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Migration of Th1 CellsInduced−Inhibits Sphingosine-1-Phosphate

Preferentially Mycobacterium tuberculosisMannose-Capped Lipoarabinomannan from

Kornfeld and William W. CruikshankJillian M. Richmond, Jinhee Lee, Daniel S. Green, Hardy

http://www.jimmunol.org/content/189/12/5886doi: 10.4049/jimmunol.1103092November 2012;

2012; 189:5886-5895; Prepublished online 5J Immunol 

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The Journal of Immunology

Mannose-Capped Lipoarabinomannan from Mycobacteriumtuberculosis Preferentially Inhibits Sphingosine-1-Phosphate–Induced Migration of Th1 Cells

Jillian M. Richmond,* Jinhee Lee,† Daniel S. Green,* Hardy Kornfeld,† and

William W. Cruikshank*

Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflam-

mation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (Man-

LAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize

T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during my-

cobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the

lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant

increases in T cells, primarily CCR5+ (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse

T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM

pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2

cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002

inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration.

These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling

pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to

sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung. The Journal of Immunology,

2012, 189: 5886–5895.

Initiation of adaptive immune responses most often occurs intissue-draining lymph nodes where lymphocytes can becomeactivated and mature and acquire effector functions before

returning to the site of injury or infection. Normal T lymphocytemigration to lymph nodes requires binding to high endothelialvenules and a chemokine gradient of the CCR7 ligands CCL19 andCCL21 (1). Following interactions with APCs in the node, T cellsbecome activated and upregulate the sphingosine-1-phosphatereceptor 1 (S1P1) (2). A sphingosine-1-phosphate (S1P) concen-tration gradient then facilitates T cell egress from the nodes andtheir entrance back into the circulation, thus allowing them tomigrate to the affected tissues where they exert their effector

functions (3, 4). Enhanced migration to CCR7 ligands or abro-gation of migration to S1P would promote a disproportionateaccumulation of T cells in lymph tissues, resulting in a reductionof primed effector cells entering the circulation and tissues. Thismechanism is thought to account for the immunosuppressiveeffects of drugs that induce lymphadenopathy by down regulatingS1P1 such as FTY720, which has been shown to be therapeuticallyeffective in transplant and multiple sclerosis clinical trials (5–7).Induction of T lymphocyte responses against pulmonary My-

cobacterium tuberculosis infection are initiated in lung draininglymph nodes (8). Following activation, T cells exit the lymph nodeand migrate to the infected lung (8). However, it has been notedthat T cell responses against M. tuberculosis are delayed as com-pared with other pulmonary pathogens. Several hypotheses havebeen provided to explain this delayed T cell response, includinginhibition of APC maturation by M. tuberculosis, and altered ki-netics of leukocyte migration to and from the lung tissue (9). Ourlaboratory has previously shown that a component of the M. tuber-culosis cell wall, mannose-capped lipoarabinomannan (ManLAM),is able to direct T cell migration. Because of the growing body ofevidence that chemokine receptor cross-desensitization accountsfor another layer of regulation of leukocyte recruitment, we in-vestigated the ability of ManLAM to desensitize T lymphocytes tochemoattractants involved in migration to and from lymph tissue.In vivo infection studies in mice and humans have demonstrated

that the development of anti-ManLAM Abs appears to be bene-ficial, because the levels correlate with decreased dissemination,lower bacterial loads, prolonged survival, and better disease out-comes (10, 11). This is likely due to the prevention of immuno-modulatory effects that ManLAM can exert on host cells. DuringM. tuberculosis infection, ManLAM is secreted from infected hostmacrophages and dendritic cells in the form of lipid bodies (12).

*Pulmonary Center, Boston University School of Medicine, Boston, MA 02118; and†Department of Medicine, University of Massachusetts Medical School, Worcester,MA 01655

Received for publication October 27, 2011. Accepted for publication October 11,2012.

This work was supported by National Institutes of Health Grants RO1 HL081149(to H.K.) and RO1 CA122737 (to W.W.C.). J.M.R. was supported by a fellowshipfrom National Science Foundation Grant 0538608. All tuberculosis materials wereobtained through collaboration with Colorado State University under National Insti-tutes of Health, National Institute of Allergy and Infectious Diseases ContractHHSN266200400091C entitled “Tuberculosis Vaccine Testing and Research Mate-rials.”

Address correspondence and reprint requests to Dr. William W. Cruikshank, Pulmo-nary Center, Boston University School of Medicine, 715 Albany Street, Boston, MA02118. E-mail address: bcruiksh@bu.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: i.t., intratracheal(ly); LAM, lipoarabinomannan;ManLAM, mannose-capped lipoarabinomannan; PIM6, hexamannosylated phos-phatidylinositol; S1P, sphingosine-1-phosphate; S1P1, sphingosine-1-phosphate re-ceptor 1.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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Once secreted, ManLAM can interact with host cell surface re-ceptors such as C-type lectins or the mannose receptor (13–15).Alternatively, ManLAM can incorporate directly into lipid rafts onPBMCs (16). ManLAM’s interactions with host cell receptorsand membranes results in altered cellular signaling and responses.This is thought to be achieved through a steric inhibition mech-anism or through direct binding of host proteins to the acyl tailsof ManLAM itself, which resemble mammalian phosphatidylino-sitol-3,4,5-trisphosphate (17–19). A recent study demonstratedManLAM’s ability to inhibit CD4+ T cell activation via inhibitionof p56lck phosphorylation and signaling from the TCR (20).Furthermore, ManLAM stimulation prevents phagolysosomal fu-sion inM. tuberculosis–infected macrophages via a PI3K-dependentpathway (21, 22). Mechanistically, S1P-directed migration in en-dothelial cells and T lymphocytes has previously been shown torely upon PI3K/AKT signaling pathways (23–25).In this study, we investigated the ability of ManLAM to de-

sensitize human and mouse T lymphocytes to CCL21 and S1P-directed chemotaxis in vitro. Our data demonstrate that ManLAMexposure desensitizes both human and mouse T cells to S1P stim-ulation. We further identified a preferential effect on the CCR5+

(Th1) subset, which is considered to be a host-protective pop-ulation during M. tuberculosis infection. Intratracheal (i.t.) instil-lation of ManLAM confirmed the selective effect of ManLAM asCCR5+ cells were significantly increased in lung-draining lymphnodes as compared with CCR4+ (Th2) cells. We therefore testedthe response of in vitro-differentiated mouse Th1 and Th2 cells toS1P-directed migration with or without ManLAM pretreatmentand found that only Th1 cell migration was inhibited. We proposethat selective inhibition of Th1 cell migration by ManLAM is re-lated to differential signaling pathway induction by S1P in the Th1versus Th2 cell subset, and furthermore, these data suggest an-other mechanism by which ManLAM produced during M. tuber-culosis infection can alter the immune response.

Materials and MethodsAgs and Abs

ManLAM, anti-ManLAM Ab (CS-35, 1:250 titer), whole irradiatedH37Rv, and hexamannosylated phosphatidylinositol (PIM6) were ob-tained through the Colorado State University.

Mice

Eight- to 12-wk-old female C57BL/6J for ManLAM experiments (Jax000664) were housed at the Laboratory Animal Science Center at BostonUniversity School of Medicine or the Animal Medicine facility at Uni-versity of Massachusetts Medical School. Mice were administered food andwater ad libitum. All experiments were performed in compliance with theU.S. Department of Health and Human Services Guide for the Care and Useof Laboratory Animals and were approved by either the Boston UniversityInstitutional Animal Care and Use Committee or the University of Mas-sachusetts Medical Center Institutional Animal Care and Use Committeeand Institutional Biosafety Committee, respectively.

Isolation of human peripheral blood T cells

All human cell studies have been approved by the Boston University In-stitutional Review Board and the National Institutes of Health and wereconducted in accordance with the guidelines of the World Medical Asso-ciation’s Declaration of Helsinki. Primary human T cells from healthydonors were isolated as described previously (26). Briefly, peripheral bloodwas obtained from healthy donors and the T cells isolated from the buffycoat following Hypaque Ficoll (Amersham Biosciences) density gradientcentrifugation and nylon wool adherence (Polysciences). The T cells, 95%CD3+, were cultured in complete M199 (Life Technologies) until use.

Isolation of murine T cells

Mouse T cells were isolated from mixed spleen and lymph node prepa-rations. Cells were dissociated, and RBC lysis was performed using RBCLysing Buffer (Sigma-Aldrich) prior to selection with either nylon wool or

an Invitrogen Dynal CD4-negative bead isolation kit per the manufacturer’sprotocol. Mouse T cells were incubated in RPMI 1640 medium supple-mented with 1% BSA, HEPES, and penicillin/streptomycin prior to use inassays (Life Technologies).

In vitro M. tuberculosis infections. Bone marrow–derived macrophageswere generated through culturing bone marrows isolated from C57BL/6Jmice in complete RPMI 1640 medium containing 20% L929 culture su-pernatant for 1 wk. A total of 4 3 105 cells plated in 24-well plates wereeither left in culture (control) or infected with M. tuberculosis Erdman(Trudeau Institute Mycobacterial Culture Collection) at a multiplicity ofinfection of 5. Cell culture supernatants were collected 48 h postinfectionand were sterile filtered and frozen prior to use. Anti-ManLAM Ab (CS-35)was used to preclear either control or M. tuberculosis–infected super-natants at 4˚C overnight prior to use in chemotaxis assays. After pre-clearing, the supernatants were used to pretreat murine T cells prior to usein Transwell assays.

Migration assays

Boyden chamber assays were conducted as described previously (27).Human T cells were incubated with components from the H37Rv strain ofM. tuberculosis prior to migration induced by S1P (Avanti Polar Lipids)and CCL21 (R&D Systems). Migration was conducted for 1 h using an8 mM pore nitrocellulose filter (Neuroprobe) before the filters were fixedand H&E stained. Migration through the filter was compared with baselinemigration established with media control. Quantification was accom-plished by counting 5 high-power fields, and on average, 10–15 cells/fieldwere counted for media controls. Migration was expressed as fold overmedia control.

Transwell assays were conducted using mouse T cells from spleens andlymph nodes. Cells were incubated in RPMI 1640 medium with 1% BSA,HEPES, and penicillin/streptomycin and then suspended at 1 3 106 cells/100 ml. The cells were incubated in Transwell chambers with 5-mm poly-carbonate filters for 3.5 h prior to counting cells in the bottom chamber.Directed migration was established as more cells as compared with mediacontrol wells, which usually contained 10,000–20,000 cells. Flow cyto-metric analysis of CCR4 and CCR5 expression was performed on pooledcells that migrated to the bottom chambers. Migration was expressed asfold over media control.

Flow cytometry analysis

Cells were Fc blocked with anti-CD16/32 Abs (eBioscience, San Diego,CA) for 10 min prior to staining with selected Abs for 30 min on ice. Thefollowing anti-mouse Abs and isotype controls were purchased fromeBioscience: CD3-FITC (clone 145-2C11), CD3-PerCP-Cy5.5 (clone 145-2C11), CD4-FITC (clone GK1.5), CD8-PE (clone 53-6.7), CCR5-PE (cloneHM-CCR5 [7A4]), and CCR7-PE-Cy7 (clone 4B12). Anti-mouse CCR4-allophycocyanin (clone 2G12) and allophycocyanin Armenian hamster Igwere purchased from BioLegend. Propidium iodide was purchased fromSigma-Aldrich. Samples were run on a BD Biosciences LSR II. Data werecollected with FACSDiva software version 6.0 (BD Biosciences) and an-alyzed using FlowJo software version 7.2.2 (Tree Star, Ashland, OR).

In vivo imaging experiments

Isolated T lymphocytes were labeled with QDot 565 nanocrystals perthe manufacturer’s protocol (Invitrogen). Labeling was confirmed by flowcytometry, and 5 3 106 labeled T cells were injected via the tail vein intorecipient 8- to 12-wk-old female C57BL/6J mice. Mice were allowed torest for 1 h before PBS or 25 mg ManLAM was administered i.t. Mice wereimaged 24 h postinstillation with the Caliper Life Sciences In Vivo Imag-ing System Spectrum and Living Image software version 3.2 (Hopkinton,MA) with the following settings: excitation = 430 nm, emission = 560 nm,F stop = 1, and exposure = 30 s; image depicts counts following backgroundsubtraction.

In vivo i.t. instillation of tuberculosis Ags

Mice were i.t. instilled with the tuberculosis Ags in a volume of 100 ml.Twenty-four hours following instillation, the inguinal, cervical, mediasti-nal, and axillary lymph nodes were isolated, and cell counts were per-formed. T cell numbers were determined via flow cytometric analysis ofCD3 expression. Cells isolated from lymph nodes were also assessed forCCR4, CCR5, and CCR7 expression.

Th1/Th2 skewing

Mouse T cells were skewed using 3 mg/ml plate bound anti-CD3 (clone17A2; eBioscience) and 3 mg/ml soluble anti-CD28 (clone 37.51; eBio-science) and cultured for 3 d in RPMI 1640 medium supplemented with

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10% FCS (Atlanta Biologicals), 13 nonessential amino acids, HEPES,sodium pyruvate, 2-ME, and penicillin/streptomycin (Life Technologies).Th1 cells were cultured with 10 ng/ml IL-12 and 10 mg/ml anti–IL-4(clone 30340; R&D Systems), and Th2 cells were cultured with 10 ng/ml IL-4 and 10 mg/ml anti–IFN-g (clone H22; R&D Systems). Cells weresplit on day 3, rested in plain medium 24 h prior to harvest, and harvestedon days 7–8. Skewing was confirmed by IL-4, IL-5, and IFN-g ELISA(R&D Systems).

Western blotting

Human or mouse T cells were incubated with ManLAM or vehicle controlfor 2 h. Cells were then washed and stimulated with S1P for 5 min prior tolysis with buffer (Cell Signaling Technology) containing protease inhibitors(Roche) and phosphatase inhibitors (Sigma-Aldrich). Samples were run ona 4–12% Bis-Tris gel (Invitrogen) and transferred to polyvinylidene di-fluoride membranes (Millipore). Membranes were blocked with 4% milkin TBS with 0.1% Tween 20 and probed with the following Abs from CellSignaling Technology per the manufacturer’s protocol: phospho-AKTSer473, total AKT, phospho-p44/42 MAPK, total p44/42 MAPK, and goatanti-rabbit HRP-conjugated secondary Ab. Detection was performed withan ECL reagent kit (GE Healthcare), and films were scanned for den-sitometry analysis with ImageJ Software (National Institutes of Health).

Statistical analyses

All data are presented as the mean 6 SEM. Statistical analyses wereperformed as either Student t test or two-way ANOVA with Bonferronipost tests (Graph-Pad Prism software, version 5.0 for Windows).

ResultsHuman T cells exhibit decreased migratory responses to S1Pfollowing ManLAM treatment

To initially determine whether ManLAM could affect T lympho-cyte trafficking to or from lymph tissue, we examined the abilityof human T lymphocytes to migrate to a lymph node–specific che-mokine, CCL21, or to the lymph node egress factor S1P followingManLAM treatment. T cells were isolated from PBMCs fromhealthy human donors, pretreated with whole irradiated H37RvM. tuberculosis, ManLAM, anotherM. tuberculosis cell wall com-ponent PIM6 (16), or vehicle for 2 h, washed, and placed in Boydenchemotaxis chambers. Boyden assays were performed with humancells because of reproducibility and migratory capacity of thecells, based on prior studies and experience in our laboratory (27).S1P-induced human T cell migration, but not CCL21-induced mi-gration, was significantly inhibited following irradiated H37Rvor ManLAM treatment (*p = 0.02, **p = 0.007, optimal dosesshown; Fig. 1A, 1B; see also Supplemental Fig. 1). PIM6 stimu-lation, used as a glycolipid control, had no effect on human T cellchemotaxis following S1P stimulation (Fig. 1C). A dose titration

curve of ManLAM demonstrated that a concentration of 100 ng/ml was the lowest effective concentration of ManLAM that in-hibited S1P directed migration (Supplemental Fig. 1).

ManLAM pretreatment inhibits S1P-induced AKTphosphorylation in human T cells

To determine the mechanism for inhibition of S1P-induced mi-gration, surface expression levels of CCR7 and S1P1 were assessedvia flow cytometry following ManLAM pretreatment. It was de-termined that there was no downmodulation of receptors from thecell surface (Fig. 2A, 2B). Because ManLAM had no effect onchemokine receptor expression, we examined signaling pathwayactivation in the T cells to determine the mechanism of desensi-tization. Western blotting was performed on cell pellets that hadbeen pretreated with vehicle or ManLAM and then stimulatedwith S1P for a total of 5 min (Fig. 2C). Quantification demonstrateda 22% increase in AKT phosphorylation over baseline followingS1P stimulation, which was partially blocked by ManLAM pre-treatment (16% increase over baseline, representative blot fromtwo separate experiments). Next, we wanted to determine whetherthis phosphorylation event was biologically relevant for S1P-induced migration. A recent study demonstrated that S1P/S1P1signaling in T cells affected regulatory T cell development and sup-pressive functions through a PI3K/AKT-dependent migration path-way (23). Another study demonstrated that AKT-mediated phosphory-lation of S1P1 is required for endothelial cell migration toward S1P(24). Treatment with the PI3K/AKT inhibitor Ly294002 blockednaive human T cell migration to S1P (*p = 0.05; Fig. 2D).

ManLAM treatment induces decreased mouse T cell migratoryresponses to S1P

To determine whether the effects of ManLAM could be investi-gated in a murine model, we next examined the ability of ManLAMto affect in vitro migration of mouse T cells stimulated by eitherCCL21 or S1P. T lymphocytes were isolated from the spleen andlymph nodes of 8- to 12-wk-old female C57BL/6J mice and werepretreated with whole irradiated H37Rv M. tuberculosis for 2 hprior to use in Transwell chemotaxis assays (28). As with humancells, mouse naive T cells that had been treated with irradiatedH37Rv or ManLAM demonstrated no significant difference intheir responsiveness to CCL21 but did exhibit a significant de-crease in their response to S1P as compared with vehicle treatedcells (*p = 0.03 and 0.02, respectively; Fig. 3A, 3B). The sameconcentration of PIM6, another M. tuberculosis glycolipid, had noeffect on S1P- or CCL21-induced migratory responses (Fig. 3C).

FIGURE 1. ManLAM pretreatment desensitizes naive human T cell migration to S1P. T cells from healthy donors were treated with 1 mg/ml whole

irradiated H37Rv (A), 100 ng/ml ManLAM (B), or 100 ng/ml PIM6 (C) for 2 h before use in Boyden chamber assays for media control, S1P (10 nM), or

CCL21 (50 ng/ml). Migrating cells were fixed and stained, and the number of T cells counted at 3400 magnification from at least 10 fields was normalized

to media control. irH37Rv inhibited S1P-directed migration but not CCL21-directed migration (*p = 0.02; Student t test). (B) Pretreatment with 100 ng/ml

ManLAM also blocks S1P-directed migration (**p = 0.007; Student t test). Data in (A) and (B) were obtained from three donors assayed in duplicate (mean6SEM). (C) A dose of 100 ng/ml PIM6 had no effect on human T lymphocyte migration induced by S1P (n = 2 donors assayed in duplicate, mean 6 SEM).

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To further test the specificity of the effect of ManLAM on S1P-induced migration, we examined the ability of an anti-ManLAMAb to potentially neutralize the ManLAM effect. For these stud-ies, T cell migration to S1P was assessed following pretreatmentwith culture supernatants from M. tuberculosis Erdman–infectedmouse bone marrow–derived macrophages. Bone marrow–derivedmacrophages were cultured (control) or infected with a multiplic-ity of infection of 5 M. tuberculosis Erdman (MTB sup) for 48hprior to harvest of supernatants. Supernatants were precleared withCS-35 anti-ManLAM Ab overnight at 4˚C, before pretreatingmouse T cells for 1.5 h at 37˚C. T cells were then washed andsubjected to a Transwell assay. CS-35 alone did not affect mouseT cell migration to S1P (Fig. 3D). As expected, M. tuberculosissupernatant blocked S1P-directed migration; however, supernatantthat had been precleared to remove ManLAM had no effect (*p =0.05; Fig. 3D).To determine whether AKT was also involved in mouse T cell

migratory responses to S1P, Western blots were performed as inFig. 2C. As with human T cells, ManLAM pretreatment inhibitedAKT phosphorylation in mouse T cells (representative blot fromtwo separate experiments; Fig. 3E). Ly294002 treatment blockedmouse T cell S1P directed migration, indicating that PI3K/AKTactivation is a necessary pathway for S1P chemotaxis in naivemouse T cells (*p = 0.02; Fig. 3D).

In vivo instillation of ManLAM in C57BL/6J mice results inretention of T cells in lung draining lymph nodes

On the basis of the in vitro data, we hypothesized that ManLAMinhibition of S1P-directed migration would result in trapping ofT cells in lymph nodes. Therefore, we investigated the ability of

ManLAM to alter in vivo T cell homing patterns in mice. T cellsfrom lymph nodes and spleens of donor mice were labeled withInvitrogen 565 QDot nanocrystals. Five million labeled cells wereinjected via the tail vein into 8- to 12-wk-old recipient femaleC57BL/6J mice and were tracked using whole-body imaging. After1 h, the mice were i.t. administered ManLAM or vehicle. A dose of25 mg ManLAM was chosen to correspond to doses used in mouseAg challenge models and to correspond with levels of ManLAMdetected in sputum during live M. tuberculosis infection (29, 30).Homing patterns and lymph node accumulation of labeled cells24 h postinstillation were determined with the In Vivo ImagingSystem Spectrum. On the basis of these heat map images, it wasdetermined that ManLAM instilled i.t. induced accumulation ofT cells in areas of the chest, which likely corresponded with themediastinal and cervical lymph nodes (Fig. 4A). To more accu-rately localize the recruited cells, mice were sacrificed, and thelymph nodes were excised. The cervical and mediastinal lymphnodes were visibly enlarged, whereas the inguinal and axillarynodes had no detectable difference (Fig. 4B).To more accurately quantify cell numbers and to assess the rate

of accumulation, T cell kinetics studies were performed to deter-mine onset and extent of the effect of a single bolus of ManLAM onT cell homing to secondary lymph tissue. Inguinal, axillary, cer-vical, and mediastinal lymph nodes were harvested from mice 12,24, and 48 h following instillation of ManLAM or vehicle. TotalT cell numbers were determined via CD3 staining, and maximalaccumulation was observed at 24 h postinstillation (only the 24-htime point is shown; Fig. 4C). There was a 3-fold increase inT lymphocytes in the cervical lymph nodes and a 2-fold increasein T cells in mediastinal lymph nodes of mice that received

FIGURE 2. Naive human T cell migration to S1P requires intact AKT signaling. (A) Cells pretreated with ManLAM do not modulate surface CCR7 or

S1P1 expression as determined by flow cytometry (gray filled histogram, isotype control; black, PBS treated; gray, ManLAM treated; representative image).

(B) Quantification of surface receptor expression as in (A) (n = 3 donors assayed in duplicate, mean6 SEM). (C) Western blotting analysis of human T cells

pretreated with ManLAM or PBS for 2 h and then stimulated with S1P or vehicle for 5 min. Percent-phosphorylated AKT was determined by creating

a ratio of densitometry of phospho-AKT Ser473 to total AKT (representative blot; n = 2 separate experiments with three donors). (D) Pretreatment with

10 mM Ly294002 for 1 h inhibits human T cell migration to S1P (*p = 0.05; Student t test, n = 2 donors assayed in duplicate, mean 6 SEM).

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ManLAM as compared with mice that received vehicle 24 h fol-lowing treatment (*p = 0.04 cervical and 0.03 mediastinal, re-spectively; Fig. 4C). In contrast, there was no significant dif-ference in the number of cells present in either the inguinal oraxillary lymph nodes (Fig. 4C), suggesting that i.t. administrationof ManLAM altered homing patterns of cells only within lymphnodes that directly drain the lungs. Cell numbers from animalstreated with PIM6 were not significantly different from vehicle-treated animals (Fig. 4D). In addition, mice were instilled withLPS. LPS is a likely contaminant, and it has been shown to haveeffects on recruitment and or activation of immune cells in thelungs (31, 32). As shown in Fig. 4E, instillation of LPS induceda slight decrease in the number of T cells in lung-draining lymphnodes, although this was not statistically significant.The increase in T cell numbers in the lymph nodes could be a

result of increased proliferation. To ascertain whether the increasein T cells in lung-draining lymph nodes following ManLAM in-stillation was as a result of T cell proliferation, cell samples fromManLAM and vehicle-treated mice were stained with propidiumiodide and were subjected to cell cycle analysis. As shown in Fig.4F, there was no difference in proliferative profiles of cells isolated

from vehicle or ManLAM-treated animals, indicating that Man-LAM was not inducing proliferation of these cell populations.These data agree with other published studies demonstrating thatManLAM does not have a proliferative effect and in many casesinhibits T cell proliferation (33).

LAMs from other mycobacterial species

We also tested LAMs from other mycobacterial strains to determinewhether the effects we observed were unique to M. tuberculosis.Mycobacterium smegmatis LAM was assayed and found to inhibitT cell migration to both S1P and CCL21 (Supplemental Fig. 2A).Similar to ManLAM, LAM from Mycobacterium leprae inhibitedT cell migration to S1P to the same extent as M. tuberculosisManLAM (Supplemental Fig. 2A) as well as having no effect onCCL21-induced migration.We also tested LAM from M. leprae and M. smegmatis in the

in vivo i.t. model. LAM from M. smegmatis was unable to inducean effect on the number of T cells in the lymph nodes (Supple-mental Fig. 2C), whereas LAM from M. leprae did induce a sig-nificant increase in cervical lymph node T cells, but the effect wasnot as large as that seen with M. tuberculosis ManLAM (*p =

FIGURE 3. Migratory responses of mouse T lymphocytes after exposure to tuberculosis Ags. Mouse T cells were preincubated with 1 mg/ml

whole irradiated H37Rv (A), 100 ng/ml ManLAM (B), or 100 ng/ml PIM6 (C) for 2 h. Cells were washed and placed in Transwell chambers and

stimulated for migration induced by media control, S1P (10 nM), or CCL21 (50 ng/ml). The assay was terminated after 3.5 h, and migration was

expressed as fold change over baseline (media control expressed as 1). Data in (A)–(C) are from three independent experiments performed in triplicate

(mean 6 SEM; *p = 0.03, *p = 0.02; Student t test). (D) Culture supernatants from control or M. tuberculosis-infected mouse bone marrow–derived

macrophages were harvested 48 h postinfection. Supernatants were precleared with CS35 anti-ManLAM Abs at 4˚C overnight. Primary mouse T cells were

pretreated with the supernatants for 1.5 h prior to Transwell assay. The data depicted shows migration over baseline to S1P at 10 nM. (Duplicate wells from

two separate experiments; mean 6 SEM;*p = 0.04; Student t test). (E) Western blotting analysis of mouse T cells pretreated with ManLAM or PBS for 2 h

and then stimulated with S1P or vehicle for 5 min. Percent-phosphorylated AKTwas determined by creating a ratio of densitometry of phospho-AKT Ser473

to total AKT. (Representative blot, n = 2 separate experiments). (F) Mouse T lymphocytes were pretreated with 50 mM of the PI3K/AKT inhibitor

Ly294002 for 1 h prior to Transwell migration. S1P migration was significantly inhibited, whereas migration to CCL21 was not significantly affected (n = 3

experiments performed in triplicate;*p = 0.02; Student t test).

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0.04; Supplemental Fig. 2D, 2-fold increase in cervical lymphnode T cells following M. leprae LAM treatment versus up to 4-fold increase in cervical lymph node T cells following M. tuber-culosis LAM treatment). Although M. smegmatis LAM was ca-pable of inhibiting S1P-induced migration, its inhibitory effecton CCL21-induced migration may have reduced the number ofT cells in vivo that were recruited to the lymph nodes following i.t.administration. Therefore, the equal numbers of T cells may resultfrom the balance of both a net reduction of newly recruited cellsand egressing cells, thereby resulting in the little increase in de-tectable lymphadenopathy.

ManLAM preferentially inhibits CCR5+T cell migration to S1Pin vitro and in vivo

To better understand the inhibitory effect of ManLAM, we assessedthe phenotype of lymph node–sequestered cells following in vivoManLAM treatment. Cells were dissociated from lymph tissue ofmice that had been treated i.t. with PBS or ManLAM for 24 h andwere assessed for expression of CCR4 (predominantly expressedby Th2 cells) and CCR5 (predominantly expressed by Th1 cells)by flow cytometry. The ratios of CCR5+ to CCR4+ (Th1:Th2) cellsisolated from cervical and mediastinal lymph nodes of mice thathad received ManLAM were higher than that of mice that receivedvehicle, with statistically significant increases in total CCR5+

but not CCR4+ cell numbers accumulating in lymph nodes (*p =0.007 and 0.005, Fig. 5A, 5B; see also Supplemental Fig. 2D, 2E).These data indicate that ManLAM exposure in the lung results inpreferential sequestration of CCR5+ cells in lung-draining lymphnodes.To determine whether changes in the percentage of CCR5+ cells

could be attributed exclusively to altered migration patterns orwhether ManLAM stimulation could induce alteration in surfaceexpression of CCR4 or CCR5, T cells were incubated in vitro withManLAM for 2, 5.5, and 24 h prior to surface receptor analysis(these time points correspond with pretreatment, migration assays,

and in vivo ManLAM administration, respectively). For all con-centrations and time points investigated, there was no significantchange in expression of CCR4 or CCR5 (representative datashown; Fig. 5C). These findings indicate that the significant re-duction in CCR5+ cell numbers responding to S1P was due todirect effects on migration and not on the alteration of CCR5expression.We also examined total cell numbers (Supplemental Fig. 2B, 2C)

and ratios of CCR5+ to CCR4+ (Th1 to Th2) cells (SupplementalFig. 2D, 2E) from mice that had been administered LAM from M.smegmatis or M. leprae and found no significant increases. Thisindicates that the preferential sequestration of CCR5+ (Th1) cellsin lymph nodes is a selective effect for M. tuberculosis ManLAM.

Th1 and Th2 cells require different signaling pathways tomigrate toward S1P

The in vivo migration studies indicated that ManLAM was havinga greater effect on CCR5+ than CCR4+ cell S1P migration. Tomore directly investigate this, we analyzed S1P-directed migrationin Th1 versus Th2 cell populations. Naive cells were skewedin vitro under Th1 or Th2 polarizing conditions, and subset puritywas confirmed using Th1/Th2 cytokine analysis by ELISA. Skewedcell populations were pretreated with ManLAM or vehicle, washed,and placed in Transwell chambers. ManLAM pretreatment was ableto inhibit Th1 cell migration to S1P but not Th2 cell migration (**p =0.009; Fig. 6A). These data are consistent with reports showing thatManLAM stimulation can enhance Th2 cytokine production butdiminish Th1 cytokine production (34).ManLAM has been shown to preferentially desensitize the AKT

pathway (18, 19), thereby providing a potential mechanism forthe selective inhibitory effect of ManLAM on Th1 cell migration.To examine differences in AKT pathway activation, Western blot-ting was performed on cell pellets of Th1 and Th2 cells that hadbeen pretreated with PBS or ManLAM, followed by stimulationwith S1P (representative blots shown with average percentage of

FIGURE 4. In vivo instillation of MTB ManLAM results in retention of T cells in lung draining lymph nodes. (A) In vivo imaging of 5 3 106 QDot 565-

labeled T lymphocytes 24 h after i.t. administration of PBS (left) or 25 mg ManLAM (right) in 8- to 12-wk-old female C57BL/6J mice (representative

image, n = 3 per group). (B) 340 image of inguinal, axillary, cervical, and mediastinal lymph nodes isolated from PBS-treated (left) or ManLAM-treated

(right) mice 24 h postinstillation (representative images from two experiments). (C) Average number of CD3+ T cells per lymph node 24 h postinstillation;

n = 6 mice/group from two separate experiments (mean 6 SEM; *p = 0.04 cervical and *p = 0.03 mediastinal, respectively; Student t test). PIM6 (D) or

LPS (E) treatment does not induce sequestration-associated lymphadenopathy 24 h postinstillation; n = 4 or 5 mice/group from two separate experiments

(mean 6 SEM). (F) Cell cycle analysis of the T cells from the cervical lymph nodes of PBS (solid)- or ManLAM (dashed)-treated mice as determined

by propidium iodide staining. The lack of change from control mice indicates that the cells in the lung draining lymph nodes following ManLAM treatment

do not have an increased proliferative index (representative plot from two independent experiments).

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phosphorylated protein from two separate experiments; Fig. 6B,6C). Strikingly, Th1 cells exhibited basal phospho-AKT, whereasTh2 cells exhibited basal phospho-ERK. ManLAM pretreatmentalone induced an increase in phospho-AKT over baseline but

prevented cells from achieving the same extent of AKT phos-phorylation as control cells stimulated with S1P (Fig. 6B). Con-versely, ManLAM pretreatment resulted in enhanced ERK phos-phorylation as compared with S1P stimulation alone in Th2 cells,

FIGURE 5. ManLAM preferentially sequesters

CCR5+ (Th1) cells in lymph nodes. (A) CCR5:CCR4

numbers of T lymphocytes isolated from cervical

lymph nodes of mice that received i.t. ManLAM or

PBS. Average Th1:Th2 ratio was 1.6:1 for PBS treat-

ment and 2.62:1 for ManLAM treatment; n = 7 mice/

group (mean 6 SEM; **p = 0.007; Student t test). (B)

CCR5:CCR4 numbers of T cells from mediastinal

lymph nodes. Average Th1:Th2 ratio was 1.21:1 for

PBS treatment and 1.42:1 for ManLAM treatment, n =

3 mice/group (mean 6 SEM; **p = 0.005; Student

t test). (C) ManLAM treatment does not alter chemo-

kine receptor expression. Mouse T cells were incu-

bated in vitro with ManLAM for 24 h and analyzed via

flow cytometry. There was no difference between PBS

(black) and ManLAM (gray) histograms (representa-

tive from two separate experiments; filled gray histo-

gram, isotype).

FIGURE 6. ManLAM inhibits Th1, but not Th2, cell migration induced by S1P because of differential signaling requirements. (A) Spleen and lymph

node T cells were isolated from 7- to 12-wk-old C57BL/6J female mice and skewed in vitro before treatment and Transwell assay. There was a statistically

significant decrease in Th1 cell migration to 10 nM S1P following 100 ng/ml ManLAM treatment for 2 h, and no significant difference in Th2 cell migration

(n = 3 experiments performed in triplicate; mean 6 SEM; **p = 0.009; Student t test). (B and C) Western blotting analysis of Th1 and Th2 cells following

ManLAM pretreatment and S1P stimulation for phosphorylated AKT (B) or phosphorylated ERK (C). Insets depict percent-phosphorylated protein av-

eraged from blots from two separate experiments. Th1 (D) and Th2 (E) cell migration to 10 nM S1P following a 1-h treatment with DMSO vehicle, 10 mM

PI3K inhibitor Ly294002, or 10 mM MEK1/2 inhibitor U0126. S1P-directed Th1 cell migration was inhibited by Ly294002 and S1P-directed Th2 cell

migration was inhibited by U0126 pretreatment (n = 3 separate experiments performed in triplicate; mean 6 SEM; *p = 0.02, **p = 0.005; Student t test).

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indicating that the cells were still responsive to the stimulus fol-lowing ManLAM pretreatment (Fig. 6C).We next investigated the functionality of the AKT and ERK

pathways, which have both been shown to be important for T cellchemotaxis, in Th1 and Th2 cell migration induced by S1P. Asshown in Fig. 6D, 6E, the AKT inhibitor Ly294002 significantlyinhibited Th1 migration to S1P, whereas the ERK inhibitor U0126significantly inhibited Th2 migration to S1P, with neither inhibitorsignificantly affecting the migratory response of the other T cellsubset (*p = 0.02 and **p = 0.005). Taken together, these dataindicate that S1P-induced migration of Th1 cells requires activa-tion of the AKT pathway, whereas S1P-induced migration of Th2cells is primarily dependent on the ERK pathway. Furthermore,M.tuberculosis ManLAM preferentially affects the AKT pathway,thereby inhibiting S1P-induced migration of Th1 cells.

DiscussionIn this study, we demonstrate that a M. tuberculosis virulencefactor, ManLAM, is capable of inhibiting T cell migration to thelymph tissue egress signal S1P. This was accomplished by inter-ference with PI3K/AKT pathway activation. Th1 populations werepreferentially affected because of their dependence on AKT acti-vation for S1P-induced migration. It is possible that the effect ofManLAM on T cells could delay and potentially reduce initiationof Th1 effector responses in lung tissue, where even transientdelays in initiation of adaptive immunity could contribute to long-term impairment for control of infection (8, 35, 36). Furthermore,these findings demonstrate a potential mechanism by which M.tuberculosis could foster a favorable infection environment andsuggest that therapeutics that target ManLAM, such as benzo-thiazinones, may significantly alter Th1 cell skewing and re-cruitment to the lung, thereby reducing the bacterial burden (37).We have identified that naive and Th1 cell S1P-induced mi-

gration requires the AKT pathway and that ManLAM pretreatmentis able to inhibit S1P-directed migration of these populations.The selective effect of ManLAM on Th1 cells seems to rely on theirpropensity to use the AKT pathway as opposed to the ERKpathway: we found that Th1 cells have basal levels of phospho-AKT that are absent in Th2 populations, and conversely, Th2cells have basal phospho-ERK that is absent in Th1 populations.These data corroborate previous studies that have shown distinctsignaling events in human Th1 and Th2 cells (38) and that AKTand ERK contribute to Th1 and Th2 differentiation, respectively(39, 40). Additional studies are required to examine in more detailthe mechanisms by which ManLAM desensitizes the AKT path-way.A number of chemoattractants have been reported to induce

desensitization of cells to subsequent migratory stimuli. Examplesof this would be cross-desensitization of CCR5 by IL-16 and MIP-1b (41), CD4-induced desensitization of CXCR3 and CCR5 fol-lowing IL-16 stimulation (42), and desensitization of CXCR2 andC5aR by fMLP stimulation (43, 44). ManLAM has previouslybeen shown to induce T cell migration (45), and we now dem-onstrate that pretreatment with ManLAM renders T cells refrac-tory to S1P stimulation. Most of the chemokine receptor cross-desensitization studies involved receptor mediated desensitization,and ManLAM has been shown to interact with some cell surfacereceptors including C-type lectins and the mannose receptor (13–15). Future studies could determine whether ManLAM interactswith a receptor on the surface of T cells that could mediate thiseffect. Another potential mechanism is the direct incorporation ofManLAM into the membranes of PBMCs: Ilangumaran et al. (16)showed that ManLAM preferentially incorporates into lipid raftswhere AKTactivation following S1P1 stimulation normally occurs.

Our data suggest that ManLAM can selectively inhibit the AKTpathway without abrogating ERK signaling. ERK signaling hasbeen shown to be caveolae independent and therefore potentiallyless affected by membrane integration by ManLAM (46). Thisdifference may explain why ManLAM preferentially inhibits thePI3K pathway; however, additional studies are required to deter-mine the mechanism by which ManLAM binds to T cells andinitiates the inhibitory effect on S1P receptor signaling.Our studies did not address effects of ManLAM on other S1P

receptors expressed by other immune cells. Of note, there are fivedifferent S1P receptors that are differentially expressed on immunecells (47). Our studies focused on T lymphocytes (48), whichexpress S1P1 and S1P4 as do B lymphocytes; however, mast cells,macrophages, and dendritic cells express S1P2 and ManLAMstimulation either through membrane integration or receptor in-teraction could affect cellular responses. Further examinationof this phenotype could reveal preferential use of different S1Preceptors on different leukocytes (49). It has also been shown thatMTB inhibits sphingosine kinase activity, the enzyme responsiblefor production of S1P (50, 51). It is possible that reduced pro-duction of S1P in vivo, a consequence of MTB infection, couldcontribute to the phenotype we observe.Previous studies have shown that 100 ng/ml ManLAM rep-

resents the lower end of a dose response curve used to inhibitCD4+ T cell activation and cytokine secretion (20, 34). In ad-dition, human M. tuberculosis patient sputum has been shownto contain 1 ng/ml to 1 mg/ml ManLAM (30), although the preciseconcentration in lymph nodes is unknown. It has been reportedthat ManLAM incorporates into lipid rafts on the surface of PBMCsas rapidly as 30 min, with maximal incorporation occurring withinseveral hours (16). Concentrations and time points used in thisstudy correspond well with these studies, supporting the conceptthat ManLAM can alter the immune response in the nanogramrange and within a short period of time. During the course of alive infection, it is likely that ManLAM is constantly secretedby infected macrophages, thus potentiating the sequestration ofcells for an extended period of time. The spread of infection to thelymph nodes and subsequent ManLAM secretion within the con-fines of the lymph tissue could further contribute to inhibition ofTh1 cell egress with reduced immune responses in the lung. In-terestingly, M. tuberculosis–infected CCR52/2 mice demonstrateincreased lymphocytic infiltrates in their lungs and increasedproinflammatory cytokine production (52), which would be con-sistent with ManLAM’s ability to efficiently and selectively in-hibit CCR5+/Th1 cell subsets but not CCR4+/Th2 subsets.There is evidence that indicates a shift in Th1/Th2 cytokines

during M. tuberculosis infection. Infected mice initially produceboth IFN-g and IL-4 in the lungs, but IFN-g levels decrease,whereas IL-4 levels increase over the course of infection (53).This finding is consistent with our data indicating that ManLAMproduction would alter normal Th1 cell homing patterns. Man-LAM has also been shown to promote secretion of Th2-relatedcytokines while simultaneously decreasing release of Th1 cyto-kines from T lymphocytes (34). As IFN-g production by Th1 cellsis considered to be host-protective, our data would support thehypothesis that shedding of ManLAM during M. tuberculosis in-fection promotes an environment that is more favorable for in-fection and suggests further that antibiotics targeting ManLAM,such as benzothiazinones, may significantly alter Th1 cell skewingin the lung thereby reducing the bacterial burden (37). It is likelythat live MTB produce other factors that are immunoregulatory.Our data indicate that shedding of ManLAM represents a majorcomponent for induction of lymphadenopathy with selectivity forCCR5+ T cells; however, additional studies are required to more

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fully understand how bacterial products can modify the immuneresponse and promote the infection process.

AcknowledgmentsWe thank Nathalie Ayahi for assistance with Th1/Th2 skewing methods,

Matthew Jones for assistance with Western blotting methods, Matthew

Blahna for assistance with propidium iodide staining methods, Marina

Tuzova for assistance with cell culture, and Elizabeth Duffy for assistance

with ELISAs for assessing Th1/Th2 skewing. All flow cytometric data were

acquired using equipment maintained by the Boston University Medical

Campus and University of Massachusetts Worcester Flow Cytometry Core

Facilities. Whole-body animal imaging was acquired using equipment

maintained by the Boston University Medical Campus Animal Imaging

Core Facilities.

DisclosuresThe authors have no financial conflicts of interest.

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