TLR-2 Antigen Presentation Pathways Through Regulates CD1 Mycobacterium Tuberculosis

Mycobacterium tuberculosis Regulates CD1 Antigen Presentation Pathways through TLR-2

This information is current as Carme Roura-Mir, Lisheng Wang, Tan-Yun Cheng, Isamu of October 2, 2021. Matsunaga, Christopher C. Dascher, Stanford L. Peng, Matthew J. Fenton, Carsten Kirschning and D. Branch Moody J Immunol 2005; 175:1758-1766; ; doi: 10.4049/jimmunol.175.3.1758 http://www.jimmunol.org/content/175/3/1758 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mycobacterium tuberculosis Regulates CD1 Antigen Presentation Pathways through TLR-21

Carme Roura-Mir,2* Lisheng Wang,2,3* Tan-Yun Cheng,* Isamu Matsunaga,* Christopher C. Dascher,* Stanford L. Peng,4* Matthew J. Fenton,5† Carsten Kirschning,‡ and D. Branch Moody6*

Mycobacterium tuberculosis remains a major pathogen of worldwide importance, which releases lipid Ags that are presented to human T cells during the course of tuberculosis infections. Here we report that cellular infection with live M. tuberculosis or exposure to mycobacterial cell wall products converted CD1؊ myeloid precursors into competent APCs that expressed group 1 CD1 proteins (CD1a, CD1b, and CD1c). The appearance of group 1 CD1 proteins at the surface of infected or activated cells occurred via transcriptional regulation, and new CD1 protein synthesis and was accompanied by down-regulation of CD1d

transcripts and protein. Isolation of CD1-inducing factors from M. tuberculosis using normal phase chromatography, as well as Downloaded from the use of puriﬁed natural and synthetic compounds, showed that this process involved polar lipids that signaled through TLR-2, and we found that TLR-2 was necessary for the up-regulation of CD1 protein expression. Thus, mycobacterial cell wall lipids provide two distinct signals for the activation of lipid-reactive T cells: lipid Ags that activate T cell receptors and lipid adjuvants that activate APCs through TLR-2. These dual activation signals may represent a system for selectively promoting the presentation of exogenous foreign lipids by those myeloid APCs, which come into direct contact with pathogens. The Journal of

Immunology, 2005, 175: 1758–1766. http://www.jimmunol.org/

he discovery of CD1 Ag presentation pathways provides phosphatidylinositols, sulfatides, and isoglobosides activate CD1- a new perspective on microbial immunity by showing restricted T cells (12–16). Thus, mammalian APCs normally en- T how T cells can recognize lipid Ags from pathogens. CD1 counter and present both foreign and self lipid Ags to T cells, proteins present a variety of pathogen-derived lipids to T cells in raising basic questions about how such responses are regulated. vitro (1–6), and T cells reactive to mycobacterial lipid Ags have A role for CD1-expressing APCs in controlling differential T been isolated from the bloodstream of tuberculosis patients, pro- cell responses to self and foreign Ags has been suggested by stud- viding evidence that CD1 and lipid-reactive T cells normally func- ies showing that dendritic cells (DCs)7 and B cells have highly tion in the host response during infections with Mycobacterium regulated subcellular pathways for internalizing exogenous lipid by guest on October 2, 2021 tuberculosis (7–9). However, CD1-mediated T cell responses are Ags into endosomal compartments for loading onto CD1 proteins. not limited only to foreign pathogens, as endogenous mammalian For example, myeloid dendritic cells use the mannose receptor phosphatidylinositols normally bind to cellular CD1 proteins as (CD206) and Langerhans cells use langerin (CD207) to deliver they traverse the secretory pathway (10, 11), and self gangliosides, exogenous lipid Ags to endosomes (17, 18), where saposin lipid- transfer proteins and a low pH environment promote binding of lipid Ags to CD1 proteins (19–22). Such endosomal loading path- *Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hos- pital and Harvard Medical School, Boston, MA 02115; †The Pulmonary Center, Bos- ways are necessary for efﬁcient T cell activation by most or all ton University School of Medicine, Boston, MA 02118; and ‡Institute of Medical known bacterial lipid Ags, including mycolic acid, lipoarabino- Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany mannan (LAM), glucose monomycolate (GMM), mannosyl phosphomycoketides, and didehydroxymycobactins (DDM) (1, 4, 23– Received for publication April 6, 2005. Accepted for publication May 16, 2005. 25). A general implication of the discovery of endosomal pathways The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance for loading lipid Ags onto CD1 proteins is that APCs can actively with 18 U.S.C. Section 1734 solely to indicate this fact. control which Ags are displayed to T cells by controlling which 1 This work was supported by grants from the Cancer Research Institute, the Amer- lipids are taken up into endosomal compartments (26). ican College of Rheumatology Research and Education Foundation, the Pew Scholars Related to this, there is evidence that myeloid DCs, which are Program in the Biomedical Sciences, the Mizutani Foundation for Glycoscience, and the National Institutes of Health (Grants AR48632 and AI49313). the only peripheral APCs that are known to be capable of express- 2 C.R.-M. and L.W. contributed equally to this study. ing all human CD1 isoforms (CD1a, CD1b, CD1c, CD1d, CD1e), selectively express CD1 proteins and initiate lipid Ag processing 3 Current address: Stem Cell Biology and Regenerative Medicine, Robarts Research Institute, University of Western Ontario, 100 Perth Drive, London, Ontario, Canada pathways in response to certain activating stimuli. For example, N6A 5K8. group 1 CD1 isoforms (CD1a, CD1b, CD1c) are not detectably 4 Current address: Washington University School of Medicine, Campus Box 8045, expressed on the surface of blood-derived monocytes, but these CSRB 6617, 660 South Euclid Avenue, St. Louis, MO 63110. three CD1 proteins can be induced in vitro by high concentrations 5 Current address: Division of Pulmonary and Critical Care Medicine, University of Maryland School of Medicine, 685 West Baltimore Street, MSTF-800, Baltimore, MD 21201. 7 Abbreviations used in this paper: DC, dendritic cell; LAM, lipoarabinomannan; 6 Address correspondence and reprint requests to Dr. D. Branch Moody, Division of GMM, glucose monomycolate; DDM, didehydroxymycobactins; RT-PCR, real-time Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Har- PCR; CHO, Chinese hamster ovary; M. tb-GFP, M. tuberculosis expressing GFP; vard Medical School, Smith Building Room 514, 1 Jimmy Fund Way, Boston, MA CWS, cell wall skeleton; PIM, phosphatidylinositol mannoside; araLAM, LAM with 02115. E-mail address: [email protected] arabinosylated termini.

of the cytokines that promote the early phases of differentiation to mix reagent kit in the ABI PRISM 7700 System (Applied Biosystems). DCs, GM-CSF, and IL-4 (1, 27). Related to this, studies of MHC CD1 mRNA levels were determined by the comparative Ct method relative class II-mediated Ag processing have shown that the transition of to the expression of the housekeeping gene GAPDH. Primers are: CD1a (5Ј-TGTTAGCTGTTCTCCCAGGTGA-3Ј forward, 5Ј-AGGATGC- myeloid precursors into immature DCs in response to GM-CSF GATCCAGATGACAT-3Ј reverse), CD1b (5Ј-CCATTTCAACTGT- and IL-4 is associated with increasing rates of macropinocytosis TAGCTGTTCTCTT-3Ј forward, 5Ј-GAAAGGAGGTCGGCCCC-3Ј re- and related mechanisms of bulk flow Ag uptake (28). This raises verse), CD1c (5Ј-TGGTGACAATGCAGACGCA-3Ј forward, 5Ј-GGTTG the possibility that monocytes in the peripheral blood generally ACAAATGAGAAGATCTGGA-3Ј reverse), and CD1d (5Ј-AGCGCTG AAGTCCCGCA-3Ј forward, 5Ј-TATTGGCGAAGGACGAGATCTG-3Ј lack the ability to present lipids on group 1 CD1 proteins, until reverse). The fluorogenic probes are: CD1a (5Ј-FAM-AGACGGGCTCA they traffic to local sites of inflammation or infection, where as yet AGGAGCCTCTCTCCTTAMRA-3Ј), CD1b 5Ј-FAM-CCTGGTGGTAA undefined stimuli lead to CD1 expression. CAGTGAACATGCCTTCC-TAMRA-3Ј), CD1c (5Ј-FAM-CCCAGGAA In these studies, we considered the possibility that microbial CACGTCTCCTTCCATGTCTAMRA-3Ј), and CD1d (5Ј-FAM-AGGCTT Ј pathogens might modulate CD1 expression and Ag-presenting TTCCCCCTCCGCTGCCTAMRA-3 ). The PCR cycling parameters were 40 cycles of 95°C denaturation and 60°C annealing and extension. function in human myeloid cells. We initially focused on M. tuberculosis because this prevalent and deadly pathogen produces Cellular assays many types of lipid Ags that are presented by the CD1 system, IL-2 release by the human CD1b-restricted T cell line LDN5 was measured including mycolic acids, GMM, LAM, mannosyl phosphomy- after 1 day in culture at a 1:1 ratio with lipid-treated monocytes (6). Ac- coketides, acylated sulfotrehaloses, and DDM lipopeptides (3, 4, tivation of J.RT-3 cells transfected with the TCR ␣- and ␤-chains from the 5–9, 29). Furthermore, prior studies had shown that human my- human CD1a-restricted T cell line CD8-2 (34, 35) was measured by flow eloid cells that migrate to the sites of cutaneous infection by My- cytometry of cells treated with the calcium-sensitive fluorophores Fluo-4

and Fura-red (23). Glycolipid uptake was measured by incubating mono- Downloaded from cobacterium leprae or pulmonary infection by M. tuberculosis in- cytes at 100,000 cells per well in 96-well plates with M. tuberculosis lipids fection express CD1a, CD1b, or CD1c (30–32). (10 ␮g/ml) or medium alone for 3 days followed by addition of 14C-labeled To test the hypothesis that M. tuberculosis itself might regulate GMM as described (23). Macropinocytosis was measured by culturing 106 ␮ CD1 function, we developed systems to study the mechanism of monocytes/ml with M. tuberculosis lipids (10 g/ml) for 3 days and washing before further culture with FITC-dextran (1 mg/ml; m.w. 40,000 Da; CD1 expression and Ag-presenting function in human monocytes Sigma-Aldrich) for 1 h. Cells were washed and then subjected to flow infected with M. tuberculosis. We found that M. tuberculosis or its cytometric analysis. cell wall lipids strongly induced the expression of all three group http://www.jimmunol.org/ 1 CD1 isoforms (CD1a, CD1b, and CD1c), but not the only mem- Live infection ber of human group 2 CD1 proteins, CD1d. CD1 induction was M. tuberculosis strain H37Ra or strain H37Rv (Colorado State University, accompanied by increases in the rate of lipid Ag uptake and was Fort Collins, CO) or M. tuberculosis H37Ra or H37Rv expressing GFP sufficient to convert monocytes into fully competent APCs. Isola- (36) was grown in 7H9 medium with albumin-dextrose-catalase supple- ment, glycerol, and Tween 80. Mid-log-phase cultures were washed with tion of the mycobacterial factors that controlled this process medium and filtered through a 5-␮m filter. Aliquots were thawed and used showed that the stimuli were polar lipids shed from the mycobac- to infect monocytes at 2 or 10 bacteria per cell as determined by counting terial cell wall, which signal through TLR-2. Furthermore, we in Petroff-Hauser chambers or from colony forming unit determinations on found that TLR-2 was necessary for the induction of CD1 by my- Middlebrook 7H11 plates. After extensive washing to remove extracellular bacteria, cells were cultured 1–3 days in medium, stained with anti-CD1 by guest on October 2, 2021 cobacteria. These studies define a new immunological function for mAb, fixed with 2% paraformaldehyde, and analyzed by flow cytometry. TLR-2 by showing that primary activation of this innate pattern For confocal microscopy, cells stained with anti-CD1 Abs were examined recognition receptor controls the subsequent functions of group 1 as cytospin preparations on glass slides using a Zeiss Axiovert S100 mi- CD1 proteins. Furthermore, theses studies suggest a model in croscope equipped with a Bio-Rad Mrc 1024 confocal imaging system. which microbial cell walls provide two signals that lead to CD1- Microbial extracts and glycolipids mediated T cell activation: Ags that bind to CD1 and adjuvants that trigger CD1 expression through activation of TLR-2. Total lipid extracts of M. tuberculosis H37Ra, M. tuberculosis H37Rv (Colorado State University), Mycobacterium avium (H. Remold, Harvard University, Cambridge, MA), Mycobacterium phlei, Staphylococcus au- Materials and Methods reus, Candida albicans, and Aspergillus niger (American Type Culture Cells and flow cytometry Collection), promastigotes from Leishmania donovani (J. Amprey, Weill College of Medicine, Cornell University, Ithaca, NY, and S. Porcelli, Al- Human monocytes were isolated from peripheral blood using Ficoll and bert Einstein College of Medicine, New York, NY) and Leishmania tropica adherence to tissue-culture flasks and were cultured in RPMI 1640 medium (American Type Culture Collection), and Schistosoma mansoni (L. with 10% FBS (33). In selected experiments, monocytes were further de- Tielens, Universiteit Utrecht, Utrecht, The Netherlands, and D. van der pleted of residual lymphocytes using anti-CD20 (L26), anti-CD3 (TCS3), Kleij, Leiden University Medical Center, Leiden, The Netherlands) were anti-CD56 (B159), and goat anti-mouse Ig-coated magnetic beads (Dynal) prepared by extracting lyophilized organisms at 5 mg/ml in chloroform/ such that lymphocytes constituted Ͻ2% of total cells as determined by flow methanol (2:1) for 2 h. M. tuberculosis H37 Rv was further fractionated by cytometric staining with non-cross-reactive lymphocyte markers. These ad- drying under nitrogen, resuspending in chloroform, and eluting from an ditional purification steps to remove lymphocytes did not substantially in- open silica column (6). In selected experiments, M. tuberculosis polar lip- fluence CD1 induction on myeloid cells. Cell surface protein expression ids were further purified by one-dimensional TLC using 200-␮m silica was measured using murine mAb against human CD1a (OKT6), CD1b plates with 10 mg of polar lipids, resolved in chloroform-methanol-water (BCD1b3.1 or 4A7), CD1c (F10/21A3.1), CD1d (CD1d4.2), CD80 (60:16:2, v:v:v), and extracted with chloroform-methanol (2:1, v:v). The (L307.4), CD83 (HB15e), CD86 (FUN-1), CD11c (B-ly6), and CD14 molecular weights and purity of lipids were assessed by analytical TLC (M5E2) from BD Biosciences; CD64 (10.1; Caltag Laboratories); CD209 with known standards or electrospray ionization mass spectroscopy (Finni- (eB-h209; eBioscience); HLA-DR (L243; American Type Culture Collec- gan LCQ Advantage with MSn). Ag preparations were tested for LPS using tion); and IgG1 (P3) or IgG2a (MOPC-21; Sigma-Aldrich) isotype controls the Limulus lysate assay (Charles River Laboratories). Ј followed by a FITC-conjugated goat anti-mouse IgG F(ab )2 or anti-mouse IgG PE. TLR analysis Real-time PCR analysis Monocytes were incubated in the presence or absence of different concentrations of poly(I:C) (Sigma-Aldrich), Painz (EMC Microcollections) un- Total RNA from monocytes was isolated using the RNeasy kit (Qiagen) methylated CpG oligonucleotides (Integrated DNA Technologies). For in- and was retrotranscribed using Superscript II H-Reverse Transcriptase (In- hibition of the TLR-2-dependent activity, monocytes were incubated with vitrogen Life Technologies). The relative mRNA for CD1 expression was an anti-TLR2 Ab (T2.5) (37) or isotype control Ab for 1 h before adding analyzed by real-time PCR (RT-PCR) using a Taqman universal master the M. tuberculosis lipids (10 ␮g/ml). Cells were extensively washed after 1760 MICROBIAL ADJUVANTS FOR CD1 FUNCTION

2 h, resuspended in fresh medium containing 20 ␮g/ml of the TLR-2 Ab or fected (39), moderately down-regulated (40), or down-regulated to control Ab, and further cultured for 3 days. CD1 expression was measured undetectable levels after infection (41). The question of whether by flow cytometry using fluorescein-labeled CD1a (CB-T6), CD1b (SN13/ mycobacteria can induce CD1 expression in the first instance had K5-1B8), CD1c (M241), or isotype control Abs (Ancell). TLR-2 agonism was tested using Chinese hamster ovary (CHO) cells cotransfected with not been previously addressed, and the data shown in Fig. 1A in- TLR-2 and a CD25 expression vector under the control of NF-␬B, as de- dicated that whatever inhibitory signals might be provided by M. scribed (38). tuberculosis, these were not sufficient to block the initial induction of CD1a, CD1b, and CD1c on fresh monocytes. Nevertheless, be- Results cause not all the monocytes in the culture up-regulated CD1, it M. tuberculosis induces group 1 CD1 expression remained possible that cells with live intracellular bacteria might To test the possibility that mycobacteria could modulate CD1 ex- have been prevented from expressing CD1 and that CD1 might has pression, we infected human monocytes with M. tuberculosis and been present predominantly on noninfected bystander cells. measured CD1 expression by flow cytometry over a period of sev- To separately measure CD1 expression on cells with and with- eral days. Whereas fresh monocytes and control cells treated with out bacteria, we infected monocytes with M. tuberculosis express- medium for 3 days failed to detectably stain with mAbs specific for ing GFP (M. tb-GFP) (36). Two-color immunofluorescence mi- group 1 CD1 proteins, a large number of cells in infected cultures croscopy of monocytes showed bright staining for CD1a on cells stained brightly with Abs against CD1a, CD1b, and CD1c. Each of with and without overlying fluorescent bacteria 3 days after infec- these group 1 CD1 proteins was induced to high levels by day 2 of tion (Fig. 1B). Confocal microscopy showed bright staining for infection, and expression levels were typically maximal at day 3 CD1a, CD1b, and CD1c on infected cells as well as colocalization (Fig. 1A). of CD1b within compartments that contained bacteria (Fig. 1C and Downloaded from To investigate the cellular mechanisms by which M. tuberculo- data not shown). In addition, flow cytometric analysis of the entire sis induced CD1 cell surface expression, we first sought to deter- population of infected cells showed clear evidence for CD1a, mine whether CD1 staining was found on cells that were infected CD1b, and CD1c expression on a large subpopulation of both high low with bacteria, uninfected bystander cells, or both. This was of in- GFP and GFP cells (Fig. 1D). These experiments provided terest because the subset of cells with productive intracellular in- strong evidence that group 1 CD1 proteins are up-regulated over a fection had the greatest contact with bacteria and might have re- period of 2–3 days on infected and bystander cells alike. ceived the highest doses of CD1-inducing products. In contrast, http://www.jimmunol.org/ prior studies of mycobacteria-infected cells conducted with mixed CD1 induction is mediated by mycobacterial lipids agonist and antagonist signals suggested that live mycobacteria The appearance of group 1 CD1 proteins on uninfected bystander can inhibit CD1 expression. In particular, previous studies had cells suggested that the mechanism of CD1 induction involved found that when monocytes were first treated with GM-CSF and secretion or cell-to-cell transfer of stimulatory factors that were IL-4 so that they expressed high levels of CD1 before mycobac- derived from the mycobacterium, the monocytes, or the interaction terial infection, group 1 CD1 protein expression was either unaf- of the mycobacterium with the monocytes. Further studies showed by guest on October 2, 2021

FIGURE 1. Mycobacterial infection of monocytes induces group 1 CD1 proteins on the surface of infected and bystander cells. A, Fresh human monocytes were infected with M. tuberculosis H37Ra (10 bacteria/cell) for 4 h, washed extensively, and cultured before flow cytometric detection of cell surface CD1. The percentage of cells staining with mAb against CD1a (OKT6), CD1b (BCD1b3.1), or CD1c (F10/21A3.1) above background levels seen with an isotype-matched mAb (P3) is shown. B, Monocytes were infected with M. tb-GFP, washed, and cultured for 3 days before staining with anti-CD1a and anti-mouse Ig PE (red). Representative phase contrast and overlay immunofluorescence micrographs show four cells, three of which express CD1a and one of which is infected. C, The upper panel is a representative confocal micrograph of a human monocyte 2 days after M. tb-GFP infection and is stained with anti-CD1b (red) with colocalization shown in yellow. The lower panel is a composite image of the Z plane corresponding to the superimposed line in the upper panel. D, Two-color flow cytometric analysis of monocytes after 2 days of culture in medium or infection with M. tb-GFP and staining with Ј Ͼ anti-CD1a, anti-CD1b (4A7.6.5), anti-CD1c, or control (P3) Ab and FITC-labeled goat anti-mouse F(ab )2. These experiments are representative of 10 experiments performed. The Journal of Immunology 1761

tuberculosis infection in terms of the kinetics of induction, percentage of cells involved, and absolute levels of cell surface expression (Figs. 1A and 2). CD1 induction involves new protein synthesis Flow cytometric analysis of cells treated with M. tuberculosis lipids showed that CD1d expression was not up-regulated, in contrast with the results seen for CD1a, CD1b, and CD1c (Fig. 3A). Com- pared with medium-treated cells, B7 costimulatory molecules (CD80, CD86) were up-regulated in parallel with group 1 CD1 proteins. Similar to previous reports of an inability of M. tuberculosis to promote the complete maturation of DCs (41), cells did not express CD83 (Fig. 3A). To further characterize the phenotype of the subset of monocytes that differentiated into CD1-expressing cells, we conducted two-color flow cytometry of cells for markers of myeloid cell differentiation on cells that fell into a gate defined by high CD1a expression. CD1aϩ cells were found to down-regulate CD14 and CD64 in response to M. tuberculosis lipid treatment, so they did not have a surface phenotype corresponding to activated macrophages (Fig. 3B). Instead, CD1aϩ cells uniformly Downloaded from up-regulated CD11c to high levels and showed modest increases in DC-specific ICAM-3-grabbing nonintegrin (CD209) expression and high levels of MHC class II. Thus, mycobacteria lipid-treated FIGURE 2. Polar lipids from M. tuberculosis induce cell surface expres- cells that came to express group 1 CD1 proteins also expressed sion of group 1 CD1 proteins by human myeloid cells. Whole M. tuberculosis many markers that are also found on immature myeloid DCs. strain H37Rv was lyophilized and extracted with chloroform:methanol (2:1) to Next, we sought to determine the cellular mechanism for the http://www.jimmunol.org/ obtain soluble M. tuberculosis lipid and a pellet composed of CWSs. The M. appearance of group 1 CD1 proteins at the surface of cells. Al- tuberculosis lipid fraction was loaded onto a silica chromatographic column though it was known that group 1 CD1 protein expression on my- that was eluted sequentially with chloroform, acetone, and methanol to yield eloid cells is dependent on activation (1), the precise cellular fractions enriched in neutral lipids, glycolipids, and polar lipids, respectively (6). Fractions were dried, sonicated into medium at 10 ␮g/ml (A and C)orthe mechanisms by which CD1 proteins come to be expressed on the indicated concentration (B), cultured with monocytes for 3 days or the indi- surface had not been previously investigated. For MHC Ag-pre- cated time, and stained as described in Fig. 1. senting molecules, peptide ligands can stabilize Ag complexes and thereby increase steady state levels of cell surface expression. The up-regulation of MHC class II during myeloid DC maturation also by guest on October 2, 2021 that killed mycobacteria and filtered supernatants of bacteria-con- prominently involves redirected transport of these complexes from ditioned culture medium could substitute for live M. tuberculosis late endosomes and lysosomes to the cell surface (42). Therefore, in inducing CD1 expression (data not shown). Therefore, this pro- we investigated these, as well as transcription, as candidate mech- cess did not require growth of live bacteria within cells and could anisms underlying the appearance of group 1 CD1 proteins on the be mediated by products physically shed from the cell wall. To cell surface. purify those factors, intact M. tuberculosis organisms were ex- M. tuberculosis lipid extracts that induced group 1 CD1 expres- tracted with chloroform:methanol to yield fractions that contained sion contained lipid ligands of CD1, but these did not account for organic solvent-soluble lipids that were not covalently attached to increased CD1 expression at the cell surface, as purified Ags pre- the cell wall (M. tuberculosis lipid) and pellets of insoluble cell sented by CD1a (DDM), CD1b (mycolic acid, GM1 ganglioside, wall skeletons (CWS) containing protein, hydrophilic small mol- and GMM), or CD1c (mannosyl phosphomycoketides) did not re- ecules, peptidoglycan, and the arabinogalactan complex. Whereas sult in detectable levels of CD1 protein at the cell surface, even CWS induced only trace levels of group 1 CD1 expression, the M. after 3 days of exposure to high Ag concentrations (Fig. 4A). tuberculosis lipid fraction potently mediated a dose-dependent re- This study (Fig. 1A) and prior studies have generally found that sponse that was similar to the CD1 induction seen during live M. monocytes do not have any detectable group 1 CD1 proteins at the

FIGURE 3. Differentiation markers of monocytes treated with M. tuberculosis lipids. A, After 3 days of culture with total M. tuberculosis lipids (10 ␮g/ml), cells were stained with mAb speciﬁc for CD80 (L307.4), CD86 (FUN-1), CD83 (HB15e), or CD1 (solid lines) or an isotype-matched mAb (P3, dotted line) and FITC-labeled Ј goat anti-mouse IgG F(ab )2. B, Fresh monocytes and monocytes treated with M. tuberculosis lipids were subjected to ﬂow cytometry to analyze the expression of CD64 (10.1), CD14 (M5E2), CD209 (eB-h209), CD11c (B-ly6), and HLA-DR (L243) on the CD1aϩ cells. These results are representative of more than three experiments. 1762 MICROBIAL ADJUVANTS FOR CD1 FUNCTION

of CD1d mRNA in fresh monocytes declined on the ﬁrst day of lipid treatment and then partially recovered by day 3 (Fig. 4B). The reduction of CD1d expression correlated with the reduction of cell surface CD1d protein expression under the same conditions (Fig. 3A). Therefore, we conclude that levels of group 1 and group 2 CD1 protein expression are oppositely regulated by exposure to mycobacterial cell wall products.

M. tuberculosis lipid stimulates lipid Ag uptake and processing Previous studies had shown that CD1 Ag presentation required not only cell surface expression of CD1 but also an efficient transport of exogenous lipid Ags into endosomes, where CD1-lipid complexes are formed (1, 4, 23, 25, 43). Therefore, we sought to determine whether M. tuberculosis lipids, in addition to inducing CD1 translation, could also promote Ag uptake and other cellular functions related to the processing of lipid Ags in the endosomal network. We found that untreated monocytes did not rapidly in- ternalize fluorescent dextran particles that serve as a marker for macropinocytosis. However, monocytes incubated with M. tuber- Downloaded from culosis total lipids for 3 days efficiently internalized both FITC-

dextran and radiolabeled C32 GMM, a CD1b-presented lipid Ag (Fig. 5, A and B).

FIGURE 4. CD1 up-regulation by M. tuberculosis lipids involves new

protein synthesis. A, Monocyte expression of CD1 was measured by ﬂow http://www.jimmunol.org/ cytometry after coculture for 3 days with lipids (10 ␮g/ml except for MPI that was used at a 10-fold excess of the concentration that stimulates T cells) that were known from previous studies to be presented by CD1a

(DDM), CD1b (GMM, mycolic acid, GM1 ganglioside), or CD1c (mannosyl phosphoisoprenoid (MPI)). B, After treatment of monocytes with M. tuberculosis lipids (10 ␮g/ml) for the indicated time, total RNA was extracted, retrotranscribed, and analyzed by RT-PCR using complementary oligonucleotide ﬂuorescent probes (Taqman reagent). CD1 mRNA levels were determined by the comparative Ct method relative to the expression of the housekeeping gene GAPDH. These results are representative of by guest on October 2, 2021 those seen in four experiments completed in triplicate and are expressed as the mean Ϯ SD.

surface (1), but it remained possible that preformed CD1 proteins might be present in intracellular compartments and that cell surface expression might have resulted from their release from lysosomal compartments and redirected traffic to the cell surface. How- ever, we found that fresh monocytes did not detectably stain with mAb against CD1, even when they were permeabilized such that high levels of MHC class II and the intracellular marker HLA-DM could be detected (data not shown). The lack of detectable group 1 CD1 proteins within and on the surface of monocytes suggested that the appearance of CD1 on the cell surface of lipid-activated monocytes resulted from new protein synthesis. To quantitatively measure CD1 gene transcription after exposure to M. tuberculosis lipid, we designed RT-PCR primers to FIGURE 5. Mycobacterial lipids have separate adjuvant and antigenic amplify isotype-specific sequences of the otherwise homologous effects on CD1-restricted T cells. A and B, Monocytes were cultured for 3 human CD1a, CD1b, CD1c, and CD1d genes. Primers for each days in medium alone (dashed line) or M. tuberculosis lipid (solid line; 10 CD1 isoform failed to cross-prime any other CD1 isoform and ␮g/ml) and then were incubated with FITC-labeled dextran for 1 h, showed similar sensitivity and amplification efficiency (data not washed, and analyzed by flow cytometry. Alternatively, monocytes were 14 shown). Compared with untreated or medium-treated monocytes, incubated with [ C]-labeled C32 GMM for 6 h, washed, and subjected to monocytes cultured with M. tuberculosis lipid were consistently scintillation counting to measure internalization. C, Monocytes were treated with medium or M. tuberculosis lipids (10 ␮g/ml), washed, and found to have 100- to 3000-fold increases in mRNA encoding ␮ incubated for 24 h with C80 GMM (2 M) and the CD1b-restricted T cell CD1a, CD1b, and CD1c. Comparison of group 1 CD1 mRNA and line LDN5. IL-2 in supernatants was measured by [3H]thymidine incorpo- protein production showed that they occurred with similar kinetics ration by the IL-2-dependent T cell line HT-2. D, Monocytes were treated and that the mRNA slightly preceded the appearance of CD1 pro- as in C and then cultured with DDM, CD1a-presented lipopeptide. These tein at the cell surface (Figs. 1A and 4B). In contrast with the cells were washed to remove Ag and were mixed with J.RT-3/CD8.2 cells increase in group 1 CD1 mRNA, we found that the absolute levels and subjected to flow cytometric analysis for calcium flux (7). The Journal of Immunology 1763

FIGURE 6. Monocyte response to human, mycobacterial, bacterial, protozoan, and fungal lipids. Total lipid extracts of human C1R B lymphoblastoid cells (human) or the indicated microbe were prepared by extraction of intact organisms with chloroform:methanol (2:1). Total lipid extracts were dried, sonicated into medium at 10 ␮g/ml, cocultured with human monocytes for 3 days, and subjected to ﬂow cytometry. These results are typical of more than three experiments. Downloaded from

To determine whether M. tuberculosis lipids could convert GMM, a CD1b-presented Ag that is processed in late endosomal monocytes into cells that have all the features necessary to present compartments (23), and DDM, a CD1a-presented Ag that requires exogenous lipid Ags to T cells, monocytes were treated with M. internalization into early endosomal compartments before being tuberculosis lipids for 3 days and were tested for presentation of recognized (5, 25). Monocytes that were pretreated with M. tuber-

lipid Ags that were known to require endosomal processing. The culosis lipid for 3 days and then pulsed with GMM or DDM Ags http://www.jimmunol.org/ mixture of M. tuberculosis lipids might have activated T cells by were found to activate Ag-specific T cells, but untreated mono- supplying cognate lipid Ags that bind CD1 and contact the TCR, cytes pulsed with Ag did not (Fig. 5, C and D). The failure of by stimulating CD1 Ag processing pathways or via both effects. It untreated monocytes to present GMM or DDM suggested that was possible to distinguish between these potential mechanisms by these two lipid Ags did not have CD1-inducing properties that using T cells, which specifically recognize lipid Ags that are made could substitute for the mixture of all cell wall lipids, and this was by M. tuberculosis in vivo, but are not produced at detectable confirmed in separate experiments in which these and other known levels under the nonpermissive conditions of in vitro growth used CD1-presented Ags failed to induce CD1 when applied at high

to prepare these M. tuberculosis lipid extracts. These Ags were C80 concentrations for 3 days (Fig. 4A). Thus, certain lipids within the by guest on October 2, 2021

FIGURE 7. TLR-2 mediates the induction of CD1 expression on monocytes. A, CHO cells (3E10) transfected with human TLR-2 and a reporter plasmid controlling CD25 expression (CHO-TLR-2) were treated overnight with medium, Pam3Cys (100 ng/ml), or polar M. tuberculosis lipids isolated as in Fig. 2A and further puriﬁed by one-dimensional silica TLC (1.5 ␮g/ml). Results are shown as the mean ratio of duplicate samples of CD25 expression by CHO-TLR-2 compared with mock-transfected CHO cells cultured in the same way. B, Monocytes were treated for 3 days with GM-CSF (300 U/ml) and ␮ ␮ IL-4 (200 U/ml) or M. tuberculosis total lipids (10 g/ml), M. tuberculosis dimannosyl phosphatidylinositol mannoside (PIM2;5 g/ml), Mycobacterium smegmatis araLAM (5 ␮g/ml), or M. tuberculosis mannose-capped LAM (Man LAM; 5 ␮g/ml). C, Monocytes were cultured for 3 days with medium alone

or with different concentrations of synthetic Pam3Cys (100 and 10 ng/ml) and were analyzed as in A. D, Monocytes were treated for 3 days in medium, in the presence of M. tuberculosis total lipids (10 ␮g/ml) or with unmethylated CpG oligonucleotides or poly(I:C) at the indicated concentrations. E, Monocytes were cultured for 3 days with M. tuberculosis lipids (10 ␮g/ml) in the absence or presence of a TLR-2 neutralizing Ab (T2.5) or an isotype control Ab (30 ␮g/ml). Data in this figure are representative of at least three experiments. 1764 MICROBIAL ADJUVANTS FOR CD1 FUNCTION mycobacterial cell wall function as Ags that bind to CD1, and our findings identify a new function for TLR-2 as an initiator of others function as adjuvants that initiate CD1 expression. group 1 CD1 Ag presentation pathways (38, 44–46, 50, 51). All natural and synthetic TLR-2 agonists tested were sufficient to stim- CD1 induction is mediated by microbial agonists of TLR-2 ulate CD1 expression, and inhibition of TLR-2 function with To identify the lipid stimulants of CD1, we first screened lipid blocking Abs provided evidence that signaling through TLR-2 was extracts from a variety of microbial pathogens and human cells. necessary to initiate the differentiation of human monocytes in Lipids from human cells, protozoa (S. mansoni, L. donovani), and response to mycobacterial lipids (Fig. 7). Because M. tuberculosis fungi (C. albicans, A. niger) did not induce group 1 CD1 expres- lipids also promoted lipid Ag uptake and were sufficient to allow sion, and a Gram-negative bacterium (Salmonella typhimurium) monocytes to mediate T cell activation by Ags that require endo- induced only trace levels of CD1. Lipid mixtures from S. aureus somal processing (Fig. 5), this appears to be a coordinated pathway and all four tested species of mycobacteria (M. tuberculosis of cellular differentiation that converts monocytes into fully com- H37Ra, M. tuberculosis H37Rv, M. phlei, and M. avium) stimu- petent lipid APCs. In terms of their cell surface phenotype lated high levels of CD1a, CD1b, and CD1c expression (Fig. 6). (CD14lowCD11chigh, intermediate DC-specific ICAM-3-grabbing None of the lipid preparations up-regulated CD1d expression. This nonintegrin), high Ag uptake capacity, and their ability to secrete initial screen provided evidence that CD1-inducing factors were IL-12 (data not shown), these mycobacterial lipid-treated cells not likely to be abundant lipids that compose the membranes of have acquired the phenotype of DCs, which have not reached full human or other eukaryotic cells. Instead, any potent stimulatory maturity such that they express CD83 (27). These results are in factors were conserved among a more limited range of mycobac- agreement with prior studies showing that M. tuberculosis does not terial and bacterial species. fully mature DCs such that they express CD83 and are able to Furthermore, several features of these patterns of reactivity im- strongly prime MHC class II-restricted cells (52). However, any Downloaded from plicated TLR-2 in mediating the response. The staphylococcal and potential maturation arrest induced by mycobacteria is not suffi- mycobacterial species that induced CD1 represented two types of cient to block the expression or function of group 1 CD1 proteins. pathogens that were known to produce TLR-2 agonists (44–46), Both infected and lipid-activated cells showed increases in cell and the monocytes under study expressed TLR-2 (data not shown). surface expression of group 1 CD1 and MHC class II protein, but In addition, we found that CD1-inducing lipid extracts from M. our data show that the cellular mechanisms of up-regulation are tuberculosis also had TLR-2 agonistic properties, as CHO cells distinct. MHC class II normally increases from intermediate to http://www.jimmunol.org/ expressing CD25 under an NF-␬B reporter construct were stimu- high levels via a mechanism that involves redistribution of pep- lated to express CD25 by mycobacterial lipids only when the cells tide-loaded MHC class II complexes from lysosomes to the cell were cotransfected with TLR-2 (CHO/TLR-2) (Fig. 7A). These surface over a period of 5 or more days (53, 54). In contrast, group observations led us to further purify the stimulatory factors from 1 CD1 proteins increased from undetectable to high levels within M. tuberculosis total lipids using open silica columns, and we 2 days after the contact with mycobacterial products (Fig. 1). The found that the CD1-inducing factors were detected exclusively in latter situation represents a more rapid, all or nothing mode of polar lipid fractions that are highly enriched for phospholipids induction that is mediated predominantly or wholly by increases in (Fig. 2A). M. tuberculosis produces two phospholipids that were transcription (Fig. 4). The acquisition of peptide and lipid Ag pro- by guest on October 2, 2021 known to agonize TLR-2: phosphatidylinositol mannoside (PIM) cessing pathways by maturing DCs may represent interrelated pro- and LAM with arabinosylated termini (araLAM) (47, 48). Highly cesses. For example, the appearance of group 1 CD1 proteins rel- purified preparations of araLAM and PIM induced group 1 CD1 atively early in the maturation process is consistent with their expression (Fig. 7B), but CD1 was not up-regulated in response to proposed function in activating CD1 autoreactive T cells that se- mannosylated LAM, a phospholipid that is structurally related to crete cytokines to promote the terminal phases of DC maturation araLAM. (55). In addition, the ability of M. tuberculosis lipids to up-regulate These results provided strong correlative evidence suggesting bulk flow mechanisms for acquisition of large antigenic particles that TLR-2 mediates CD1 induction, so we sought to more directly likely serves the dual purpose of bringing both lipids and proteins test this hypothesis with selective agonists and antagonists of TLR into endosomes for processing by DCs (Fig. 5, A and B) (44). function. A synthetic agonist of TLR-2, 3-bis(palmitoyloxy)-(2- The lack of expression of group 1 CD1 proteins on blood-de-

RS)-propyl-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,(P3Cys), rived monocytes, along with new evidence that TLR-2 selectively which mimicked the end-terminal lipopeptides of Borrelia burg- up-regulates group 1 CD1 but not group 2 CD1 expression, con- dorferi, potently induced group 1 CD1 proteins (Fig. 7C) (49). tributes to emerging ideas about the separate roles of group 1 and CD1 induction was not seen in response to unmethylated CpG group 2 CD1 proteins in immune responses. It has been convinc- oligonucleotides or polyinosinic-polycytidylic acid (poly(I:C)), ingly argued that the constitutive expression of CD1d on myeloid which agonize TLR-9 and TLR-3, respectively (Fig. 7D). As with cells and B cells, as well as its rapid presentation of endogenous intact Gram-negative bacteria, purified LPS, a TLR-4 agonist, only Ags to invariant NK T cells in vivo, points to a function in innate slightly induced CD1 when applied at 10 ng/ml, and higher con- immunity (56). However, human CD1a, CD1b, and CD1c do not centrations (Ͼ100 ng/ml) did not detectably induce CD1 (data not as clearly fit into this paradigm because their expression on DCs is shown). Last, CD1 induction by M. tuberculosis lipids was signif- delayed, and these isoforms present a more diverse array of self icantly inhibited in the presence of a neutralizing anti-TLR-2 Ab, and foreign Ags to T cells with diverse TCRs (35). The cellular demonstrating that TLR-2 activation is necessary for CD1 induc- signaling mechanisms by which TLR-2 agonism leads to CD1 ex- tion by mycobacterial lipids (Fig. 7E). Thus, these cellular studies pression likely involved NF-␬B, but have not yet been compre- indicate that CD1a, CD1b, and CD1c represent an inducible Ag hensively investigated. The 24- to 48-h delay in the appearance of processing system, whose function is regulated by microbial prod- high levels of group 1 CD1 transcripts and proteins after exposure ucts and an innate pattern recognition receptor, TLR-2. to M. tuberculosis suggests the possibility that this process may involve cytokine secretion. Discussion Our results provide evidence that group 1 CD1 expression is TLR-2 plays a central role in mediating interactions between my- controlled by an innate pattern recognition receptor for bacterial eloid cells and mycobacterial and staphylococcal pathogens, and products, and they show how group 1 CD1-restricted T cells can be The Journal of Immunology 1765 most efficiently stimulated after APCs are preactivated by local Hermann Wagner and Guangxun Meng helped in producing the TLR-2 contact with microbial cell wall products (Figs. 5 and 7). The abil- neutralizing Ab. Joel Ernst and Lalita Ramakrishnan kindly provided M. ity of microbial products and innate receptors to activate CD1 Ag tb-GFP, and Laurie Glimcher supported development of the RT-PCR processing can be compared with other systems in which anteced- system. ent activation of the innate system promotes subsequent adaptive responses, such as the role of CD19 ligation in B cells or the role Disclosures of TLR-mediated priming of MHC-restricted T cells (57, 58). The authors have no financial conflict of interest. Viewed in this way, the regulated and inducible expression of CD1a, CD1b, and CD1c on maturing DCs can be contrasted with References that of CD1d-restricted NK T cells, as the former likely represents 1. Porcelli, S., C. T. Morita, and M. B. Brenner. 1992. CD1b restricts the response of human CD4Ϫ8Ϫ T lymphoyctes to a microbial antigen. Nature 360: 593–597. a delayed pathogen recognition system rather than an innate re- 2. Fischer, K., E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, sponse. Furthermore, the ability of a single microbial stimulus to R. Hurwitz, M. Kursar, M. Bonneville, S. H. Kaufmann, and U. E. Schaible. oppositely regulate group 1 and 2 protein expression points to 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA 101: 10685–10690. distinct roles for the two groups of CD1 proteins in immune 3. Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, and responses. M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted ␣␤ T cells. This kind of system suggests a model in which mycobacterial Nature 372: 691–694. 4. Sieling, P. A., D. Chatterjee, S. A. Porcelli, T. I. Prigozy, R. J. Mazzaccaro, cell walls are complex mixtures of lipids that have two separate T. Soriano, B. R. Bloom, M. B. Brenner, M. Kronenberg, and P. J. Brennan. functions in simultaneously inducing CD1 Ag processing path- 1995. CD1-restricted T cell recognition of microbial lipoglycan antigens. Science ways and providing lipids that enter cellular Ag processing path- 269: 227–230.

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