Mycobacterium tuberculosis Cell Wall Glycolipids Directly ...identiﬁed that M. tuberculosis cell...

INFECTION AND IMMUNITY, Oct. 2009, p. 4574–4583 Vol. 77, No. 100019-9567/09/$08.00�0 doi:10.1128/IAI.00222-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis Cell Wall Glycolipids Directly InhibitCD4� T-Cell Activation by Interfering with Proximal

T-Cell-Receptor Signaling�

Robert N. Mahon,1* Roxana E. Rojas,2 Scott A. Fulton,2 Jennifer L. Franko,1,2

Clifford V. Harding,1† and W. Henry Boom1,2,3†Department of Pathology, Case Western Reserve University and University Hospitals, Case Medical Center, Cleveland, Ohio 441061;

Department of Medicine, Case Western Reserve University and University Hospitals, Case Medical Center, Cleveland,Ohio 441062; and Tuberculosis Research Unit, Case Western Reserve University and University Hospitals,

Case Medical Center, Cleveland, Ohio 441063

Received 25 February 2009/Returned for modification 14 May 2009/Accepted 29 July 2009

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust adaptive CD4� T-cellresponses. We have previously shown that M. tuberculosis can indirectly inhibit CD4� T cells by suppressing themajor histocompatibility complex class II antigen-presenting cell function of macrophages. This study was under-taken to determine if M. tuberculosis could directly inhibit CD4� T-cell activation. Murine CD4� T cells werepurified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4� T cells wereactivated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence of M. tuberculosis andits subcellular fractions. CD4� T-cell activation was measured by interleukin 2 production, proliferation, andexpression of activation markers, all of which were decreased in the presence of M. tuberculosis. Fractionationidentified that M. tuberculosis cell wall glycolipids, specifically, phosphatidylinositol mannoside and mannose-capped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-likereceptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin.ZAP-70 phosphorylation was decreased in the presence of M. tuberculosis glycolipids, indicating that M. tuberculosisglycolipids directly inhibited CD4� T-cell activation by interfering with proximal T-cell-receptor signaling.

Aerosolized Mycobacterium tuberculosis infects alveolar andlung parenchymal macrophages, where it replicates unre-strained in the face of innate responses until T-cell immunitycontrols its growth. Despite robust activation of innate andadaptive immunity, M. tuberculosis survives and persists as alatent infection (15, 18). CD4� T cells have a central role incontrolling M. tuberculosis during acute and latent infections(53). Animal studies have shown that depletion or absence ofCD4� T cells during primary infection results in unchecked M.tuberculosis growth in the lung and decreased survival (37, 38,41). Depletion of CD4� T cells during latent infection alsoworsens disease and survival (46). In humans, loss of CD4� Tcells from progressive human immunodeficiency virus infectionis directly responsible for the high rates of tuberculosis inhuman immunodeficiency virus-infected persons (48).

Much is known about how M. tuberculosis manipulates mac-rophages for its survival (19, 29, 44). However, the way inwhich M. tuberculosis interferes with adaptive T-cell immunityis not well understood. Our recent studies have demonstratedthat M. tuberculosis can modulate CD4� T-cell function bothindirectly and directly. M. tuberculosis, through Toll-like recep-tor 2 (TLR-2), inhibits gamma-interferon-regulated genes that

result in decreased major histocompatibility complex class II(MHC-II) antigen processing by macrophages for effector andmemory CD4� T cells (22, 23, 40, 43). M. tuberculosis can alsoinduce increased adhesion to fibronectin through �5�1 integrinon CD4� T cells (45).

M. tuberculosis molecules responsible for modulating CD4�

T-cell function reside in the mycobacterial cell wall and includethe lipoproteins LpqH, LprG, and LprA as well as the glyco-lipid phosphatidylinositol mannoside (PIM). The lipoproteinsbind to TLR-2 on macrophages, and PIM binds to VLA-5(�5�1) on CD4� T cells. The M. tuberculosis cell wall alsocontains lipoarabinomannan (LAM); complex lipids, such asphthiocerol dimycocerosate, and cord factor/dimycolytreha-lose; and sulfolipids that are both targets of the immune re-sponse (e.g., glycolipids presented by CD1 to T cells) andagonists of host cell receptors (9, 10, 32). Although M. tuber-culosis bacilli largely reside within macrophages, mycobacterialcell wall components, including glycolipids, can traffic outsideinfected macrophages through the production of exosomes.These exosomes can then deliver M. tuberculosis molecules toT cells and other host cells that are not directly interacting withM. tuberculosis-infected cells and thereby affect host immuneresponses (3, 4).

The purpose of this study was to determine if M. tuberculosiscan directly (i.e., independently from its effect on MHC-IIantigen processing) interfere with CD4� T-cell activation and,if so, what the M. tuberculosis molecules(s) and mechanism(s)are. Highly purified murine CD4� T cells devoid of antigen-presenting cells (APCs) were activated by CD3 and CD28monoclonal antibodies (MAbs) in the presence or absence of

* Corresponding author. Mailing address: Division of InfectiousDiseases, Department of Medicine, Case Western Reserve Universityand University Hospitals, Case Medical Center, 10900 Euclid Avenue,BRB 1010B, Cleveland, OH 44106-4984. Phone: (216) 368-4855. Fax:(216) 368-2034. E-mail: [email protected].

† W. Henry Boom and Clifford V. Harding share senior authorship.� Published ahead of print on 3 August 2009.

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M. tuberculosis and biochemical fractions. We have found thatM. tuberculosis bacilli directly inhibited CD4� T-cell activation.Biochemical fractionation identified cell wall glycolipids as po-tent inhibitors of signaling through the T-cell receptor (TCR)complex by interfering with ZAP-70 phosphorylation.

MATERIALS AND METHODS

Mice. Eight- to 10-week-old female C57BL/6 mice were purchased fromCharles River Laboratories (Wilmington, MA). D011.10 TCR transgenic micethat express TCRs specific for the OVA323–339 peptide presented in the contextof I-Ad (39) were a gift from Alan Levine (Case Western Reserve University,Cleveland, OH). TLR2�/� and MyD88�/� mice were generously provided byShizuo Akira (Research Institute for Microbial Disease, Osaka University,Osaka, Japan) and were backcrossed to C57BL/6 mice a minimum of eight times.Mice were housed under specific-pathogen-free conditions. Studies were ap-proved by the Institutional Animal Care and Use Committee at Case WesternReserve University.

Antibodies and reagents. The following MAbs were purchased for analysis ofsurface marker expression: phycoerythrin (PE)-conjugated CD3ε (145-2C11),fluorescein isothiocyanate-conjugated CD4 (GK1.5) from BD Biosciences (SanJose, CA), APC-conjugated CD28 (37.1), PE-conjugated interleukin 2R� (IL-2R�; PC61.5), PE-Cy7-conjugated CD62L (MEL-14) from eBioscience (SanDiego, CA), PeCy5-conjugated CD69 (H1.2F3), PE-conjugated CD44 (IM7.8.1)from Invitrogen (Carlsbad, CA), and conjugated isotype controls. For T-cellactivation, hamster anti-mouse CD3ε (145-2C11), anti-mouse CD28(L3T4), andmouse anti-hamster secondary immunoglobulin G1 (IgG1) were purchased fromBD Biosciences. Rabbit MAbs against ZAP-70 and phosphorylated ZAP-70(Tyr319) were purchased from Cell Signaling Technologies (Danvers, MA).Horseradish peroxidase (HRP)-conjugated secondary MAb (Jackson Immu-noResearch, West Grove, PA) was used for detection. Mannose-capped LAM(ManLAM) and PIM from M. tuberculosis H37Rv were obtained through theTuberculosis Vaccine Testing and Research Materials Contract (NIAIDHHSN266200400091C) at Colorado State University.

Culture and fractionation of M. tuberculosis. M. tuberculosis H37Ra (AmericanType Culture Collection, Manassas, VA) was grown to log phase in Middlebrook7H9 medium (Difco, Detroit, MI) with albumin, dextrose, and catalase enrich-ments (Difco); harvested; and frozen at �80°C as previously described (40).Bacterial titer was determined by counting CFU on 7H10 medium (Difco). M.tuberculosis lysate was prepared by passing bacterial pellets resuspended indeionized water containing 7.5 mM EDTA, 0.7 �g/ml leupeptin (Sigma, St.Louis, MO), 0.2 mM phenylmethylsulfonyl fluoride (Sigma), 0.7 �g/ml pepstatinA (Sigma), 10 U/ml DNase (Sigma), and 25 U/ml RNase (Sigma) through aFrench press four times (40). Intact cells and cellular particulate were removedby centrifuging the suspension for 15 min at 3,000 rpm. Protein concentrationswere estimated by bicinchoninic acid protein assay (Thermo Scientific, Rockford,IL). Lysate was stored at �80°C.

Purification of glycolipids from M. tuberculosis H37Ra was performed using amodified protocol previously described (22, 23, 43). M. tuberculosis lysate wasextracted with 6% Triton X-114 (Sigma). The detergent layer was washed threetimes with Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.5) and oncewith cold water and then precipitated by overnight incubation at �20°C with coldacetone (Fischer Scientific, Pittsburgh, PA). The pellet was washed once withcold acetone and resuspended in phosphate-buffered saline (PBS). Acetoneprecipitate was then mixed with an equal volume of PBS-saturated phenol(Fischer Scientific) and vigorously shaken overnight at room temperature. Themixture was then centrifuged at 15,000 � g for 30 min and the aqueous layerremoved and replaced with fresh PBS. Following multiple washes with PBS, theorganic phase was mixed with PBS and vigorously shaken overnight. The aque-ous layer was once again removed and the organic phase washed multiple timeswith PBS. The organic phase was dialyzed (1-kDa cellulose acetate dialysismembrane; Fischer Scientific) against running water for 48 h. After dialysis, thesupernatant was lyophilized, weighed, reconstituted in PBS, stored at �80°C, anddesignated M. tuberculosis glycolipids.

Purification of CD4� T cells. Unless otherwise specified, all experiments wereperformed at 37°C in a 5% CO2 atmosphere and in serum-free HL-1 medium(BioWhittaker, East Rutherford, NJ) supplemented with 1 �M 2-mercaptoeth-anol, 10 mM HEPES buffer, nonessential amino acids, 2 mM L-glutamine, 100 �gof streptomycin, and 100 U of penicillin (complete HL-1; BioWhittaker). Spleno-cytes from 8- to 10-week-old C57BL/6 mice were isolated, and red blood cellswere lysed in hypotonic lysis buffer (10 mM Tris-HCl and 0.83% ammoniumchloride). Splenocytes were plated in 100-mm tissue culture plates and allowed

to adhere for 1 h at 37°C. Untouched CD4� T cells were purified from nonad-herent splenocytes using a CD4� T-cell-negative isolation kit (Miltenyi Biotec,Germany) by following the manufacturer’s instructions. The purity of CD4� Tcells was confirmed by flow cytometry and ranged between 88 and 95%. CD4� Tcells then were further purified by staining with anti-CD4 monoclonal antibodyand positive selection through fluorescence-activated cell sorting using the BDAria fluorescence-activated cell sorting system (BD Biosciences). The resultingCD4� T-cell populations were 98 to 99% pure. For purification of naïve CD4�

T cells, negatively selected CD4� T cells were positively selected for CD4 andthen sorted by their CD44 and CD62L surface levels.

To test for CD4� T-cell viability, cells were assayed for mitochondrial metab-olism with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT; Sigma). CD4� T cells were harvested, counted, and resuspended in HL-1(1 � 106 cells/ml). One hundred microliters of cells was added to a 96-well,flat-bottomed-well plate (Becton Dickinson, Franklin Lakes, NJ) along with 25�l of MTT (5 mg/ml in PBS) for 2 h at 37°C. The reaction was ceased by theaddition of 100 �l of stop solution (5 g sodium dodecyl sulfate, 25 ml of dimethylformamide, 1 ml acetic acid, 1.25 ml 1 N HCl in water). The plate was incubatedovernight, and the absorbance was read at a 570-nm wavelength using the VersaMax turntable microplate reader and the Soft Max Pro LS computer analysissoftware. Wells with medium alone were used to subtract background absor-bance.

Generation of effector CD4� T cells. Splenocytes from naïve DO11.10 trans-genic mice were isolated, and red blood cells were lysed with a hypotonic lysisbuffer. Effector cells were generated by 24 h of stimulation with 100 nMOVA32–339 peptide (AnaSpec, San Jose, CA) followed by a 7-day culture in 100U of IL-2 in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker)supplemented as indicated above for complete HL-1 with the addition of 10%heat-inactivated fetal bovine serum (HyClone, Logan, UT). Nonviable cells wereremoved by density gradient centrifugation using Ficoll-Paque (GE Healthcare).Remaining cells rested for 24 h in complete DMEM and were used in T-cellactivation assays. As assessed by flow cytometry, �92% of the cells were CD4�

and 72 to 80% had the high-level CD44 (CD44high) and low-level CD62L(CD62Llow) phenotype.

Assays of CD4� T-cell activation. CD4� T cells (1 � 105 cells/well) wereactivated in 96-well, flat-bottomed microtiter plates with 1 �g/ml of soluble CD28MAb in wells precoated with 1 �g/ml of CD3 MAb. Cells were stimulated in thepresence or absence of M. tuberculosis or its fractions for 48 h, when IL-2 andproliferation were measured. Supernatants from activated CD4� T cells wereassayed for IL-2 production using a set of capture and detection MAbs fromeBioscience previously described (11). Briefly, diluted supernatants were addedto Immulon 4HBX flat-bottom microtiter plates (Thermo) coated with purifiedIL-2 MAb (1 �g/ml). Biotinylated IL-2 MAb (1 �g/ml) was added, followed byalkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch) andphosphatase substrate (Sigma). Plates were read using a Versa Max turntablemicroplate reader and data analyzed with the Soft Max Pro LS computer analysissoftware.

To measure CD4� T-cell proliferation, cells were pulsed with 1 �Ci of[3H]thymidine (MP Biomedicals, Solon, OH) for an additional 16 h. Cells wereharvested onto glass fiber filters (Packard, The Netherlands) with a Filtermate196 Harvester (Packard), and 3[3H]thymidine incorporation was measured witha Matrix 96 direct beta counter (Perkin Elmer, Waltham, MA).

For activation of CD4� T cells by phorbol 12-myristate 13-acetate (PMA) andionomycin, T cells were cultured in 96-well, flat-bottomed microtiter plates withincreasing concentrations of PMA (0.05 to 5 ng/ml; Sigma) and 0.1 �g/ml ofionomycin (Sigma) for 6 h in the presence or absence of M. tuberculosis glyco-lipids (10 �g/ml). For overnight activation, CD4� T cells were activated with 0.05ng/ml of PMA and 0.01 �g/ml of ionomycin.

Flow cytometry. For surface receptor expression on CD4� T cells by flowcytometry, cells (1 � 106 cells/well) were stimulated with plate-bound CD3 andsoluble CD28 MAbs in 24-well plates (Falcon) for either 16 h (CD69) or 48 h(CD3, CD28, CD25) with or without M. tuberculosis glycolipids (10 �g/ml).Following stimulation, harvested cells were incubated with the appropriateMAbs, fixed with 2% paraformaldehyde, and analyzed with a FACSCaliburinstrument (BD Biosciences).

Measurement of ZAP-70 phosphorylation. CD4� T cells were rested overnightin complete DMEM supplemented with 1% fetal bovine serum. T cells (3 � 107

cells/ml) were resuspended in complete HL-1 medium in 1.5 ml Eppendorf tubes(LPS, Rochester, NY) in a 100-�l total volume. Glycolipid (10 �g/ml) or mediumalone was either added at the time of activation or incubated for 1 h at 37°Cbefore activation. The TCR complex was activated by adding a cross-linkingmouse anti-hamster IgG1 (10 �g/ml) for 2 min before the addition of anti-CD3ε(10 mg/ml) for 2 min. CD4� T-cell activation was stopped by adding 100 �l of 2�

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Laemmli sample buffer and boiling the samples for 10 min. Unstimulated cells,incubated with mouse anti-hamster IgG1 alone, served as the control for non-specific TCR-CD3 activation.

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis on a 10% gel under reducing conditions and electrotransferred tonitrocellulose membranes (Bio-Rad, Hercules, CA) in a buffer containing 25 mMTris, 192 mM glycine, and 20% methanol. After transfer, membranes wereincubated at room temperature for 1 h in Super Block (Thermo Scientific).Primary and secondary antibodies were diluted in 1% nonfat milk, 0.05% Tween20 in PBS at the concentrations recommended by the manufacturer. Membraneswere incubated with primary antibody at 4°C overnight. Following multiplewashes with 0.05% Tween 20, membranes were incubated with HRP-conjugatedsecondary antibody for 1 h at room temperature. Detection of HRP-conjugatedAbs was performed using West Pico Supersignal (Thermo Scientific). Chemilu-minescence for all membranes was detected using BioMax film (Kodak). Bandintensities were determined using the free ImageJ 1.41 software (http://rsb.info.nih.gov/ij) from the National Institutes of Health (Bethesda, MD). Phosphory-lated ZAP-70 band intensities were normalized for loading differences based oncorresponding ZAP-70 bands.

Statistical analysis. All statistical analyses were performed by using a one-tailed Student t test. A P value of �0.05 was considered statistically significant.

RESULTS

M. tuberculosis bacilli directly inhibit CD4� T-cell prolifer-ation and IL-2 production. To determine whether M. tubercu-losis bacilli could directly regulate CD4� T-cell activation in-dependently of effects on APCs, highly purified CD4� T-cellpopulations were required. To this end, CD4� T cells were firstisolated from murine spleens by negative selection with MAb-coated magnetic beads. This was followed by CD4 staining andsorting by flow cytometry, resulting in CD4� T-cell purities of98% or greater (Fig. 1A). Purified CD4� T cells were stimu-lated with plate-bound CD3 and soluble CD28 MAbs for 48 hin the presence or absence of M. tuberculosis bacilli. T-cellproliferation (Fig. 1B and C) and IL-2 production (Fig. 1D andE) were measured. M. tuberculosis inhibited CD4� T and IL-2production by 25 to 100% at M. tuberculosis bacillus/CD4�

T-cell ratios ranging from 0.2 to 2. Under these conditions,CD4� T-cell viability at 48 h was not affected by M. tubercu-losis, as measured by counting of viable cells through trypanblue exclusion and MTT assay (data not shown). These resultsindicate that M. tuberculosis can directly inhibit CD4� T-cellactivation.

M. tuberculosis cell wall glycolipids inhibit CD4� T-cell ac-tivation. Studies have shown that M. tuberculosis cell wall gly-colipids have immunomodulatory effects on multiple compo-nents of host immunity, and we postulated that they may playa role in our observed inhibition (5, 45, 49). We first verifiedthat intact or viable M. tuberculosis bacilli were not required forinhibition by testing lysates of H37Ra. IL-2 production wassignificantly inhibited (P � 0.05) at a concentration rangebetween 0.1 and 30 �g/ml, with toxicity not observed at con-centrations less than 100 �g/ml (data not shown).

Glycolipids were purified from H37Ra lysate by TritonX-114 and phenol extraction, followed by dialysis of the or-ganic phase. As shown in Fig. 2A, M. tuberculosis glycolipidsinhibited IL-2 consistently at concentrations of �1 �g/ml; how-ever, in some experiments, significant inhibition was seen at100 ng/ml. Maximal inhibition was observed between 10 and 30�g/ml. Thin-layer chromatography and Western blot analysesdemonstrated that the M. tuberculosis glycolipid preparationcontained both PIM and ManLAM (data not shown). To verifyour results and determine whether suppression was specific for

either molecule, we tested purified PIM and ManLAM fromH37Rv (obtained through the TB Vaccine Testing and Re-search Materials Contract at Colorado State University). Us-ing concentrations similar to those used with the whole-glyco-lipid fraction, ManLAM significantly inhibited IL-2 productioncompared to medium alone (Fig. 2B). Inhibition was also ob-served with PIM, however, when both molecules were com-pared on a molar basis, ManLAM was 10-fold more potent asan inhibitor than PIM (data not shown). These results dem-onstrated that M. tuberculosis glycolipids can directly inhibitCD4� T-cell activation. In addition, since the highly purifiedManLAM came from a virulent strain of M. tuberculosis,H37Rv, the observed inhibition was not unique to the avirulentH37Ra strain of M. tuberculosis.

Naïve and effector CD4� T-cell activation is inhibited by M.tuberculosis glycolipids. Resting splenic CD4� T cells consist ofmultiple T-cell subsets (e.g., naïve and effector cells) that ex-press different receptors and have unique mechanisms of acti-vation. To test whether M. tuberculosis glycolipid inhibition wassubset specific, we stimulated enriched populations of bothnaïve and effector CD4� T cells. CD44 and CD62L surfaceexpression was used to positively select naïve (CD44low

CD62Lhigh) from resting CD4� T cells. Naïve CD4� T cellsbehaved similarly to total resting CD4� T cells, with significantinhibition observed at M. tuberculosis glycolipid concentrationsof 0.1 �g/ml and over 60% suppression observed with 10 �g/ml(Fig. 2C).

Since sufficient numbers of effector CD4� T cells (CD44high

CD62Llow) were not present within the resting CD4� T-cellpopulation, we generated effector CD4� T cells by stimulatingD011.10 splenocytes with the OVA323–339 peptide for 24 h andexogenous IL-2 for 1 week. By flow cytometry, activated spleencells were 92% positive for CD4, with 72 to 80% of the cellsbeing of the CD44high CD62Llow phenotype. As shown in Fig.2C, effector CD4� T cells incubated with M. tuberculosis gly-colipid (10 �g/ml) were inhibited to the same degree as naïveCD4� T cells. These results indicate that inhibition by M.tuberculosis glycolipids is not restricted to a CD4� T-cell subsetand imply a common mechanistic pathway.

TLR signaling is not required for M. tuberculosis glycolipid-induced CD4� T-cell activation. Since murine CD4� T cellsexpress TLRs that modulate T-cell responses (24, 52) and M.tuberculosis glycolipids are known TLR-2 agonists (28), we firstdetermined if TLR-2 had a role in the M. tuberculosis glyco-lipid-mediated inhibition of T-cell responses. CD4� T cellsfrom TLR-2 knockout (KO) (Fig. 3A) and MyD88 KO (Fig.3B) mice were activated in the presence of increasing concen-trations of M. tuberculosis glycolipids (0.1 to 10 �g/ml), andIL-2 responses were compared to those of wild-type (WT)mice. CD4� T cells from both strains of mice were inhibited tothe same degree as those from WT mice by M. tuberculosisglycolipids as measured by IL-2 production (�80% for 10�g/ml). Thus, M. tuberculosis glycolipid-mediated inhibition ofCD4� T-cell activation is not dependent on TLR-2 or MyD88signaling.

M. tuberculosis glycolipids inhibit CD4� T-cell activationthrough TCR-mediated signaling pathways. Studies with M.tuberculosis and other pathogens have shown that surface ex-pression of CD3 and CD28 on T cells can be downregulated bymicrobial products (47, 54). Thus, in order to exclude the

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possibility that M. tuberculosis glycolipids affect the expressionof CD3 and CD28, we measured their surface levels. Expres-sion levels of CD3 and CD28 on CD4� T cells treated with M.tuberculosis glycolipids (10 �g/ml) were identical to those of

cells in medium alone after 48 h of incubation (Fig. 4A and B).Anti-CD3 and -CD28-activated CD4� T cells also did notexpress decreased levels of either CD3 or CD28 in the pres-ence of M. tuberculosis glycolipids (data not shown).

FIG. 1. M. tuberculosis bacilli inhibit CD4� T-cell activation. In complete HL-1 medium, purified CD4� T cells (1 � 105 cells/well) wereincubated with or without plate-bound anti-CD3 (1 �g/ml) and/or soluble anti-CD28 (1 �g/ml) for 48 h in the presence of M. tuberculosis bacillior medium alone. Supernatants were harvested for an IL-2 enzyme-linked immunosorbent assay (ELISA), and the cells were pulsed with[3H]thymidine for 16 h to measure proliferation. (A) Purity of CD4� T-cell preparations (see Materials and Methods). (B and C) Effect of M.tuberculosis bacilli on T-cell proliferation. (D and E) Effect of M. tuberculosis bacilli on IL-2 production. Data points represent the means of resultsfor triplicate wells with standard deviations. Results shown are representative of at least three experiments. *, P � 0.05; **, P � 0.01. MTB, M.tuberculosis bacilli; FITC, fluorescein isothiocyanate; TdR, thymidine.



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In contrast, expression of CD25 (IL-2 receptor � chain) onactivated CD4� T cells was inhibited by M. tuberculosis glyco-lipids (Fig. 4C). To determine if M. tuberculosis glycolipidsinterfered with IL-2 binding or function, we tested the re-sponse of CTLL-2 cells to IL-2 in the presence or absence ofM. tuberculosis glycolipids. Glycolipids at 10 �g/ml did notaffect the CTLL-2 response to IL-2 at concentrations of 1 to100 ng/ml (data not shown).

The activation inducer molecule CD69 is rapidly expressedupon CD4� T-cell activation, with peak expression detectedafter 16 h (50). As shown in Fig. 4D, in the presence of M.tuberculosis glycolipids, CD69 expression was reduced com-pared to that on activated CD4� T cells cultured without M.tuberculosis glycolipids. The average decrease in CD69 expres-sion was 68% 13% in the presence of glycolipids (10 �g/ml).Since CD69 expression occurs soon after TCR stimulation andis not dependent on the production of IL-2 (58), this suggestedthat M. tuberculosis glycolipids affect CD4� T-cell activationthrough the TCR signaling pathway.

M. tuberculosis glycolipids do not inhibit activation of CD4�

T cells by PMA and ionomycin. To determine where in the

FIG. 2. M. tuberculosis glycolipids inhibit IL-2 production byCD4� T cells. (A and B) In complete HL-1 medium, CD4� T cells(1 � 105 cells/well) were activated with plate-bound anti-CD3 (1�g/ml) and soluble anti-CD28 (1 �g/ml) for 48 h in the presence ofmedium alone or increasing concentrations of M. tuberculosis(MTB) glycolipids (A) and purified ManLAM (B). Following incu-bation, supernatants were harvested to measure IL-2 production byELISA. Data points represent the means of results for triplicatewells with standard deviations. All data points in the presence of M.tuberculosis glycolipids and ManLAM were statistically significant,with P values of �0.005. (C) Purified naïve (CD44low CD62Lhigh)CD4� T cells and generated effector (CD44high CD62Llow) CD4� Tcells were activated with plate-bound anti-CD3 (1 �g/ml) and sol-uble anti-CD28 (1 �g/ml) for 48 h in the presence of M. tuberculosisglycolipids (10 �g/ml) or medium alone (control). Following incu-bation, supernatants were harvest to measure IL-2 production byELISA. Percent inhibition by M. tuberculosis glycolipid was calcu-lated as follows: 1 � (amount of IL-2 produced by M. tuberculosisglycolipid treated cells/amount of IL-2 produced by medium-alonecontrol cells) � 100%. Results are the means and standard devia-tions from three independent experiments.

FIG. 3. TLR signaling is not necessary for M. tuberculosis glycolipidinhibition of CD4� T-cell activation. In complete HL-1 medium,CD4� T cells (1 � 105 cells/well) purified from WT (A and B), TLR-2KO (A), and MyD88 KO (B) mice were activated with plate-boundanti-CD3 (1 �g/ml) and soluble anti-CD28 (1 �g/ml) for 48 h in thepresence or absence of increasing concentrations of M. tuberculosis(MTB) glycolipids. Following incubation, supernatants were harvestedfor an IL-2 ELISA. WT CD4� T cells in the absence of M. tuberculosisglycolipid produced more IL-2 than both TLR-2 and MyD88 KOCD4� T cells (�4,000 pg/ml compared to �1,000 pg/ml). Percentinhibition by M. tuberculosis glycolipid was calculated as follows: 1 �(amount of IL-2 produced by M. tuberculosis glycolipid-treated cells/amount of IL-2 produced by medium-alone control cells) � 100%.Data points are means of results from triplicate wells with standarddeviations. Results shown are representative of two experiments.



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TCR signaling pathway M. tuberculosis glycolipids inhibitedCD4� T-cell activation, we first examined whether the inhibi-tion could be bypassed by activating secondary signaling path-ways with PMA and ionomycin. CD4� T cells were stimulatedwith PMA (0.005 to 0.05 ng/ml) and ionomycin (1 �g/ml) in thepresence of increasing concentrations of M. tuberculosis glyco-lipids or medium alone for 6 h. Even at high concentrations(10 �g/ml), M. tuberculosis glycolipids did not inhibit CD4�

T-cell activation (Fig. 5A). To ensure that the lack of inhibitionwas not caused by decreasing the incubation period from 16 to6 h, CD4� T cells were stimulated with CD3 and CD28 MAbsor with PMA and ionomycin overnight. PMA and ionomycinconcentrations were reduced to 0.05 ng/ml and 0.01 �g/ml,respectively, to allow for this longer incubation time withoutaffecting T-cell viability. M. tuberculosis glycolipids did not af-fect PMA-ionomycin-induced IL-2 production but did inhibitIL-2 production by anti-CD3- and CD28-stimulated CD4� Tcells (Fig. 5B). This suggested that M. tuberculosis glycolipidsinhibit CD4� T-cell activation by blocking proximal TCR sig-nal transduction.

ZAP-70 phosphorylation inhibited by M. tuberculosis glyco-lipids. To determine if M. tuberculosis glycolipids inhibitedproximal TCR signaling, we focused on changes in tyrosinephosphorylation. Proximal TCR signaling is driven primarilyby tyrosine phosphorylation of a series of proteins, includingZAP-70 and LAT (linker of activated T cells) (31), that can besuppressed by other pathogens (25, 51). Lysates from CD4� Tcells either in medium alone or in the presence of M. tubercu-losis glycolipids (10 �g/ml) had no observable changes in theiroverall tyrosine phosphorylation levels (data not shown). Thus,we postulated that M. tuberculosis glycolipids may need a pe-riod of preincubation prior to activation to induce inhibition.Incubation of M. tuberculosis CD4� T cell glycolipids for up to1 h prior to stimulation led to reduced tyrosine phosphoryla-tion levels compared to that in cells preincubated in mediumalone (data not shown).

One of the bands that was markedly decreased by M. tuber-culosis glycolipids was in the 70-kDa range, which we postu-lated was ZAP-70. To test this hypothesis, we analyzed thephosphorylation levels of ZAP-70 using an antibody specific to

FIG. 4. M. tuberculosis glycolipids inhibit CD25 and CD69 expression on anti-CD3/CD28-activated CD4� T cells. (A and B) CD4� T cells (1 �106 cells/well) were cocultured with M. tuberculosis glycolipids (GL; 10 �g/ml) or medium alone for 48 h without anti-CD3 (� CD3) and -CD28stimulation. Cells were harvested and stained with PE-conjugated anti-CD3 MAb (A), APC-conjugated anti-CD28 MAb (B), or an isotype controlMAb. (C) CD4� T cells (1 � 106 cells/well) were activated with plate-bound anti-CD3 (1 �g/ml) and soluble anti-CD28 (1 �g/ml) for 48 h in thepresence or absence of GL (10 �g/ml). Following incubation, cells were harvested and stained with fluorescein isothiocyanate-conjugatedanti-IL-2R� (CD25) MAb or an isotype control MAb. (D) CD4� T cells (1 � 106 cells/well) were activated with plate-bound anti-CD3 (1 �g/ml)and soluble anti-CD28 (1 �g/ml) for 16 h in the presence or absence of GL (10 �/ml). Cells were harvested and stained with PE-Cy5-conjugatedanti-CD69 MAb or an isotype control MAb. Baseline levels of CD69 were determined by staining unstimulated CD4� T cells incubated in mediumalone for 16 h. All histograms are representative of at least two independent experiments.



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the Tyr-319 residue required for LAT phosphorylation. Asshown in Fig. 6A, ZAP-70 phosphorylation was reduced whenCD4� T cells were incubated with M. tuberculosis glycolipidsprior to activation. In addition to our M. tuberculosis glycolipidpreparation, purified ManLAM reduced ZAP-70 phosphory-lation when administered to CD4� T cells prior to activation(Fig. 6A). Densitometry analysis of multiple experiments forboth M. tuberculosis glycolipids and ManLAM-treated cellsshowed no significant difference in their rates of suppression ofphosphorylation, with both showing significant decreases inrelative intensity of around 60% (Fig. 6B). These results sug-gest that M. tuberculosis glycolipids, including ManLAM, in-hibit CD4� T-cell activation by blocking the phosphorylationof ZAP-70.

DISCUSSION

This report presents the novel observation that M. tubercu-losis bacilli, and M. tuberculosis glycolipids, including PIM andManLAM, can directly inhibit CD4� T-cell activation as mea-sured by expression of T-cell activation markers, IL-2 produc-tion, and proliferation. M. tuberculosis glycolipids did not affectsurface expression of either CD3 or CD28 and did not blockIL-2 signaling by T cells. Inhibition of CD4� T-cell activationby M. tuberculosis glycolipids was seen with multiple subsetsand was not mediated through TLR-2 or MyD88. M. tubercu-losis glycolipids did not affect the downstream T-cell signalingpathways activated by PMA and ionomycin; however, they didsuppress ZAP-70 phosphorylation, critical to proximal TCR

FIG. 5. M. tuberculosis glycolipids do not inhibit IL-2 production by PMA- and ionomycin-activated CD4� T cells. (A) CD4� T cells wereactivated by increasing concentrations of PMA (0.05 to 0.5 ng/ml) and ionomycin (1 �g/ml) for 6 h. Cells were cocultured with glycolipids (10�g/ml) or medium alone. Following incubation, supernatants were harvested for IL-2 production by ELISA. (B) CD4� T cells were activated witheither anti-CD3 and -CD28 (1 �g/ml each) or PMA (0.05 ng/ml) and ionomycin (iono; 0.01 �g/ml) for 16 h. Activated cells were cocultured withglycolipids (10 �g/ml) or medium alone. Following incubation, supernatants were harvested for IL-2 production by ELISA. Data points and valuesare means of results from triplicate wells with standard deviations. Figures shown are representative of at least three similar experiments. *, P �0.003.

FIG. 6. M. tuberculosis glycolipids suppress ZAP-70 phosphorylation. (A) CD4� T cells were preincubated with 10 �g/ml of M. tuberculosisglycolipids (lane 3) or ManLAM (lane 4) for 1 h. Cells (3 � 106) were stimulated for 4 min with anti-CD3 and cross-linking hamster anti-mouseAb (10 �g/ml each). Samples were immunoblotted for phosphorylated ZAP70-Tyr319 (top) and unphosphorylated ZAP-70 as a loading control(bottom). (B) Relative band intensities were quantified by densitometry. The density of the phosphorylated ZAP-70 band was divided by that ofthe unphosphorylated ZAP-70 control. Results are the means and standard deviations of results from four independent experiments. unstim.,unstimulated; �CD3, anti-CD3; GL, glycolipid. �, P � 0.02.



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signal transduction. All studies using our M. tuberculosis gly-colipid fraction were reproducible using a highly purified sam-ple of the major cell wall glycolipid ManLAM. To our knowl-edge this is the first demonstration that major M. tuberculosisglycolipids such as ManLAM can directly inhibit CD4� T-cellactivation by interfering with proximal TCR signaling.

ManLAM, found in the cell wall of M. tuberculosis, readilyinteracts with host immune cells. ManLAM binds to receptorssuch as the mannose receptor, dendritic-cell-specific intercel-lular adhesion molecule 3-grabbing nonintegrin, and TLR-2 inand on macrophages and dendritic cells, resulting in the pro-duction of cytokines and chemokines that can affect CD4�

T-cell function (10, 12). Macrophages exposed to ManLAMcan produce IL-10 and transforming growth factor �, whichcan inhibit T-cell activation, along with prostaglandins, whichcontribute to the expansion of regulatory T cells (20). How-ever, these inhibitory activities of ManLAM are mediated bymolecules secreted by APCs and do not require a direct effectof ManLAM on T cells.

Less is known about the direct interactions of M. tuberculosisglycolipids such as ManLAM and PIM with T cells. A study byShabaana et al. suggested that mycobacterial LAMs can mod-ulate cytokine production by human CD4� Th1- and Th2-cellclones (49). Non-mannose-capped LAMs inhibited Th1-cyto-kine production, including IL-2, while ManLAM had littleeffect. However, in our experimental system, ManLAM had aninhibitory effect on IL-2 production. There are two significantdifferences that may explain these divergent results. First, ourstudy used primary resting CD4� T cells, which differ fromCD4� T-cell clones in activation status and distribution in lipidrafts of molecules necessary for T-cell activation, such as CD3(36). Second, those authors used PMA to activate the CD4�

T-cell clones, whereas we have shown that the inhibition can bebypassed when CD4� T cells are stimulated with PMA. Thus,it is possible that Shabaana et al. did not observe the inhibitiondue to their T-cell stimulation strategy. This novel mechanismfor modulating CD4� T-cell function is also distinct from re-sults of our earlier studies that determined that PIM, but notManLAM, can directly interact with VLA-5 on CD4� T cells,promoting activation and binding to fibronectin. Overall, thesestudies suggest that M. tuberculosis glycolipids can directly af-fect CD4� T-cell function above and beyond the effects thatglycolipids such as ManLAM and PIM have on the APCsrequired for the activation of M. tuberculosis-specific CD4� Tcells in vivo.

How might M. tuberculosis glycolipids directly access CD4�

T cells? The intracellular localization of M. tuberculosis mightpreclude direct interaction between mycobacterial glycolipidsand CD4� T cells. However, in infected cells, mycobacterial-glycolipid-containing vesicles can traffic outside phagosomes asexosomes into the extracellular environment and are thenavailable for fusion with plasma membranes of nearby cells,including CD4� T cells (3, 4). These exosomes are capable ofactivating T cells but only in the presence of an APC, leavingthe possibility that they have additional inhibitory effects di-rectly on T cells (6, 26, 55). Since local concentrations ofglycolipids will be highest around infected cells, direct inhibi-tion of CD4� T-cell activation most likely will occur in theimmediate vicinity of these M. tuberculosis-infected APCs.

Through their glycosylphosphatidylinositol (GPI) anchor,

PIM and ManLAM can integrate into regions of cell mem-branes rich in GPI (30). These regions include lipid rafts,highly organized structures containing sphingolipids and cho-lesterol that allow for quick rearrangement of the cell mem-brane, such as occurs during immune synapse formation (2,49). Insertion of ManLAM into macrophage lipid rafts isnecessary for inhibition of phagosomal maturation (57). Inaddition to the insertion of ManLAM, we have observed sup-pression of T-cell activation with both PIM and non-mannose-capped LAM (data not shown), indicating the shared GPIanchor as the likely inducer of inhibition. We postulate thatManLAM exerts its suppressive effect on proximal TCR sig-naling by inserting into the plasma membrane or lipid raft ofCD4� T cells. The mechanism linking lipid raft insertions witha TCR signaling defect is not known.

While direct inhibition of CD4� T-cell activation is a novelobservation for M. tuberculosis, it has been shown to occur withother pathogens. These include bacteria, viruses, and proto-zoan parasites. Bacterial toxins, including those from Helico-bacter pylori and Bacillus anthracis, can suppress TCR signalingat various points. H. pylori produces the exotoxin VacA, whichblocks calcium influx and induces the activation of the stresskinase p38 (7, 21). B. anthracis lethal and edema toxins sup-press downstream T-cell signaling through the mitogen-acti-vated protein kinase and calcium signaling pathways, respec-tively (14, 17, 42). Salmonella enterica may also express aprotein(s) that can down-modulate TCR expression (56). Yer-sinia pestis produces YopH, a tyrosine phosphatase that candephosphorylate multiple components of the proximal TCRsignaling pathway, including Lck (1), LAT, and SLP-76 (25).Whole Neisseria gonorrhoeae and outer membrane vesicles in-teract with CEACAM1, a coinhibitory receptor that inducesSHP-1 and -2 phosphatase activity (8, 33, 34). The herpesfamily of viruses also can inhibit proximal TCR signaling. Her-pes simplex virus reduces LAT phosphorylation (51), whileherpesvirus saimiri inhibits ZAP-70 phosphorylation by se-questering Lck (13).

Whereas mechanisms used by pathogens to directly inhibitT-cell function appear to be both diverse and not uncommon,few appear to use glycolipids as mediators. A surface glyco-lipid, glycoinositolphospholipid on Trypanosoma cruzi, candownregulate T-cell activation (27). While not directly inhibi-tory of T-cell function, Leishmania spp. produce a glycolipid,lipophosphoglycan, that blocks phagosome maturation (35)and respiratory burst in macrophages by blocking signal trans-duction (16). As these examples indicate, pathogens do notutilize one universal inhibitory pathway but instead can blockT-cell activation at various points in the TCR signaling path-way and can activate more than one suppressive pathway at atime. Collectively, these studies indicate that direct suppres-sion of T cells may play a fundamental role in a pathogen’sresistance to adaptive host immunity. The different mecha-nisms used likely reflect the unique biological niche and dis-tinct evolution of each pathogen.

For a chronic pathogen, such as M. tuberculosis, survival inmacrophages and evasion of adaptive T-cell responses are es-sential for long-term survival in the human host. The ability ofM. tuberculosis to directly interfere with CD4� T-cell activationthrough the release of its abundant cell wall glycolipids Man-LAM and PIM provide it with an additional mechanism to



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evade host immunity. This novel mechanism is likely one ofmany used by M. tuberculosis to modulate innate and adaptiveimmune responses to its advantage as it seeks to survive inmacrophages in the face of robust CD4� T-cell responses.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants AI-27243 and HL-55967 (to W.H.B.); contract no. HHSN266200700022C/NO1-AI-70022 for the Tuberculosis Research Unit (TBRU) (to W.H.B.);NIH grants AI069085, AI034343, and AI035726 to C.V.H.; AmericanLung Association grants RG48786N to R.E.R. and RG-8407-N to S.A.F.;and grant HHSN26620040091C.

We thank the Cytometry Facility of the Comprehensive CancerCenter at CWRU (P30 CA43703) for their assistance in purifyingCD4� T cells by flow cytometry, Karen Dobos from Colorado StateUniversity for thin-layer chromatography analysis of our M. tubercu-losis glycolipid sample, and Alan Levine, Mursalin Anis, and ScottReba for technical advice.

REFERENCES

1. Alonso, A., N. Bottini, S. Bruckner, S. Rahmouni, S. Williams, S. P. Schoen-berger, and T. Mustelin. 2004. Lck dephosphorylation at Tyr-394 and inhi-bition of T cell antigen receptor signaling by Yersinia phosphatase YopH.J. Biol. Chem. 279:4922–4928.

2. Alonso, M. A., and J. Millan. 2001. The role of lipid rafts in signalling andmembrane trafficking in T lymphocytes. J. Cell Sci. 114:3957–3965.

3. Beatty, W. L., E. R. Rhoades, H. J. Ullrich, D. Chatterjee, J. E. Heuser, andD. G. Russell. 2000. Trafficking and release of mycobacterial lipids frominfected macrophages. Traffic 1:235–247.

4. Beatty, W. L., H. J. Ullrich, and D. G. Russell. 2001. Mycobacterial surfacemoieties are released from infected macrophages by a constitutive exocyticevent. Eur. J. Cell Biol. 80:31–40.

5. Berman, J. S., R. L. Blumenthal, H. Kornfeld, J. A. Cook, W. W. Cruikshank,M. W. Vermeulen, D. Chatterjee, J. T. Belisle, and M. J. Fenton. 1996.Chemotactic activity of mycobacterial lipoarabinomannans for human bloodT lymphocytes in vitro. J. Immunol. 156:3828–3835.

6. Bhatnagar, S., K. Shingawa, F. J. Castellino, and J. S. Schorey. 2007. Exo-somes released from macrophages infected with intracellular pathogensstimulate a proinflammatory response in vitro and in vivo. Blood 110:3234–3244.

7. Boncristiano, M., S. R. Paccani, S. Barone, C. Ulivieri, L. Patrussi, D. Ilver,A. Amedei, M. M. D’Elios, J. L. Telford, and C. T. Baldari. 2003. TheHelicobacter pylori vacuolating toxin inhibits T cell activation by two inde-pendent mechanisms. J. Exp. Med. 198:1887–1897.

8. Boulton, I. C., and S. D. Gray-Owen. 2002. Neisserial binding to CEACAM1arrests the activation and proliferation of CD4� T lymphocytes. Nat. Im-munol. 3:229–236.

9. Brennan, P. J. 2003. Structure, function, and biogenesis of the cell wall ofMycobacterium tuberculosis. Tuberculosis (Edinburgh) 83:91–97.

10. Briken, V., S. A. Porcelli, G. S. Besra, and L. Kremer. 2004. Mycobacteriallipoarabinomannan and related lipoglycans: from biogenesis to modulationof the immune response. Mol. Microbiol. 53:391–403.

11. Canaday, D. H., S. Chakravarti, T. Srivastava, D. J. Tisch, V. K. Cheruvu, J.Smialek, C. V. Harding, and L. Ramachandra. 2006. Class II MHC antigenpresentation defect in neonatal monocytes is not correlated with decreasedMHC-II expression. Cell. Immunol. 243:96–106.

12. Chatterjee, D., and K. H. Khoo. 1998. Mycobacterial lipoarabinomannan: anextraordinary lipoheteroglycan with profound physiological effects. Glycobi-ology 8:113–120.

13. Cho, N. H., P. Feng, S. H. Lee, B. S. Lee, X. Liang, H. Chang, and J. U. Jung.2004. Inhibition of T cell receptor signal transduction by tyrosine kinase-interacting protein of Herpesvirus saimiri. J. Exp. Med. 200:681–687.

14. Comer, J. E., A. K. Chopra, J. W. Peterson, and R. Konig. 2005. Directinhibition of T-lymphocyte activation by anthrax toxins in vivo. Infect. Im-mun. 73:8275–8281.

15. Cosma, C. L., D. R. Sherman, and L. Ramakrishnan. 2003. The secret livesof the pathogenic mycobacteria. Annu. Rev. Microbiol. 57:641–676.

16. Descoteaux, A., S. J. Turco, D. L. Sacks, and G. Matlashewski. 1991. Leish-mania donovani lipophosphoglycan selectively inhibits signal transduction inmacrophages. J. Immunol. 146:2747–2753.

17. Fang, H., R. Cordoba-Rodriguez, C. S. Lankford, and D. M. Frucht. 2005.Anthrax lethal toxin blocks MAPK kinase-dependent IL-2 production inCD4� T cells. J. Immunol. 174:4966–4971.

18. Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu. Rev.Immunol. 19:93–129.

19. Flynn, J. L., and J. Chan. 2003. Immune evasion by Mycobacterium tuber-culosis: living with the enemy. Curr. Opin. Immunol. 15:450–455.

20. Garg, A., P. F. Barnes, S. Roy, M. F. Quiroga, S. Wu, V. E. Garcia, S. R.Krutzik, S. E. Weis, and R. Vankayalapati. 2008. Mannose-capped lipoara-binomannan- and prostaglandin E2-dependent expansion of regulatory Tcells in human Mycobacterium tuberculosis infection. Eur. J. Immunol. 38:459–469.

21. Gebert, B., W. Fischer, E. Weiss, R. Hoffmann, and R. Haas. 2003. Helico-bacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science301:1099–1102.

22. Gehring, A. J., K. M. Dobos, J. T. Belisle, C. V. Harding, and W. H. Boom.2004. Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligandthat inhibits human macrophage class II MHC antigen processing. J. Immu-nol. 173:2660–2668.

23. Gehring, A. J., R. E. Rojas, D. H. Canaday, D. L. Lakey, C. V. Harding, andW. H. Boom. 2003. The Mycobacterium tuberculosis 19-kilodalton lipoproteininhibits gamma interferon-regulated HLA-DR and Fc gamma R1 on humanmacrophages through Toll-like receptor 2. Infect. Immun. 71:4487–4497.

24. Gelman, A. E., J. Zhang, Y. Choi, and L. A. Turka. 2004. Toll-like receptorligands directly promote activated CD4� T cell survival. J. Immunol. 172:6065–6073.

25. Gerke, C., S. Falkow, and Y. H. Chien. 2005. The adaptor molecules LATand SLP-76 are specifically targeted by Yersinia to inhibit T cell activation.J. Exp. Med. 201:361–371.

26. Giri, P. K., and J. S. Schorey. 2008. Exosomes derived from M. bovis BCGinfected macrophages activate antigen-specific CD4� T cells and CD8� Tcells in vitro and in vivo. PLoS One 3:e2461–e2470.

27. Gomes, N. A., J. O. Previato, B. Zingales, L. Mendonca-Previato, and G. A.DosReis. 1996. Down-regulation of T lymphocyte activation in vitro and invivo induced by glycoinositolphospholipids from Trypanosoma cruzi. Assign-ment of the T cell-suppressive determinant to the ceramide domain. J. Im-munol. 156:628–635.

28. Heldwein, K. A., and M. J. Fenton. 2002. The role of Toll-like receptors inimmunity against mycobacterial infection. Microbes Infect. 4:937–944.

29. Hestvik, A. L., Z. Hmama, and Y. Av-Gay. 2005. Mycobacterial manipulationof the host cells. FEMS Microbiol. Rev. 29:1041–1050.

30. Ilangumaran, S., S. Arni, M. Poincelet, J. M. Theler, P. J. Brennan, D.Nasirud, and D. C. Hoessli. 1995. Integration of mycobacterial lipoarabino-mannans into glycosylphosphatidylinositol-rich domains of lymphomono-cytic cell plasma membranes. J. Immunol. 155:1334–1342.

31. Iwashima, M. 2003. Kinetic perspectives of T cell antigen receptor signaling.A two-tier model for T cell full activation. Immunol. Rev. 191:196–210.

32. Karakousis, P. C., W. R. Bishai, and S. E. Dorman. 2004. Mycobacteriumtuberculosis cell envelope lipids and the host immune response. Cell. Micro-biol. 6:105–116.

33. Lee, H. S., I. C. Boulton, K. Reddin, H. Wong, D. Halliwell, O. Mandelboim,A. R. Gorringe, and S. D. Gray-Owen. 2007. Neisserial outer membranevesicles bind the coinhibitory receptor carcinoembryonic antigen-related cel-lular adhesion molecule 1 and suppress CD4� T lymphocyte function. Infect.Immun. 75:4449–4455.

34. Lee, H. S., M. A. Ostrowski, and S. D. Gray-Owen. 2008. CEACAM1 dy-namics during Neisseria gonorrhoeae suppression of CD4� T lymphocyteactivation. J. Immunol. 180:6827–6835.

35. Lodge, R., and A. Descoteaux. 2005. Modulation of phagolysosome biogen-esis by the lipophosphoglycan of Leishmania. Clin. Immunol. 114:256–265.

36. Marwali, M. R., M. A. MacLeod, D. N. Muzia, and F. Takei. 2004. Lipid raftsmediate association of LFA-1 and CD3 and formation of the immunologicalsynapse of CTL. J. Immunol. 173:2960–2967.

37. Mogues, T., M. E. Goodrich, L. Ryan, R. LaCourse, and R. J. North. 2001.The relative importance of T cell subsets in immunity and immunopathologyof airborne Mycobacterium tuberculosis infection in mice. J. Exp. Med. 193:271–280.

38. Muller, I., S. P. Cobbold, H. Waldmann, and S. H. Kaufmann. 1987. Im-paired resistance to Mycobacterium tuberculosis infection after selective invivo depletion of L3T4� and Lyt-2� T cells. Infect. Immun. 55:2037–2041.

39. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigenof intrathymic apoptosis of CD4� CD8� TCRlo thymocytes in vivo. Science250:1720–1723.

40. Noss, E. H., R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock,W. H. Boom, and C. V. Harding. 2001. Toll-like receptor 2-dependent inhi-bition of macrophage class II MHC expression and antigen processing by19-kDa lipoprotein of Mycobacterium tuberculosis. J. Immunol. 167:910–918.

41. Orme, I. M., and F. M. Collins. 1983. Protection against Mycobacteriumtuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients. J. Exp. Med. 158:74–83.

42. Paccani, S. R., F. Tonello, R. Ghittoni, M. Natale, L. Muraro, M. M. D’Elios,W. J. Tang, C. Montecucco, and C. T. Baldari. 2005. Anthrax toxins suppressT lymphocyte activation by disrupting antigen receptor signaling. J. Exp.Med. 201:325–331.

43. Pecora, N. D., A. J. Gehring, D. H. Canaday, W. H. Boom, and C. V. Harding.2006. Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 thatregulates innate immunity and APC function. J. Immunol. 177:422–429.

44. Pieters, J., and J. Gatfield. 2002. Hijacking the host: survival of pathogenicmycobacteria inside macrophages. Trends Microbiol. 10:142–146.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

45. Rojas, R. E., J. J. Thomas, A. J. Gehring, P. J. Hill, J. T. Belisle, C. V.Harding, and W. H. Boom. 2006. Phosphatidylinositol mannoside from My-cobacterium tuberculosis binds �5�1 integrin (VLA-5) on CD4� T cells andinduces adhesion to fibronectin. J. Immunol. 177:2959–2968.

46. Scanga, C. A., V. P. Mohan, K. Yu, H. Joseph, K. Tanaka, J. Chan, and J. L.Flynn. 2000. Depletion of CD4(�) T cells causes reactivation of murinepersistent tuberculosis despite continued expression of interferon gammaand nitric oxide synthase 2. J. Exp. Med. 192:347–358.

47. Seitzer, U., K. Kayser, H. Hohn, P. Entzian, H. H. Wacker, S. Ploetz, H. D.Flad, J. Gerdes, and M. J. Maeurer. 2001. Reduced T-cell receptor CD3zeta-chain protein and sustained CD3epsilon expression at the site of mycobac-terial infection. Immunology 104:269–277.

48. Sepkowitz, K. A., J. Raffalli, L. Riley, T. E. Kiehn, and D. Armstrong. 1995.Tuberculosis in the AIDS era. Clin. Microbiol. Rev. 8:180–199.

49. Shabaana, A. K., K. Kulangara, I. Semac, Y. Parel, S. Ilangumaran, K.Dharmalingam, C. Chizzolini, and D. C. Hoessli. 2005. Mycobacterial li-poarabinomannans modulate cytokine production in human T helper cells byinterfering with raft/microdomain signalling. Cell. Mol. Life Sci. 62:179–187.

50. Simms, P. E., and T. M. Ellis. 1996. Utility of flow cytometric detection ofCD69 expression as a rapid method for determining poly- and oligoclonallymphocyte activation. Clin. Diagn. Lab. Immunol. 3:301–304.

51. Sloan, D. D., J. Y. Han, T. K. Sandifer, M. Stewart, A. J. Hinz, M. Yoon, D. C.

Johnson, P. G. Spear, and K. R. Jerome. 2006. Inhibition of TCR signalingby herpes simplex virus. J. Immunol. 176:1825–1833.

52. Sobek, V., N. Birkner, I. Falk, A. Wurch, C. J. Kirschning, H. Wagner, R.Wallich, M. C. Lamers, and M. M. Simon. 2004. Direct Toll-like receptor 2mediated co-stimulation of T cells in the mouse system as a basis for chronicinflammatory joint disease. Arthritis Res. Ther. 6:R433–446.

53. Stenger, S., and R. L. Modlin. 1999. T cell mediated immunity to Mycobac-terium tuberculosis. Curr. Opin. Microbiol. 2:89–93.

54. Swigut, T., N. Shohdy, and J. Skowronski. 2001. Mechanism for down-regulation of CD28 by Nef. EMBO J. 20:1593–1604.

55. Thery, C., L. Duban, E. Segura, P. Veron, O. Lantz, and S. Amigorena. 2002.Indirect activation of naïve CD4� T cells by dendritic cell-derived exosomes.Nat. Immunol. 3:1156–1162.

56. van der Velden, A. W., J. T. Dougherty, and M. N. Starnbach. 2008. Down-modulation of TCR expression by Salmonella enterica serovar Typhi-murium. J. Immunol. 180:5569–5574.

57. Welin, A., M. E. Winberg, H. Abdalla, E. Sarndahl, B. Rasmusson, O.Stendahl, and M. Lerm. 2008. Incorporation of Mycobacterium tuberculosislipoarabinomannan into macrophage membrane rafts is a prerequisite forthe phagosomal maturation block. Infect. Immun. 76:2882–2887.

58. Willerford, D. M., J. Chen, J. A. Ferry, L. Davidson, A. Ma, and F. W. Alt.1995. Interleukin-2 receptor alpha chain regulates the size and content of theperipheral lymphoid compartment. Immunity 3:521–530.

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