Cells from Bone Marrow Dendritic Induce the Differentiation of Antigens Mycobacterium tuberculosis

of April 4, 2015.This information is current as

Bone Marrowthe Differentiation of Dendritic Cells from

Antigens InduceMycobacterium tuberculosis

Krishnamurthy NatarajanVinoth K. Latchumanan, Balwan Singh, Pawan Sharma and

http://www.jimmunol.org/content/169/12/6856doi: 10.4049/jimmunol.169.12.6856

2002; 169:6856-6864; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2002 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Mycobacterium tuberculosis Antigens Induce the Differentiationof Dendritic Cells from Bone Marrow1

Vinoth K. Latchumanan, Balwan Singh, Pawan Sharma, and Krishnamurthy Natarajan2

We show in this study that incubation of freshly isolated bone marrow cells with Mycobacterium tuberculosis (M. tb) secretory Ag(MTSA), in the absence of any growth or differentiation-inducing factor, differentiates them into dendritic cell (DC)-like APCs.These DCs expressed moderate to high levels of various markers typical of DCs. These included T cell costimulatory moleculesCD80, CD86, CD40, and CD54 and high levels of surface MHC class I and II on CD11c� cells. The levels and the kinetics ofup-regulation of these molecules were comparable with those of GM-CSF-differentiated DCs. Furthermore, these DCs exhibitedmorphology characteristics to DCs like the presence of dendritic processes. These DCs were also potent stimulators of allogeneicT cells and preferentially induced the secretion of IFN-� over IL-10 from the interacting T cells. Interestingly, the differentiationof bone marrow cells into DC-like APCs was obtained with many other M. tb Ags, including whole cell extract of M. tb. Furthercharacterization of MTSA-differentiated DCs showed that they were immature in nature, as stimulation of these DCs with TNF-�,anti-CD40, or LPS further up-regulated the surface levels of various molecules together with an increase in their T cell stimulatorycapacity. The Ag-specific T cell responses of MTSA-differentiated DCs were mainly contributed by the CD4� subset, indicatingthat MTSA was largely MHC II restricted. Furthermore, stimulation of bone marrow cells with MTSA induced the nucleartranslocation of the transcription factor NF-�B, thereby indicating its role during MTSA-induced differentiation of DCs. TheJournal of Immunology, 2002, 169: 6856–6864.

Mycobacterium tuberculosis (M. tb)3 continues to causemortality and morbidity throughout the world, resultingin 3 million deaths and over 8 million new cases of

tuberculosis each year (1–3). This problem is further compoundedby the varied efficacy of immunizations withMycobacterium bovisbacillus Calmette-Guerin (BCG), still considered to be the goldstandard against which all other vaccines are measured (4–7). Thisthus underscores the need to identify and elucidate factors that playa determinant role in generating protective immune responsesagainst this pathogen (8, 9). Mycobacteria persist in macrophageswithin the granuloma in the organs of infected hosts (10). Numer-ous proteins secreted from the phagosomes of infected macro-phages serve as targets for APCs recruited at sites of infection(11–15).

Different subsets of dendritic cells (DCs) are among the mostpotent APCs of the innate immune system that have the ability tostimulate quiescent, naive, or memory T lymphocytes (16). DCsexist at various states of development, activation, and maturationthat are defined by distinct phenotypic and functional modalities(17). For instance, DCs develop in the bone marrow (BM) and aresubsequently transported to the periphery, such as lung epithelia,

mucosae, and the like. These DCs and their precursors also patrolbody fluids such as blood and lymph and are primarily immaturein nature, meaning whereby that they are programmed for Ag cap-ture and display very low levels of T cell stimulatory properties.Upon contact with various stimuli, such as LPS, TNF-�, CD40ligand (by way of cognate interactions with T cells), and certainAgs (16, 17), they undergo a process of maturation. In this study,they up-regulate their MHC (class I and II) and costimulatory mol-ecules (CD80, CD86, CD40, CD54) and are very efficient T cellstimulators (18, 19).

In this study, we looked at the interactions of a recently de-scribed 10-kDaM. tb-specific Ag (hereafter referred as MTSA)(20) with leukocyte precursors, with a view to understand the in-teractions of mycobacterial secretory Ags with DC precursors. Ourresults indicate that MTSA and otherM. tb Ags induce the differ-entiation of BM cells into DC-like APCs. The possible implica-tions of M. tb-induced DC differentiation are discussed.

Materials and MethodsAnimals

Female BALB/c mice 4–6 wk of age were used in the study for all ex-periments involving DCs. For enrichment of T cells, either BALB/c orC57BL/6 mice were used. All the animals were maintained under patho-gen-free environment-controlled conditions in the small animal facility ofour institute. The study was undertaken after prior approval from the In-stitutional Animal Ethics Committee.

Materials

FITC-tagged mAbs against mouse cell surface molecules CD80 (clone1G10), CD86 (clone GL-1), CD40 (clone 3/23), CD54 (clone 3E2), I-Ad

(clone AMS-32.1), H-2Dd (clone 3-25.4), CD16/CD32 (FC�R, clone2.4G2), and biotin-conjugated Abs to CD11c (clone HL3), CD11b (cloneM1/70), CD25 (clone 7D4), CD45R (B220) (clone RA6-3B2), CD43(clone S7), CD90 (clone 53-2.1), and PE-conjugated CD4 (clone GK 1.5)and CD8a (clone 56-6.1), and purified CD16/CD32 (FC�R, clone 2.4G2),anti-CD40 (clones HM-40), and isotype-matched control Abs were pur-chased from BD PharMingen (San Diego, CA). FITC-conjugated Ab to

Immunology Group, International Center for Genetic Engineering and Biotechnology,Aruna Asaf Ali Marg, New Delhi, India

Received for publication May 16, 2002. Accepted for publication October 16, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Defense Research and Development OrganizationGrant DALS/48222/LSRB/22/ID/RD/-81 to K.N. and P.S.2 Address correspondence and reprint requests to Dr. Krishnamurthy Natarajan, Im-munology Group, International Center for Genetic Engineering and Biotechnology,Aruna Asaf Ali Marg, New Delhi 110 067, India. E-mail address: [email protected] Abbreviations used in this paper:M. tb, Mycobacterium tuberculosis; BCG, bacillusCalmette-Guerin; BM, bone marrow; DC, dendritic cell; MTSA,M. tb secretory Ag.

The Journal of Immunology

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F4/80 (clone CI: A3-1) and DEC 205 (clone NLDC 145) were obtainedfrom Serotec (Oxford, U.K.). Anti-CD4-, anti-CD8-, anti-CD90 (Thy-1.2)-, anti-B220-, anti-CD11b-, anti-CD11c-, anti-I-A-, and anti-CD19-coated magnetic beads were obtained from Miltenyi Biotec (Auburn, CA).Mouse rGM-CSF and ELISA kits for the estimation of mouse cytokineswere purchased from R&D Systems (Cambridge, MA). RecombinantTNF-�, LPS, and polymixin B sulfate and E-Toxate endotoxin detectionkit were obtained from Sigma-Aldrich (St. Louis, MO). Ab to NF-�B p65subunit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Purified M. tb Ags ESAT-6, Ag85b, and MPT64, and M. tb whole cellextract were obtained from J. T. Belisle (Colorado State University, PortCollins, CO) under the National Institutes of Health, National Institute ofAllergy and Infectious Diseases Contract AI-75320, entitled TuberculosisResearch Materials and Vaccine Testing. The details of their preparationand composition can be viewed at http://www.cvmbs.colostate.edu/micro-biology/tb. The purity of these Ags obtained were further analyzed bySDS-PAGE, followed by silver staining. Furthermore, possible MTSApresence in the whole cell extract of M. tb was removed by incubation withrabbit anti-MTSA Ab, followed by immunoprecipitation with protein G-conjugated agarose beads.

Expression and purification of MTSA

MTSA (M. tb Rv3874) was PCR amplified from the genomic DNA of alocal clinical isolate of M. tb, and the recombinant protein was expressedas a polyhistidine-tagged protein (GenBank Accession AF419854) in Esch-erichia coli using the pQE31 vector (Qiagen, Valencia, CA). To excludethe possibility of endotoxin contamination in the recombinant protein, en-dotoxin levels were estimated in all the batches of MTSA (and other M. tbproteins) used in the study by utilizing the E-Toxate (Limulus amebocytelysate; Sigma-Aldrich) kit. The endotoxin levels in all the proteins werefound to be below 0.03 endotoxin units (data not shown). Furthermore, wealso cultured BM cells with either heat-inactivated MTSA or MTSA pre-treated with polymyxin B sulfate, known to inactivate LPS and its effects(21). Polymyxin B sulfate-treated MTSA, but not heat-inactivated MTSA,differentiated BM cells into DCs (data not shown). These results confirmedthat the observed effects were a result of MTSA and not by any contam-ination by endotoxin(s) in the recombinant protein.

Enrichment of DC precursors from BM

A total of 3 � 106 lymphoid and I-A�-depleted BM cells from the tibiasand femurs of BALB/c mice were cultured in six-well culture plates inRPMI 1640 medium containing 10% FCS, 0.05 M 2-ME, and 1 mM so-dium pyruvate, and stimulated with Ags for various lengths of time. Forsome experiments, cells were cultured with 15 ng/ml of GM-CSF for 4days. Cells at the end of incubation in all sets were either analyzed for thelevels of surface molecules by flow cytometry, as described before (22), orcocultured with either allogeneic or Ag-primed syngeneic T cells, as de-scribed below. The cell yield and viability of each culture were estimatedby trypan blue exclusion and counting.

Microscopy

BM cells cultured with various Ags or GM-CSF were observed under aNikon DiaPhot phase-contrast microscope and photographed on KodakDX400 film using Nikon U-III camera. In some cases, cells were firststained for surface CD11c using biotinylated Ab, followed by streptavidin-PE. Aliquot of cells was mounted on glass slides and observed under mi-croscope using Nikon G2A filter. The fluorescent cells thus observed werethen photographed.

Enrichment of T lymphocytes

This was done as described previously (23). Briefly, either inguinal lymphnodes or splenocytes from 4- to 6-wk-old BALB/c or C57BL/6 mice, re-spectively, were first depleted of adherent cells by panning over plasticplates. From this, B lymphocytes were then removed by two rounds ofincubation with anti-CD19- and anti-CD45R-coated magnetic beads, fol-lowed by separation through MACS columns. The purity of the resultingpopulation of T cells obtained in this fashion was 95–98%, as determinedby CD90-PE-stained cells by flow cytometry. For some experiments, en-riched T cells from the lymph nodes of BALB/c mice were further nega-tively selected as CD4� or CD8� populations using anti-CD4- or anti-CD8-coated magnetic beads and purification over MACS columns. Thepercentage of I-A� cells in all the fractions was found to be less than 0.5%.

T cell stimulation: allogeneic MLRs and T cell proliferation

Allogeneic C57BL/6 T cells were enriched from spleens, and 3 � 106

enriched T cells were cocultured with irradiated (3000 rad) MTSA-differ-

entiated DCs at various DC:T cell ratios in 24-well plates for a period of48 h. Culture supernatants were then screened for the presence of cyto-kines, as described below. For measuring T cell proliferation, differentnumbers of irradiated MTSA-differentiated DCs were cocultured with 3 �105 allogeneic C57BL/6 T cells for 3 days in 96-well U-bottom culturesdishes. The cells were pulsed with 1.0 �Ci/well of [3H]thymidine 16 hbefore harvesting and counting.

Syngeneic T cell stimulation

BALB/c mice were immunized s.c. at base of tail with MTSA (50 �g/mouse) in IFA for 7 days and boosted with a repeat immunization for anadditional 7 days. Inguinal lymph nodes from these mice were removed,and T cells (both CD4� and CD8�) were enriched, as described above.Enriched T cells were cocultured with irradiated DCs at a 5:1 ratio for 48 h,and culture supernatants were analyzed for cytokines. T cell proliferationof Ag-primed T cells cocultured with MTSA-differentiated DCs was mea-sured, as described above.

Estimation of cytokines

Culture supernatants of DC-T cell cocultures at the end of each incubationperiod were analyzed for the levels of IL-2, IFN-�, or IL-10 using a sand-wich ELISA, as recommended by the manufacturer. The sensitivity rangefor the cytokines was as follows: IL-2, 15–1000 pg/ml; IFN-�, 31.2–2000pg/ml; and IL-10, 31.2–2000 pg/ml. Quantitation was made against a stan-dard curve obtained for individual cytokine standards provided by the man-ufacturer. Samples were correspondingly diluted to obtain values withinthe linear range of the standards.

Analysis of NF-�B activation

BM cells were stimulated with either 10 �g/ml of MTSA or 15 ng/ml ofGM-CSF for varying periods of time. At the end of the incubation, cellswere chilled on ice and washed once with ice-cold PBS and lysed in lysisbuffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 M EGTA,0.5% Nonidet P-40, and 2 �g/ml each of aprotinin, leupeptin, and pepsta-tin). The resultant nuclear pellet was then extracted in buffer containing 20mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 2�g/ml each of aprotinin, leupeptin, and pepstatin. Nuclear extracts from5 � 106 cells were then resolved on 10% SDS-polyacrylamide gels andsubsequently transferred onto a nitrocellulose membrane (Hybond C pure;Amersham, Arlington Heights, IL). The blots were then probed with Ab toNF-�B p65 subunit, followed by HRP-labeled secondary Abs. The blotswere later developed by chemiluminescence using the ECL kit fromAmersham.

ResultsMTSA induces the differentiation of DC-like APCs fromBM cells

A recent report identified MTSA, which was able to prime de-layed-type hypersensitivity responses in M. tb-infected guineapigs, but not in animals infected with M. bovis BCG (20). A prod-uct of Rv3874 gene in the mycobacterial genome, MTSA is notexpressed by other members of the mycobacterial complex,namely, Mycobacterium avium and M. bovis BCG, among others.Recent reports documented the relevance of MTSA (also known asCFP-10) in generating protective immune responses against M. tb(24). CFP-10-pulsed monocyte-derived DCs were used to isolateCD8� T cell clones that interacted with M. tb, but not M. bovisBCG-infected targets (25). Furthermore, owing to its absence in M.bovis BCG strains used for vaccinations, CFP-10 has been pro-posed as an important candidate in the diagnosis of M. tb (26, 27).Therefore, in light of the above, we argued that MTSA wouldmake an ideal model to study M. tb-specific immune responses atsites of infection. To begin with, we examined the interactions ofMTSA with leukocyte lineage precursors in the BM, as reflectedby changes in the levels of cell surface molecules. For this, weincubated BM cells with various doses of MTSA and for differentperiods of time. Aliquots of cells were analyzed for the levels ofmolecules listed in Fig. 1A. As shown, culturing BM cells with 10�g/ml of MTSA up-regulated the levels of many molecules on thecell surface. These included both CD11c and CD11b. The bimodal

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nature of the peak in both CD11c and CD11b suggests that somecells (�45%) were negative for these two markers. Furthermore,the CD11c� cells were found to express high levels of CD80(B7.1) and low levels of CD86 (B7.2). Majority of the CD11c�

cells expressed high levels of MHC class I (H-2D) and MHC classII (I-A) molecules. They were positive for CD40 and expressedhigh levels of CD54 (ICAM-1). These cells also stained positivefor the DC maturation marker F4/80 Ag (28) and CD25, the IL-2R�-chain. A side-by-side comparison of MTSA-stimulated cellswas made with GM-CSF-stimulated cells, which is the conven-tional method used to generate DCs from BM cells (29). It is clearfrom Fig. 1A that the levels of MHC class I and MHC class II andcostimulatory molecules CD80, CD86, CD40, and CD54 were up-regulated to a similar degree in both MTSA- and GM-CSF-stim-ulated cultures. However, the levels of CD11c and CD11b weremuch higher in GM-CSF-stimulated cultures when compared withthose incubated with MTSA. MTSA-stimulated cultures stainednegative for FcR (CD16/CD32), which was expressed in GM-CSF-stimulated cells. Barring these minor differences, both GM-CSF- and MTSA-differentiated cells essentially expressed similar

profiles of all the markers depicted in Fig. 1A. This also includedthe absence of DEC205, CD8a, and CD4 molecules that are ex-pressed primarily by plasmacytoid DCs (16, 17), indicating thatlike GM-CSF, MTSA-differentiated DCs were of myeloid origin.The CD11c�/CD11b� cells in MTSA DCs, however, were posi-tive for all the other molecules that were expressed on CD11c�/CD11b� cells (not shown), a characteristic observed on a subset ofhuman plasmacytoid DCs (30). For the current study, both theCD11c� and CD11c� populations were collectively used.

Both MTSA- and GM-CSF-stimulated cells did not expressCD45R (B220), CD90, or CD43, markers specific for B and Tlymphocytes and macrophages, respectively. These results thus in-dicate that MTSA induces the differentiation of DCs from BMleukocyte precursors. Furthermore, the kinetics of expression ofthese markers followed a consistent pattern, wherein most of themolecules were up-regulated with similar kinetics, CD86 levelsbeing consistently low throughout the culture period in MTSA-differentiated DCs, when compared with GM-CSF-differentiatedDCs (see Fig. 1B). Up-regulation of most markers reached a peak

FIGURE 1. MTSA induces the differentiation ofDC-APCs from BM. A, Cell surface staining of indi-cated markers on BM cells cultured for 4 days either inmedium alone (None) or 10 �g/ml of MTSA or 15ng/ml of GM-CSF is shown by the thick line. The thinline depicts staining with the corresponding isotype-matched control Ab. CD11c and CD11b profiles are onpropidium iodide-excluded cells. The profiles of allother markers are on CD11c� cells. B, Shows the ki-netics of induction of the various molecules on eitherMTSA (a)- or GM-CSF (b)-stimulated BM cells as afunction of time; ‚, CD80; f, CD86; E, I-Ad; �,H2-Ld; Œ, CD40; F, CD54. Data shown are represen-tative of at least five such experiments.

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at 4 days of incubation. Dose-response profiles of MTSA (notshown) also showed a similar trend, with maximal differentiationof cells (that were also positive for markers shown in Fig. 1A)reaching a plateau at 10 �g/ml. Hence, for all subsequent exper-iments, BM cells were cultured with 10 �g/ml of MTSA for aperiod of 4 days for further processing or analyses.

We also estimated the cell yield and viability of each culture,and the results are depicted in Table I. As can be seen, the viabilityof cells cultured in medium alone decreases progressively witheach day of culture, and only 65% of the total cells initially platedwere found to be viable. In contrast, cells cultured with MTSAincreased in number by day 3, and by day 4 the total yield of viablecells went up by 30%. Similarly, the number of cells cultured withGM-CSF also increased, and were 155% of the total cells plated onday 0. The low level of cell expansion observed could possibly bea result of a single addition of either GM-CSF or MTSA as againstrepeated additions during the generation of DCs by conventional

methods (29). Furthermore, this yield and viability of MTSA-stim-ulated cultures were maintained for up to 6 days, and then startedto decrease by day 7 (not shown). The yield and viability of GM-CSF-stimulated cultures were, however, maintained beyond 7 daysof culture (not shown).

Phase-contrast microscopic examination of these cells showedthat MTSA-differentiated DCs displayed morphological character-istics typical of DCs, which included the presence of dendriticprocesses or veils (Fig. 2B). Some cells were, however, rounded inappearance. GM-CSF-differentiated DCs also displayed veils orprocesses in most cells (see Fig. 2C). Cells cultured in mediumalone did not show any processes or other features typical of DCsand were smaller and shriveled in appearance (see Fig. 2A). Fur-thermore, both MTSA- and GM-CSF-differentiated DCs stainedpositive for CD11c, as revealed by fluorescence microscopy (seeFig. 2, E and F). No fluorescence for CD11c was observed on cellscultured in medium alone (see Fig. 2D). The collective results inFigs. 1 and 2 thus suggest that MTSA induced the differentiationof cells that phenotypically and morphologically displayed fea-tures of DC-like APCs.

MTSA-differentiated DCs stimulate allogeneic T cells

One of the functional attributes of DCs is their remarkable abilityto stimulate allogeneic T cells at very low stimulator to responderratios. To ascertain that MTSA-stimulated BM cells were DC-likeAPCs, which were potent T cell stimulators, we cocultured themwith allogeneic T cells at various DC:T cell ratios, and the extentof T cell stimulation was assayed by measuring thymidine incor-poration, IL-2 production, as well as IFN-� and IL-10 secretion

FIGURE 2. MTSA-differentiated DCs display DC morphology. BM cells were cultured with MTSA or GM-CSF for 4 days. Cells were then observedunder Nikon Diaphot phase-contrast microscope and photographed (magnification �500) (see Materials and Methods). A–C, Shows morphology of cellscultured in medium alone, MTSA, and GM-CSF, respectively. An aliquot of cells from the above cultures was stained for CD11c. D–F, Depicts CD11cstaining on cells cultured in medium alone or MTSA or GM-CSF, respectively (magnification �500).

Table I. Recovery of viable cells during DC differentiationa

Group Day 1 Day 2 Day 3 Day 4

None 100 85 72 65MTSA 100 105 115 130GM-CSF 100 100 120 155

a A total of 3 � 106 cells were cultured with either MTSA or GM-CSF in RPMImedium for 4 days. Cell number and viability were estimated by counting trypanblue-excluded cells (see Materials and Methods). Values represent percentage ofviable cell recovery relative to control (100% at day 0 in all cultures). None, repre-sents cells cultured in medium alone.

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from allogeneic T cells. As shown, MTSA-differentiated DCsreadily stimulated T cells to secrete IL-2, IFN-�, and IL-10 atratios as low as 1:50 (see Fig. 3, B–D). The thymidine incorpora-tion by these T cells was also evident at ratios 1:50, thus indicatingthat MTSA-differentiated DCs were efficient T cell stimulators(Fig. 3A). Furthermore, the extent of either the thymidine incor-poration or cytokine production was comparable with GM-CSF-differentiated DCs, indicating that MTSA-differentiated DCs wereequally efficient in stimulating T cells when compared with DCsgenerated by conventional methods.

M. tb Ags induce differentiation of DCs from BM cells

To test whether the observations made in Figs. 1 and 2 were spe-cific to MTSA, we looked at the ability of various Ags to inducethe differentiation of DCs. For this, we chose three different M. tbAgs, ESAT-6, Ag 85b (Ag85b), and MPT64, and also M. tb wholecell extract. These Ags were chosen primarily on their demon-strated ability in potentiating immune responses to M. tb. Althoughboth MTSA and ESAT-6 are cotranscribed from the RD1 region ofthe Mycobacterium genome, they are translated as two separateproteins (31). ESAT-6 has been designated as an important T cellAg recognized by protective T cells in animal models of infectionwith M. tb (32). Furthermore, together with MPT64, ESAT-6 hasbeen shown to have potential in the diagnosis of M. tb, as they arerecognized by T cells in animal models (33, 34) of M. tb. LikeMTSA, both Ags have been found primarily in M. tb, but not inmost environmental mycobacteria or BCG (35, 36). MPT64 hasbeen evaluated as a skin test reagent in the guinea pig models oftuberculosis (37) and in humans (38). They have been shown to

elicit delayed-type hypersensitivity responses in guinea pig modelsof tuberculosis. Furthermore, ESAT-6 has been considered as apotential candidate for subunit-based vaccines (39). Ag85b is amember of the Ag 85 complex, a family of fibronectin-bindingproteins involved in the mycobacterial cell wall biosynthesis (40).In addition, Ag 85b has been used as a potential DNA vaccinecandidate (41). All of these Ags are secreted into the extracellularmedium in axenic cultures, and some have been identified to besecreted from the phagosomes of mycobacteria-infected macro-phages (11). Therefore, in light of the above, we thought it wouldbe worthwhile to investigate whether the observed effects ofMTSA could be reproduced by these Ags. As the cell wall com-ponents of BCG have been shown to induce the maturation of DCs(42), we also tested the ability of whole cell extract of M. tb toinduce the differentiation of DCs from BM cells.

We first ascertained the purity of these Ags by resolving themon SDS-PAGE, followed by silver staining of the gels. As shownin Fig. 4A, all these proteins were found to be essentially pure, asrevealed by the presence of a single band at the right sizes. Fur-thermore, no presence of MTSA in any of these Ags was observed.In addition, to rule out the presence of MTSA in the whole cellextract of M. tb, the same was incubated with anti-MTSA Ab,followed by immunoprecipitations using protein G-conjugatedagarose beads. Fig. 4B shows the representative FACS profiles ofsurface levels of various markers on cells cultured with differentM. tb Ags. As shown, all the four Ags reproduced the effects ofMTSA by differentiating BM cells into DC-like APCs, with highexpression of surface MHC and costimulatory molecule. The lev-els of up-regulation of all the molecules were the highest, with cellextract and relatively lowest in MPT64-differentiated DCs. Mor-phologically also, these cells resembled DCs with the presence ofdendritic processes (see Fig. 5). These results thus indicate that M.tb Ags induce the differentiation of DCs from BM precursors.

We also tested the ability of various other Ags, including thehepatitis E virus ORF3, Plasmodium falciparum MSP119, keyholelimpet hemocyanin, OVA, and chicken egg lysozyme, to differen-tiate BM cells into DCs. These Ags failed to either up-regulate thecell surface markers or induce changes in morphology of BM cells(data not shown), indicating that only M. tb Ags (at least the onestested) were able to differentiate BM cells into DCs.

MTSA-differentiated DCs are immature in nature

It is well known that DCs present at various stages of activationand maturation perform distinct functions that are characterized byAg capture vs T cell stimulation (16). Immature DCs are proficientat Ag capture, while mature DCs are effective T cell stimulators.We, therefore, investigated the maturation status of MTSA-differ-entiated DCs by further separately stimulating them with matura-tion-inducing factors such as TNF-�, anti-CD40, and LPS (17).Their effects were evaluated at the levels of modulation in the cellsurface densities of various costimulatory and MHC molecules andthe subsequent effects on allogeneic T cell responses. Addition ofall the three stimuli individually led to an increase in the levels ofcostimulatory and MHC molecules (Fig. 6A). This increase wasalso reflected in the increased expression of IL-2, IFN-�, and IL-10levels from allogeneic T cells (see Fig. 6B). The results depicted inFig. 6, therefore, indicate that MTSA-differentiated DCs were onlypartially mature. Furthermore, there was no apparent effect on themorphology of MTSA-differentiated DCs upon stimulation withany of the maturation-inducing stimuli (data not shown). However,cell yield in the LPS-stimulated MTSA-differentiated DCs did

FIGURE 3. MTSA-differentiated DCs stimulate allogeneic T cells. A,3 � 105 allogeneic C57BL/6 T cells were cocultured with different num-bers of irradiated MTSA- or GM-CSF-differentiated DCs (see Materialsand Methods) in 0.2-ml culture volume in 96-well plates for a period of72 h. Cultures were pulsed with 1.0 �Ci/well of [3H]thymidine 16 h beforeharvesting and counting. B–D, 3 � 106 C57BL/6 T cells were coculturedwith different numbers of irradiated MTSA- or GM-CSF-differentiatedDCs for 48 h, and culture supernatants were then screened for the levels ofindicated cytokines. None, represents cells cultured in medium alone. Oneof four independent experiments is shown.

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show an increase by 20% when compared with MTSA-differenti-ated DCs (not shown). The cell viability in all cultures remainedabove 98%.

MTSA-induced T cell responses are MHC class II restricted

To check whether MTSA is processed and presented during thecourse of DC differentiation, we next explored the kind of Ag-

specific T cell responses that are regulated by MTSA-differentiatedDCs. MTSA-differentiated DCs were cocultured with either un-fractionated or CD4� or CD8� MTSA-specific T cell subsets, andthe extent of T cell stimulation was monitored, as reflected inlevels of thymidine incorporation and secretion of IL-2, IFN-�,and IL-10 from the interacting T cells. As shown in Fig. 7, andconsistent with allogeneic T cell responses, MTSA-differentiated

FIGURE 5. DCs differentiated by M. tbAgs show dendritic morphology. BM cellswere cultured with M. tb Ags for 4 days andobserved under phase-contrast microscopeand photographed as before (magnification�500). A–D, Represents cells cultured withESAT-6, Ag85b, MPT64, and M. tb cell ex-tract, respectively.

FIGURE 4. MTSA induce differentiationof DCs. A, 1 �g of different Ags was elec-trophoresed on a 12.5% SDS-polyacryl-amide gel, followed by silver nitrate stain-ing. B, Thick line shows the representativeFACS profiles of indicated surface markerson BM cells cultured with 10 �g/ml of var-ious M. tb Ags for 4 days. The thin line de-picts staining with the isotype-matched con-trol Ab. Data from one of three experimentsare shown.

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DCs stimulated Ag-specific T cells with high levels of thymidineincorporation and IL-2 production (see Fig. 7, A and B). Higherlevels of IFN-� were observed when compared with IL-10 (Fig. 7,compare C with D). Furthermore, major contribution toward T cellstimulation was obtained by the CD4� T cell subset, indicatingthat MTSA was primarily MHC class II restricted. CD8� T cell-mediated responses were only marginal. This was further con-firmed in coculture experiments wherein addition of an antagonistanti-MHC class II Ab reduced the IFN-� responses to near basallevels, while the addition of anti-MHC class I Ab had marginaleffects on both IFN-� and IL-10 responses (data not shown). Theseresults are in agreement with studies that document the dominanceof CD4� over CD8� T cell responses during early infection by M.tb (10). Furthermore, as no significant proliferation or cytokinesecretion was observed in groups in which GM-CSF-differentiatedDCs were cocultured with MTSA-specific T cells, these resultsindicate that the observed responses from MTSA-differentiatedDCs were indeed Ag-specific T cell responses and not a result ofsyngeneic MLRs.

Differentiation of DCs by MTSA is mediated by NF-�B

Activation of NF-�B has been considered as a hallmark of DCmaturation (43). Furthermore, NF-�B binding sites have been re-ported in the regulatory sequences (promoters and enhancers) ofcostimulatory and MHC molecules (44, 45). In addition, it hasrecently been reported that differentiation of DCs from monocytesby calcium ionophores or LPS involves the activation of NF-�B(46). To test whether MTSA-induced differentiation of DCs in-volved the activation of NF-�B, we stimulated BM cells with ei-ther MTSA or GM-CSF for varying periods of time and looked atthe nuclear translocation of the p65 subunit of NF-�B, which is the

predominant species translocated to the nucleus upon activationand has been shown to regulate multiple immune response genes(47). It is clear from Fig. 8 that p65 is indeed translocated to thenucleus in both MTSA- and GM-CSF-stimulated BM cells, al-though with different kinetics. NF-�B p65 could be seen in thenucleus by 30 min upon MTSA stimulation and was retained therefor up to 2 h, while in GM-CSF-stimulated cultures, it was acti-vated within 15 min, but disappeared by 2 h. Nevertheless, bothstimuli activated this transcription factor, which suggests that ac-tivation of this transcription factor could be one of the pathwaysused by MTSA to induce differentiation of BM cells into DC-likeAPCs. Furthermore, incubation of potent and specific inhibitors ofNF-�B such as N-acetylcysteine and capsaicin completely pre-vented the differentiation of DCs by MTSA (data not shown), in-dicating an obligatory role of this transcription factor in mediatingdifferentiation of DCs by MTSA.

FIGURE 6. MTSA-differentiated DCs are immature. Day 4 MTSA-differ-entiated DCs were incubated with either TNF-� (20 ng/ml), anti-CD40 (cloneHM-40; 20 �g/ml), or LPS (20 �g/ml) for 24 h. A, Cells were stained for thesurface levels of various markers. The thick line depicts levels in MTSA-differentiated DCs, while the dotted line depicts levels on MTSA-differentiatedDCs stimulated with either TNF-� or anti-CD40 or LPS, respectively. The thinline depicts levels on cells cultured in medium alone. B, An aliquot of 0.6 �106 cells from the above sets was cocultured with 3 � 106 C57BL/6 enrichedT cells for 48 h. Culture supernatants were then screened for the levels of IL-2(a), IFN-� (b), and IL-10 (c), respectively.

FIGURE 7. Ag-specific T cell responses induced by MTSA-differenti-ated DCs are CD4 restricted. A, 0.6 � 105 irradiated MTSA-differentiatedDCs were cocultured with either 3 � 105 unfractionated or CD4� or CD8�

MTSA-specific T cells in 96-well plates for 72 h. Cultures were pulsedwith 1.0 �Ci/well of [3H]thymidine 16 h before harvesting and counting.B–D, 0.6 � 106 irradiated MTSA-differentiated DCs were cocultured with3 � 106 unfractionated or CD4� or CD8� MTSA-specific T cells in 24-well plates for 48 h. Culture supernatants at the end of incubation periodwere screened for the levels of indicated cytokines. UT, CD4-T, andCD8-T represent unfractionated, CD4�, and CD8� MTSA-specific T cells,respectively. One of three experiments is shown.

FIGURE 8. Differentiation of DCs by MTSA involves nuclear translo-cation of NF-�B p65. BM cells were stimulated with either 10 �g/ml ofMTSA or 15 ng/ml of GM-CSF for indicated periods of time, and nuclearextracts were prepared (see Materials and Methods). These were resolvedon a 10% SDS-PAGE, Western blotted onto nitrocellulose membranes, andprobed with Ab to p65 subunit of NF-�B, followed by HRP-conjugatedsecondary Ab. Blots were later developed with ECL reagents (Amersham).M, represents unstimulated BM cells cultured in medium alone for 60 min.

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DiscussionM. tb Ags have been in focus for more than a decade ever sincetheir identification in axenic cultures. Since then, a number of stud-ies document the potential of these Ags in regulating immune re-sponses in animal models and in humans, which among othersincludes the use of these Ags as potential vaccine candidates in theform of either adjuvants or even DNA (32–41). Despite these stud-ies, lacunae still exist regarding their physiological roles at sites ofinfection. Only very recently has their in vivo presence been dem-onstrated (11). Recently, the 19-kDa lipoprotein of M. tb has beenshown to induce the maturation of DCs (48). Secretion of Agsfrom the phagosomal complex of infected macrophages into theextracellular matrix is likely to be followed up by their uptake byAPCs such as DCs and their precursors, which are recruited toinfected areas almost immediately following any infection. There-fore, the outcome of the interactions of these proteins with theAPCs may well constitute the driving force for the nature of im-mune responses to M. tb that are subsequently generated and caneventually determine the course of an infection.

In light of the above, the present study was undertaken to ex-plore for any possible interactions of MTSA with BM leukocyteprecursors. Our results demonstrate that incubation with MTSAdid induce the differentiation of BM cells into DC-like APCs. Thiswas based on the phenotypic and morphological attributes ob-served on these cells, which included the high cell surface expres-sion of a number of markers such as the MHC class I and II andT cell costimulatory molecules, CD80, CD86, CD40, and CD54 onCD11c� cells. Furthermore, the levels and kinetics of up-regula-tion of these molecules, as well as the morphological features, suchas the presence of dendritic processes or veils, were comparable tothose observed during GM-CSF-mediated DC differentiation.

Apart from phenotypic and morphological resemblance to GM-CSF-differentiated DCs, those obtained with MTSA were alsofunctionally competent, as they were equally efficient T cell stim-ulators and induced the proliferation of and cytokine secretionfrom the interacting allogeneic T cells. Indeed, MTSA was alsoprocessed and presented during the course of DC differentiation, asthese DCs efficiently stimulated the proliferation and secretion ofcytokines from MTSA-specific T cells. Furthermore, the Ag-spe-cific responses were dominated by the CD4� T cell subset, indi-cating that MTSA was largely presented on MHC class II. Inter-estingly, the differentiation of DCs from BM precursors was alsoobtained by three other immunologically well-characterized M. tbAgs, ESAT-6, Ag85b, and MPT64, and also by M. tb whole cellextract.

Differentiation and, for that matter, activation/maturation of DCprecursors by infectious agents or their components have determi-nant effects on the subsequently elicited immune responses. Mod-ulation of DC activation and function by a large number of patho-gens has been reported in the literature. For example, EBV inhibitsthe development of DCs by causing apoptosis of their monocyteprecursors (49). Prevention of DC activation/maturation by P. fal-ciparum-infected erythrocytes has also been recorded (50). Thesecells prevent the up-regulation of costimulatory and MHC mole-cules on immature DC precursors, and thus affect DC function.Infection of human DCs by Trypanosoma cruzi prevents their mat-uration by blocking the up-regulation of various T cell stimulatorymolecules, and also drastically inhibits the secretion of cytokinessuch as TNF-�, IL-12, etc. (51).

Furthermore, in addition to subsets of immature DCs, it is nowknown that two types of DC precursors arise from stem cells,monocytes (pre-DC1) and plasmacytoid cells (pre-DC2) (52). Pre-DC1 ingest and kill various bacteria and fungi, while pre-DC2

represent the key effector cells in early antiviral innate responsesby producing large amount of IFN-�/� upon viral infection (53).Unlike other effector cells of the innate immune system, pre-DC1and pre-DC2 have the capacity to differentiate into DCs, an eventthat occurs spontaneously in the absence of exogenous cytokineaddition (53). Depending upon the kind of stimulus, the subse-quently elicited T cell responses of these DCs also differ, indicat-ing functional plasticity in regulating T cell responses that resultfrom the differentiation-inducing stimulus (52).

Therefore, in light of the above findings, and when coupled withthe enormous potential of DCs in initiating primary immune re-sponses, the differentiation of DCs by M. tb Ags assumes paramountimportance in the subsequently generated immune responses to my-cobacteria early in the infection. As the MTSA-differentiated DCswere found to be primarily immature in nature, by definition, theyshould be responsive to a second challenge by Ag(s), which mightinclude uptake of either whole bacteria or parts thereof.

Surprisingly, however, when MTSA-differentiated DCs werepulsed with M. tb cell extract and cocultured with cell extract-specific T cells, the IL-2 and IFN-� levels of these T cells becameseverely down-regulated with a concomitant increase in theirIL-10 levels, indicating the probable development of suppressor/regulatory responses (our unpublished results). These results thusindicate a putative physiological role for these secretory Ags andpossibly mimic the early events that would ensue during an infec-tion, wherein the differentiation of DCs by the secretory proteins ofM. tb Ags might be followed up by the release of bacteria frommacrophages, and eventually leads to a down-regulation of proin-flammatory responses to mycobacteria. Furthermore, the immaturenature of MTSA-differentiated DCs might perhaps be an addedadvantage to mycobacteria, possibly by serving as transport vehi-cles to migrate to secondary lymphoid organs, as has been pro-posed by others (54). However, more precise and detailed exper-iments are required to support the above hypothesis.

Toward identifying intracellular intermediates that may be mod-ulated during MTSA-mediated DC differentiation, we looked atthe activation of NF-�B that has been suggested to play a vital rolein the differentiation of DCs (43–47). Indeed, the p65 subunit ofNF-�B did translocate to the nucleus within 30 min of stimulation,indicating a possible role of this transcription factor during MTSA-induced differentiation process. Furthermore, MTSA DCs also se-creted proinflammatory cytokines TNF-�, IFN-�, IL-12p40, andIL-12p70 during the course of differentiation (data not shown).TNF-� has been shown to induce differentiation of DCs that alsoinvolves the activation of NF-�B. Therefore, activation of NF-�Bfollowed by secretion of TNF-� may be one of the mechanisms ofMTSA-induced DC differentiation. Further characterization ofMTSA-induced DC differentiation is currently being followed.

AcknowledgmentsWe gratefully acknowledge the kind gift of various M. tb Ags, Ag85b,ESAT-6, and MPT64, and whole cell extract from Dr. J. T. Belisle (Col-orado State University) under the National Institutes of Health, NationalInstitute of Allergy and Infectious Diseases Contract AI-75320, entitledTuberculosis Research Materials and Vaccine Testing. K.N. thanks Ravin-der Kumar for assistance in photography.

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