cell differentiation into memory subtypes.This correlated with the migratory behavior of naive versusmemory T cells in the presence of S1P; S1PR1 mediated at-traction of naive T cells whereas S1PR2 inhibited spontaneous orchemokine-induced memory T cell migration. The affinity of S1Pto S1PR2 appears substantially lower than its affinity to S1PR1(Kd of 27 nM versus Kd of 8 nM for review, see Ref. 30). Thissuggests that T cells are attracted toward high S1P concentrationsas long as S1PR1 expression is sufficient to overcome S1PR2activity. Our results suggest that TEM, TRM, act-T cells, and to alesser extent TCM are retained within SLO through S1PR2 sig-naling. These data challenge the classical view that S1P is amajor exit signal for T lymphocytes. S1P-mediated retention ofmemory and act-T cells within SLO may favor the re-encounterof Ag presented by dendritic cells. These data may also explainwhy, as recently shown, TEM are very abundant within SLO (2).Importantly, memory T cell subsets were found to display higherspontaneous migration than naive T cells, possibly through con-stitutive activation of integrins. This intrinsically high mobilitymay be important for their entry into SLO and to more efficientlyscan APCs. It has been postulated that egress structures in SLOlike LN may be relatively permissive to T lymphocytes, possiblythrough egress portals (15). In this context, S1PR2 may be im-portant to override the constitutive mobility of memory T cellsand promote their retention within SLO. How memory T cellsubsets reach the blood circulation remains to be determined.S1P-induced S1PR2 desensitization and attraction by other che-motactic signals such as proinflammatory or homeostatic che-mokines 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 altersthe migration of cardiac precursor cells to the midline, a phe-nomenon due to constitutive desensitization and internalization ofS1PR2 (32). CXCL12 is highly expressed in the medullar regionof LN (29) and may also contribute to promote entry of memoryT cells into lymphatic vessels.S1PR2 has been previously shown to contribute to accumulation

of germinal center B cells in the central region of the mousefollicle [Wang et al. (6); Green et al. (9)]. Sic et al. (19) alsoshowed that human tonsil germinal center and plasma B cellsspontaneous migration was inhibited by S1P. As these cells ex-press high levels of S1PR2, this was likely to be mediated byS1PR2, although this point required formal testing. Likewise,S1PR2 was shown to be critical for mouse follicular helper T cellretention in germinal centers (33) and in the proper localizationof osteoclast precursors in the bone (8). S1PR2 also inhibitsmigration in many nonhematopoietic cell types, including vas-cular endothelial and smooth muscle cells as well as tumor cells(7). Our findings therefore corroborate prior reports suggestingcounterbalancing roles of S1PR1 and S1PR2, and suggest that theantagonism between S1PR1 and S1PR2 is also important tocontrol the distribution of naive and memory T cells in human.Previous studies suggest that S1PR2 usually inhibits S1PR1signaling by activating Rho and inhibiting Rac (34–36). S1PR1 isalso known to signal through Akt (36), an event that has beencoupled to S1P responsiveness and actin polymerization in hu-man T cells (37).The S1PR2 receptor plays important roles in the physiology of

several tissues and organs, and thus, a therapeutic application ofS1PR2 antagonists or allosteric modulators will inevitably causeadverse effects if given systemically. However, local targeting ofS1PR2 may alleviate symptoms induced by overt T cell reactions,in the context of various inflammatory, allergic, or autoimmunesyndromes.

FIGURE 4. S1PR2 inhibits CXCL12-induced migration of memory T

cell subsets. (A–C) Transwell assays of the migration of the indicated tonsil

T cell subsets assessing movement toward CXCL12 (12 ng/ml) 6 S1P

(37 nM) as indicated. Results are shown as migration index. Prior to the

addition of S1P in the lower chamber, cells were treated (or not) for 2 h in

the top chamber with JTE-013 (1000 nM). Results show the mean 6 SD

of four independent experiments with n = 3 donors. Asterisks indicate

statistical significance when comparing cell migration in the control con-

dition (S1P only) with the conditions with JTE-013. *p, 0.05, **p, 0.01

(Mann–Whitney tests).

6 HUMAN T CELL RESPONSE TO S1P

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AcknowledgmentsWe acknowledge the contribution of SFR Biosciences (UMS3444/CNRS,

ENSL, UCBL, US8/INSERM) facilities, in particular the Plateau de Biol-

ogie Experimentale de la Souris, and the flow cytometry facility.

DisclosuresThe authors have no financial conflicts of interest.

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