914 • JID 2003:187 (15 March) • Natarajan et al.

M A J O R A R T I C L E

Down-Regulation of T Helper 1 Responsesto Mycobacterial Antigens Due to Maturationof Dendritic Cells by 10-kDa Mycobacteriumtuberculosis Secretory Antigen

Krishnamurthy Natarajan,1 Vinoth K. Latchumanan,1 Balwan Singh,1 Sarman Singh,2 and Pawan Sharma1

1Immunology Group, International Centre For Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, and 2Department of LaboratoryMedicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

Interactions of 10-kDa Mycobacterium tuberculosis secretory antigen (MTSA) with dendritic cells (DCs) were

investigated to elucidate the role of secretory antigens in regulating immune responses to M. tuberculosis early

in the course of infection. MTSA induced the maturation of different DC subsets. The cytokine profiles of

these DCs were characteristic to each DC subset. Of interest, coculture of M. tuberculosis whole-cell extract

(CE)–pulsed, MTSA-matured DCs with CE-specific T cells led to a marked reduction in interleukin (IL)–2

and interferon (IFN)–g production, thereby down-regulating proinflammatory responses to mycobacterial

antigens. Attenuation of IL-2 and IFN-g levels of CE-specific T cells also was obtained when M. tuberculosis

culture filtrate protein–activated DCs were employed as antigen-presenting cells, which suggests that MTSAs

induce maturation of DCs at sites of infection, probably to down-regulate proinflammatory immune responses

to mycobacteria that may subsequently be released from infected macrophages.

Infection with Mycobacterium tuberculosis continues to

be a major cause of mortality and morbidity through-

out the world, resulting in 3 million deaths and 18

million new cases of tuberculosis each year [1–3]. Al-

though immunization with M. bovis bacille Calmette-

Guerin (BCG) is still considered to be the reference

standard against which all other vaccines are measured,

its efficacy varies from 0% to 85% in different studies

Received 30 August 2002; accepted 25 November 2002; electronically published6 March 2003.

Animal studies were approved by the Institutional Animal Ethics Committee ofIndia.

Financial support: Defence Research and Development Organization, Governmentof India (grant DALS/48222/LSRB/22/ID/RD/-81 to K.N. and P.S.); Department ofBiotechnology, Government of India (grant BT/PR2423/Med/13/087/2001 to P.S.).

Reprints or correspondence: Dr. Krishnamurthy Natarajan, Immunology Group,International Centre for Genetic Engineering and Biotechnology, Aruna Asaf AliMarg, 110-067 New Delhi, India (natrajan@icgeb.res.in).

The Journal of Infectious Diseases 2003; 187:914–28� 2003 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2003/18706-0006$15.00

and in different geographic regions [4–7]. Therefore,

any step toward the development of vaccines or vaccine

candidates requires a thorough understanding of the

protective immune responses generated against this

pathogen [8, 9]. Mycobacteria persist in macrophages

within the granuloma in the organs of infected hosts

[10]. From the phagosomes, where they reside, they are

believed to secrete proteins, also called “secretory an-

tigens” [11]. Numerous proteins secreted by the bac-

teria have been described, and many have been pos-

tulated to contribute to the development of protective

immunity by serving as targets for the immune system

early in the infection where they are likely to be taken

up by antigen-presenting cells (APCs) [12–15].

Among the most potent of these APCs are the different

subsets of DCs that have the ability to stimulate quies-

cent, naive, and memory T lymphocytes [16]. DCs exist

at various states of activation and maturation that are

defined by distinct phenotypic and functional modali-

ties [17]. For example, immature DCs are programmed

for antigen capture and display very low levels of T

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 915

cell–stimulatory properties (i.e., they express low levels of surface

major histocompatibility complex [MHC] and costimulatory

molecules). After contact with various stimuli—such as lipo-

polysaccharide (LPS), tumor necrosis factor (TNF)–a, CD40 li-

gand (via cognate interactions with T cells), and certain antigens

[16, 17]—they undergo a process of maturation. During mat-

uration, they up-regulate their MHC (class I and II) and costi-

mulatory molecules (CD80, CD86, CD40, and CD54) and are

very efficient T cell stimulators [18]. Therefore, antigens that are

able to induce maturation of DCs play a major role in defining

the character of primary immune responses against the pathogen

and, thereby, have an important role in determining the course

of an infection [19].

A recent report identified 10-kDa M. tuberculosis secretory

antigen (MTSA), which was able to prime delayed-type hy-

persensitivity responses in M. tuberculosis–infected guinea pigs

but not in animals infected with M. bovis BCG [20]. MTSA is

a product of the Rv3874 gene in the mycobacterial genome

and is not expressed by other members of the mycobacterial

complex (e.g., M. avium and M. bovis BCG). Recent reports

document the importance of MTSA (also known as culture

filtrate protein [CFP]–10) in generating protective immune re-

sponses against M. tuberculosis [21]. CFP-10–pulsed, mono-

cyte-derived DCs were used to isolate CD8� T cell clones that

interacted with M. tuberculosis– but not M. bovis BCG–infect-

ed targets [22]. Furthermore, owing to its absence in M. bovis

BCG strains used for vaccinations, CFP-10 has been proposed

as an important candidate in the diagnosis of M. tuberculosis

[23, 24]. We recently showed that MTSA induces the secretion

of TNF-a in macrophages and synergizes with IFN-g to induce

nitric oxide secretion in macrophages [25]. We also recently

showed that MTSA and other mycobacterial antigens induce

the differentiation of DCs from bone marrow (BM) precursors

[26]. To further characterize the interactions of MTSA with

DCs, we examined the interactions of MTSA with DC subsets,

to understand the role of mycobacterial antigens in regulating

immune responses early in the course of infection. Our results

indicate that MTSA induces the full maturation of DCs. These

DCs, in effect, down-regulate proinflammatory responses

against M. tuberculosis whole-cell extract (CE) and, along with

other such secretory proteins, likely plays an important role in

regulation of immune responses to mycobacteria. The func-

tional implications of DC maturation by MTSA are discussed.

MATERIALS AND METHODS

Animals. Female BALB/c mice (4–6 weeks old) were used

in the study for all experiments involving DCs. For enrichment

of T cells, either BALB/c or C57BL/6 mice were used. All an-

imals were maintained under pathogen-free, environment-con-

trolled conditions in the small animal facility of the Interna-

tional Centre for Genetic Engineering and Biotechnology (New

Delhi).

Materials. Fluorescein isothiocyanate (FITC)–tagged mono-

clonal antibodies against mouse cell-surface molecules (CD80

[clone 1G10], CD86 [clone GL-1], CD54 [clone 3E2], I-Ad [clone

AMS-32.1], H-2Dd [clone 3-25.4], CD40 [clone 3/23], CD4

[clone GK 1.1], and CD8a [clone Ly-2]); biotin-conjugated

antibodies to CD11c (clone HL3) and CD90 (Thy 1.2; clone

52-2.1); purified anti-CD40 (clone HM-40) and CD16/CD32

(FCgR, clone 2.4G2); and isotype-matched control antibodies

were purchased from BD Pharmingen. FITC-conjugated anti-

body to F4/80 (clone CI: A3-1) was obtained from Serotec. Anti-

CD4, anti-CD8, anti-CD90, anti-B220, anti-CD11b, anti-CD11c,

anti–I-A, and anti-CD19–coated magnetic beads were obtained

from Miltenyi Biotec. Mouse recombinant granulocyte-macro-

phage colony-stimulating factor (GM-CSF) and ELISA kits for

the estimation of mouse cytokines were purchased from R&D

Systems. Recombinant TNF-a, LPS, polymixin B sulfate, hen egg

lysozyme, ovalbumin, polystyrene carboxylate–modified fluores-

cent beads (0.5 mm), and an E-toxate endotoxin detection kit

were obtained from Sigma. M. tuberculosis CE and M. tuberculosis

CFPs were obtained from Tuberculosis Research Materials and

Vaccine Testing (Colorado State University, Fort Collins). The

details of their preparation and composition can be viewed at

http://www.cvmbs.colostate.edu/microbiology/tb.

Expression and purification of MTSA. Expression and

purification of MTSA was done as described elsewhere [25]. In

brief, the open-reading frame Rv3874 of M. tuberculosis–en-

coding MTSA was polymerase chain reaction–amplified from

the genomic DNA of a local clinical isolate using the prim-

ers 5′-GCGGATCCCATGGCAGAGATGAAGACCG-3′ (for-

ward) and 5′-CCCAAGCTTGTCAGAAGCCATTTGCGAG-

3′ (reverse), with BamHI and HindIII as flanking enzyme

sites (underlined). The polymerase chain reaction product

(GenBank accession no. AF419854) was first directly cloned

into an intermediate vector (pGEM-T Easy; Promega) and

sequenced to ascertain the identity. After verification of the

sequence, the full-length gene was subcloned into the BamHI

and HindIII sites of the bacterial expression vector (pQE-

31; Qiagen). The recombinant protein, expressed as a poly-

histidine-tagged protein, was purified by nickel-nitrilotri-

acetic acid (Ni-NTA) metal affinity chromatography, accord-

ing to the manufacturer’s instructions (Qiagen), for purifi-

cation of recombinant protein in its native form. The purity

of MTSA was further confirmed by ion-exclusion high-per-

formance liquid chromatography (HPLC), using a POROS-

HQ column (PerSeptive Biosystems) with a matrix of cross-

linked polystyrene-divinylbenzene flow-through particles coat-

ed with fully quarternized polyethyleneimine that is complete-

ly ionized over a pH range of 1–14. One milligram of the pro-

tein was injected, and the profile was recorded.

916 • JID 2003:187 (15 March) • Natarajan et al.

Generation of DCs from BM. Generation of DCs from

BM was done as described elsewhere [26]. In brief, 63 � 10

lymphoid and I-A�-depleted BM cells from the tibias and fe-

murs of BALB/c mice were cultured in 6-well plates in RPMI

1640 medium containing 10% fetal calf serum (FCS), 0.05 M

2-mercaptoethanol, 1 mM sodium pyruvate, and 15 ng/mL

GM-CSF, with periodic changes of the culture medium. On

day 3, loosely-adherent cells were cultured in fresh medium

containing GM-CSF and, when required, stimulated with var-

ious antigens for 24 h. For some experiments, 25 mg of MTSA

was digested with varying amounts of pancreatic trypsin (Gibco

BRL) in 200 mM Tris-Cl buffer (pH 7.7) for 10 min at 25�C.

Enzyme reaction was stopped by the addition of 20% FCS. One

unit of trypsin was defined by the manufacturer as 1 mg of

enzyme required to completely digest 250 mg of target protein.

Trypsin-treated MTSA then was used for the stimulation of

BM-derived DCs (BMDCs), as described above. To check for

specificity, MTSA was incubated with the F(ab′)2 fragment of

anti-MTSA polyclonal antibody (obtained by digestion with

pepsin and subsequent purification over a protein G column)

for 60 min and later was added to cultures. The C-terminal

fragment of Plasmodium falciparum merozoite surface protein

(MSP)–119 kDa, expressed and purified in the same way as MTSA,

also was used as a negative control. Cells at the end of incu-

bation in all sets were either analyzed for the levels of surface

molecules by flow cytometry, as described elsewhere [27], or

were used in T cell stimulation experiments, as described below.

Enrichment of splenic DCs. Spleens from 6–8 mice were

pooled and cut into small fragments. These were then digested

in RPMI 1640 medium containing 10% FCS, 1 mg/mL type

III collagenase, and 325 kU/mL DNase I, with periodic pipetting

for 25 min at room temperature. EDTA (0.1 M [pH 7.2]) was

added for an additional 5 min to allow disruption of the DC–T

cell complexes. Cells were washed and resuspended in Nycod-

enz (1.077 g/mL; Sigma), overlaid on an additional layer of

Nycodenz, and centrifuged at 1700 g for 20 min. The low-

density fraction then was collected. B and T cells were removed

from the low-density fraction by incubation with titrated levels

of anti-CD19, anti-CD45R, and anti-CD90 microbeads and by

separation through magnetic cell sorter (MACS) columns (Mil-

tenyi Biotec). The percentage of CD11c� cells in the resulting

population was found to be 85%–90%, as monitored by fluo-

rescence-activated cell sorter (FACS; FACSCalibur; Becton Dick-

inson). The cells then were fractionated further into CD11c�/

CD8a� and CD11c�/CD8a� populations after MACS. Enriched

splenic DCs (90%–95%) were cultured with different antigens

for 48 h and either were analyzed for changes in the surface

expression of markers or were cocultured with allogeneic or

syngeneic T cells.

Phagocytosis by DCs. BMDCs were stimulated with 25

mg/mL either MTSA or MSP-119 kDa or 20 mg/mL LPS for 24 h.

Cells were washed in Hanks’ balanced salt solution (HBSS) and

resuspended in PBS containing 5% bovine serum albumin and

fluorescent carboxylate latex beads for 4 h at 37�C. At the end

of the incubation, cells were extensively washed in HBSS and

fixed in PBS containing 0.1% paraformaldehyde. Cells then

were analyzed by flow cytometry.

Enrichment of T lymphocytes. Enrichment of T lympho-

cytes was done as described elsewhere [28]. In brief, either

inguinal lymph nodes or splenocytes from 4–6-week-old BALB/

c and C57BL/6 mice, respectively, were first depleted of ad-

herent cells by panning over plastic plates. B lymphocytes, re-

sidual DCs, and macrophages then were removed by 2 rounds

of incubation with anti-CD19–, anti-CD45R–, anti-CD11c–,

anti-CD11b–, and anti-I-A–coated magnetic beads, followed by

separation through MACS columns. The purity of the resulting

population of T cells obtained in this fashion was 95%–98%,

as determined by anti-CD90 stained cells by flow cytometry.

For some experiments, CD4� or CD8� populations of enriched

T cells from the lymph nodes of BALB/c mice were further

negatively enriched using anti-CD4– or anti-CD8–coated mag-

netic beads and purification over MACS columns. The resulting

CD4� or CD8� T cells were 90%–95% pure, as determined by

flow cytometry. The percentage of I-A� cells in all the fractions

was found to be !0.5%.

Allogeneic mixed leukocyte reactions. For measuring T

cell proliferation, enriched allogeneic C57BL/6 T cells53 � 10

were cultured with irradiated (3000 rads) antigen-50.6 � 10

activated BM cells or splenic DCs in 96-well plates for 72 h.

[3H]-thymidine (1.0 mCi) was added 16 h before harvesting and

counting. For measuring cytokine levels, T cells were63 � 10

cocultured with irradiated DCs in 24-well plates for60.6 � 10

48 h, and culture supernatants then were screened for the pres-

ence of cytokines, as described below.

In vitro syngeneic T cell stimulation. BALB/c mice were

immunized subcutaneously (sc) at the base of the tail with

various antigens (50 mg/mouse) in incomplete Freund’s adju-

vant for 7 days and boosted with a repeat immunization for

an additional 7 days. Inguinal lymph nodes from these mice

were removed, and T cells (both CD4� and CD8�) were en-

riched as described above. Enriched T cells ( cells) were63 � 10

cocultured with antigen-stimulated irradiated DCs for60.6 � 10

48 h, and culture supernatants were analyzed for cytokines.

In vivo syngeneic T cell stimulation. MTSA- or CE-ma-

tured DC subsets ( cells) were separately injected sc into62 � 10

naive BALB/c mice at the base of the tail. This was followed

by sc transfer of an equal number of either MTSA- or CE-

specific enriched T cells at the base of the tail 24 h later. Inguinal

lymph nodes were excised 3 days later and cultured in vitro

for an additional period of 72 h, and culture supernatants were

analyzed for cytokines.

Estimation of cytokines. Culture supernatants of DCs or

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 917

DC–T cell cocultures at the end of each incubation period were

analyzed for the levels of TNF-a, IL-12p40, IFN-g, or IL-10

by use of a sandwich ELISA protocol, as recommended by the

manufacturer (R&D Systems). The sensitivity ranges of the cy-

tokines were 31.2–2000 pg/mL for IL-12p40, 15.6–1000 pg/mL

for TNF-a, 31.2–2000 pg/mL for IFN-g, and 31.2–2000 pg/mL

for IL-10. The samples were correspondingly diluted to obtain

values within the linear range of the standards. Quantitation

was made against a standard curve obtained for individual

cytokine standards provided by the manufacturer.

RESULTS

MTSA induces the maturation of BMDCs. We first examined

the ability of MTSA to induce the maturation of DCs. It is now

well established that culturing BM cells with GM-CSF differ-

entiates them into DC-like APCs that display a predominantly

immature phenotype [29, 30]. We first ascertained the purity

of MTSA on a silver-stained SDS-PAGE gel and by HPLC anal-

yses. As shown in figure 1A, the fraction eluted from the Ni-

NTA column contained a single band at 10 kDa, the size cor-

responding to MTSA. No other protein band was present at

detectable levels, even in the lane loaded with 50 mg of the

protein, which indicates that the fraction contained only a single

species of the size corresponding to MTSA. Furthermore, the

HPLC profile with 1 mg of MTSA also showed a single peak,

with no apparent detectable levels of any other species, again

indicating the homogeneity of the fraction containing MTSA.

We next stimulated GM-CSF–differentiated BMDCs with

MTSA and monitored the changes in the levels of various mol-

ecules on these cells. Figure 1B gives the FACS profiles of

CD11c� cells stained for the indicated markers. MTSA further

up-regulated the levels of both MHC and costimulatory mol-

ecules on BMDCs. In particular, CD80 levels were up-regulated

by 10-fold, whereas CD86 levels were enhanced by 4-fold. Fur-

thermore, the expression of myeloid marker F4/80 antigen,

which is known to be expressed on matured DCs [31], also

was enhanced on BMDCs after addition of MTSA. Dose and

time response profiles (data not shown) revealed that stimu-

lation with 25 mg/mL MTSA for 24 h gave the best response;

hence, all subsequent experiments with BMDCs were per-

formed with that concentration and time of exposure. Because

stimulation of allogeneic T cells at a low stimulator-to-re-

sponder ratio is a well established marker for mature DCs and

myeloid cells [16, 17], we stimulated BMDCs with MTSA and

examined the subsequent allogeneic T cell responses thus gen-

erated. As shown in figure 1C, the proliferation of allogeneic

T cells showed a 2-fold increase in both [3H]-thymidine in-

corporation and IL-2 production after stimulation with MTSA.

Furthermore, the level of IFN-g on allogeneic T cells also in-

creased by nearly 5-fold when BMDCs incubated with MTSA

were used in cocultures (figure 1C). IL-10 levels were largely

unaffected. Consistent with earlier reports [16], stimulation

with keyhole limpet hemocyanin antigen did not have any effect

on DCs, either at the level of surface markers or on the extent

of allogeneic T cell stimulation (data not shown).

Because the MTSA used in the study was recombinant ex-

pressed, it was necessary to rule out the possibility of the observed

effects being mediated by endotoxins and/or other low–

molecular-weight contaminants in the purified protein. We used

various approaches to accomplish this. First, we estimated the

endotoxins levels in all the batches of MTSA that were used in

the study by use of the E-Toxate kit from Sigma. Endotoxin levels

in every batch of MTSA used in the study were tested at 4

different doses, from 10–100 mg/mL. In all the batches, the en-

dotoxin level was !0.03 endotoxin units/mL, as determined by

the absence of a hard-gel formation in the 0.03 endotoxin unit

standard provided in the kit. Since the concentration of MTSA

used in the study was 25 mg/mL, the corresponding levels of

endotoxins would be 5-fold lower, indicating that the observed

effects in figure 1 were attributable to MTSA. We further ruled

out the effects of endotoxins by incubating MTSA with polymixin

B sulfate, which is known to inactivate LPS and related endo-

toxins [32]. BMDCs were then stimulated with polymixin B

sulfate–treated MTSA, and the levels of various markers on the

cell surface were monitored by FACS. As shown in table 1, al-

though treatment of LPS with polymixin B sulfate completely

abolished all its effects, treatment of MTSA with polymixin B

sulfate had no effect on the increase in the surface levels of various

MHC and costimulatory molecules, compared with untreated

MTSA (table 1); this result indicates that endotoxins were not

responsible for the observed effects of MTSA. Furthermore, be-

cause certain low-molecular-weight compounds, such as poly-

peptides and other bacterial components [33], may also be pre-

sent in the recombinant preparation of MTSA and have been

shown to activate DCs, we treated MTSA with increasing doses

of trypsin and examined the retention of its activity. As shown

in table 1, digestion of MTSA with trypsin led to a complete loss

of activity (as measured by the mean fluorescence intensity [MFI]

of various markers), even at a level of 0.01 U of trypsin, which

indicates that intact MTSA in its native form was required for

inducing the maturation of DCs. Incubation of MTSA with the

F(ab′)2 fragment of anti-MTSA did not result in any increase in

the surface levels of various markers on DCs, which indicates

that the observed effects were specific to MTSA. Incubation of

MTSA with preimmune serum had no effect on its activity (data

not shown). Furthermore, stimulation of DCs with similarly ex-

pressed and purified MSP-119 kDa had no effect on the levels of

various markers. The results in table 1 thus indicate that the

observed effects in figure 1 were specific to MTSA and not ob-

tained by any contaminant (such as endotoxins, low-molecular-

weight compounds, or polypeptides) in the recombinant protein.

918 • JID 2003:187 (15 March) • Natarajan et al.

Figure 1. Mycobacterium tuberculosis secretory antigen (MTSA) induces the maturation of immature dendritic cells (DCs). A, Silver-stained 12.5%SDS-PAGE gel loaded with fraction containing MTSA. Lanes 1–5, 1, 5, 10, 25, and 50 mg of MTSA, respectively. M, Molecular weight markers. Ahigh-performance liquid chromatography profile of MTSA corresponding to 1 mg of protein is depicted on the right. B, Granulocyte-macrophage colony-stimulating factor–differentiated day-3 DCs from the bone marrow (BM) were stimulated with 25 mg/mL of MTSA for 24 h. At the end of incubation,aliquots of cells were stained for the various markers and analyzed by fluorescence-activated cell sorter (see Materials and Methods). Cell-surfacelevels of indicated markers on CD11c�-gated cells in the presence (thick line) or absence (dashed line) of MTSA from 1 of 4 experiments are shown.The thin line in all the histograms indicates staining with corresponding isotype-matched control antibodies. C, MTSA-stimulated DCs were coculturedwith C57BL/6-enriched T cells in 96-well plates for 72 h for measuring [3H]-thymidine incorporation; 1.0 mCi/well of [3H]-thymidine was added 16 hbefore harvesting and counting (see Materials and Methods). For measuring cytokine levels, DCs were cocultured with T cells in 24-well plates for48 h, and culture supernatants were screened for the levels of indicated cytokines. Differences in the levels of interferon (IFN)–g between immatureand MTSA-matured DCs in panel C were significant at (Student’s t test). Data from 1 of 4 experiments are shown. BMDC, BM-derived DCs.P ! .05

Because DCs at various states of activation and maturation

perform definite functions, with respect to antigen capture ver-

sus T cell stimulation [16], we also investigated whether MTSA

induced either partial or full maturation of BMDCs. MTSA-

matured BMDCs were incubated with stimulants that induce

full maturation of DCs, such as LPS, anti-CD40, and TNF-a

[16, 17]. The responses of these DCs were evaluated for both

cell-surface phenotype and extent of allogeneic T cell stimu-

lation. We observed that the addition of these stimuli to MTSA-

matured BMDCs had no effect on either the levels of MHC

and costimulatory molecules or the extent of allogeneic T cell

stimulation (data not shown). Together with the ability to boost

the allogeneic T cell stimulation, the collective results in figure

1 indicate that MTSA induced the full maturation of BMDCs,

which is consistent with the higher levels of MHC and costimu-

latory marker expression.

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 919

Table 1. Maturation of bone marrow–derived dendritic cells (BMDCs) is specific to Mycobacteriumtuberculosis secretory antigen (MTSA).

Group

Cell marker

CD80 CD86 I-A H-2D CD40 CD54

BMDCs 80 � 0.9 50 � 4.5 35 � 3.2 55 � 14 30 � 3.1 150 � 18

BMDCs � MTSA 780 � 65 400 � 39 500 � 65 650 � 60 250 � 28 800 � 75

BMDCs � LPS 850 � 75 585 � 60 780 � 81 720 � 77 280 � 30 755 � 80

BMDCs � LPS � PBa 75 � 7.2 52 � 4.5 38 � 4.0 57 � 6.1 25 � 1.8 145 � 15

BMDCs � MTSA � PBa 750 � 65 395 � 40 520 � 60 625 � 65 275 � 29 825 � 85

MTSA � Trypsin (0.01 U)b 79 � 7.9 49 � 4.8 32 � 2.9 54 � 5.6 28 � 2.8 140 � 14

MTSA� Trypsin (10 U)b 75 � 7.2 45 � 4.3 34 � 3.5 55 � 5.7 32 � 2.5 145 � 15

MTSA� a-MTSAc 78 � 7.0 48 � 4.2 32 � 2.8 48 � 5.2 32 � 2.5 148 � 15

MSP-119 kDad 75 � 6.8 45 � 3.8 30 � 2.4 51 � 4.8 24 � 2.2 135 � 12

NOTE. Data are mean � SD fluorescence intensities of 3 independent experiments. Day-5 BMDCs were stimulated with25 mg/mL of MTSA alone or treated with various agents for 24 h, stained for the surface levels of various markers (see Materialsand Methods), and analyzed by fluorescence-activated cell sorter. a-MTSA, anti-MTSA; LPS, lipopolysaccharide; MSP-119 kDA,merozoite surface protein–119 kDA; PB, polymixin B sulfate.

a LPS (20 mg/mL) or MTSA (25 mg/mL) was incubated with 25 mg of PB for 60 min and then was added to BMDCs; 24 hlater, the cultures were analyzed for the levels of indicated markers.

b MTSA (25 mg) was digested with 0.01 or 10 U of pancreatic trypsin and added to cultures. Cell-surface densities of variousmarkers were measured as described above.

c MTSA was incubated with the F(ab′)2 fragment of a-MTSA antibody for 1 h, added to BMDCs for 24 h, and analyzed asdescribed above.

d BMDCs were stimulated with 25 mg/mL recombinant MSP-119 kDa of Plasmodium falciparum expressed in Escherichia colias a His-tagged protein for 24 h, and surface levels of various markers were measured.

MTSA induces the maturation of splenic DCs. It is well

recognized that different subsets of DCs present at various lo-

cations in the organism and respond in various ways to anti-

genic challenges [18]. Among the DC subsets reported in the

mouse spleen, the 2 most well characterized are CD8a�/CD11c�

and CD8a�/CD11c� DCs [34]. The CD8a�/CD11c� DCs are

thought to be of lymphoid origin and primarily induce Th1

responses, whereas the CD8a�/CD11c� subset of DCs induce

Th2 responses [18]. To determine whether MTSA would also

induce the maturation of splenic DCs, we carried out parallel

experiments, as were done with BMDCs. Enriched CD8a� or

CD8a� DCs from spleens were stimulated with MTSA for dif-

ferent periods of time, and the extent of modulation of cell

surface levels of costimulatory and MHC molecules was mon-

itored by flow cytometry. MTSA induced a time- and dose-

dependent increase in the levels of various molecules (data not

shown), with maximum up-regulation obtained 48 h after stim-

ulation with 25 mg/mL MTSA; hence, these conditions were

used for all subsequent experiments. Figure 2A shows the fold

increase over unstimulated controls in the MFI of various mol-

ecules of splenic DCs stimulated with MTSA for 48 h. MTSA

up-regulated all the molecules analyzed in both DC subsets. In

particular, the levels of MHC class II (I-Ad) and MHC class I

(H-2Dd) were found to be enhanced by 110–12-fold in CD8a�

DCs and by 4–10-fold in CD8a� DCs. The levels of CD40 and

CD54 were also up-regulated by 2–4-fold in both DC subsets

(figure 2A). Consistent with the increase in MFI of costimu-

latory and MHC molecules, the extent of allogeneic T cell stim-

ulation, as measured by the levels of IL-2, was enhanced by 2-

fold; levels of IFN-g and IL-10 were also enhanced by several

fold when either MTSA-stimulated CD8a� or CD8a� DCs were

used as APCs. (figure 2B). The results in figure 2 thus indicate

that MTSA induced the maturation of splenic DC subsets as

well. MTSA also induced full maturation of both CD8a� and

CD8a� DCs, but various stimuli, such as TNF-a, anti-CD40,

and LPS, did not have any significant effect on the levels of

MHC and costimulatory molecules on MTSA-matured DCs

(data not shown).

MTSA-matured DC subsets secrete distinct cytokine pat-

terns. We next examined the nature of cytokine profiles se-

creted by different DC subsets after MTSA stimulation. Table

2 gives the profiles of cytokines secreted by DCs 24 h after

stimulation. Consistent with published reports, unstimulated

BMDCs and splenic DCs secreted TNF-a and low levels of IL-

10 [16, 17]. IFN-g and IL-12p40 were below the level of de-

tection in CD8a� DCs and BMDCs. MTSA stimulation result-

ed in enhanced secretion of TNF-a by 19-fold in BMDCs and

by 2-fold in splenic DCs. IL-12p40 levels were maximally in-

duced in CD8a� DCs after MTSA stimulation, compared with

either BMDCs or CD8a� DCs. The low levels of IL-12p40 noted

in CD8a� DCs after antigen stimulation are consistent results

from a prior study that documented low-to-undetectable levels

of IL-12p40 production by this DC subset [18]. CD8a� DCs

secreted low levels of IFN-g, which was enhanced by several-

920 • JID 2003:187 (15 March) • Natarajan et al.

Figure 2. Splenic dendritic cell (DC) subsets are matured by Mycobacterium tuberculosis secretory antigen (MTSA). A, Fold increase in the relativemean fluorescence intensity (MFI) of various cell-surface molecules on CD8a� (a) or CD8a� (b) splenic DCs stimulated with 25 mg/mL of MTSA for48 h, over unstimulated DCs. Solid bars, MFIs of unstimulated DCs; hatched bars, MFIs of MTSA-stimulated DCs. Groups 1–6 in both panels representMFIs of CD80, CD86, I-A, H-2D, CD40, and CD54, respectively. All bars represent staining on propidium iodide–excluded CD11c� cells. B, MTSA-stimulated CD8a� or CD8a� DCs were cocultured with enriched C57BL/6 T cells for 48 h, and culture supernatants were monitored for the levels ofindicated cytokines at the end of the incubation period. Data from 1 of 5 experiments are shown. IFN, interferon; IL, interleukin.

fold after MTSA stimulation. IFN-g secretion was observed in

BMDCs and CD8a� DCs after MTSA stimulation. IL-10 levels

were marginally altered in BMDCs and CD8a� DCs and, in

fact, were not detected in CD8a� DCs after stimulation with

MTSA. The results in table 2 thus indicate that MTSA induced

distinct patterns of cytokine secretion by different DC subsets

that might differentially govern the nature of subsequently elic-

ited T cell responses.

MTSA-induced T cell responses are MHC class II re-

stricted. We next explored the kind of antigen-specific T cell

responses regulated by different DC subsets stimulated by

MTSA. MTSA-matured various DC subsets were cocultured

with either unfractionated or CD4� or CD8� MTSA-specific T

cell subsets, and the profiles of IL-2, IFN-g, and IL-10 pro-

duction from the interacting T cells were analyzed. MTSA-

matured DC subsets induced the proliferation of MTSA-specific

T cells, as reflected in the levels of IL-2 (figure 3). A dominance

of IFN-g secretion over IL-10 levels was observed in antigen-

specific T cells along with all 3 DC subsets, which is consistent

with allogeneic T cell responses. However, despite the fact that

CD8a� DCs secreted higher levels of IFN-g and IL-12p40 than

did CD8a� DCs and BMDCs, no significant differences were

observed in the absolute levels of either IL-2 or IFN-g in T

cell cocultures with different DC subsets; however, IL-10 levels

were found to be higher in T cells cocultured with splenic DCs,

compared with T cells cocultured with BMDCs. Nevertheless,

since IFN-g levels were far higher than IL-10 levels in all the

subsets, the differences in IL-10 levels may not be of much

significance with respect to the overall T cell response.

Major contribution toward T cell stimulation (more specif-

ically, IL-2 and IFN-g production) in all the sets was obtained

by the CD4� T cell subset, indicating that MTSA was primar-

ily MHC class II restricted. CD8� T cell–mediated responses

observed were marginal (only 10%–20% of the total). These

results are in agreement with studies that document the dom-

inance of CD4� over CD8� T cell responses during early in-

fection by M. tuberculosis [10].

MTSA-matured DCs do not respond to secondary antigenic

challenge. Maturation of DCs by antigens, particularly those

of infectious organisms, has important consequences for the

outcome of an immune response, since the functionality of

DCs can take a dramatic turn with respect to the loss or at-

tenuation of their ability to process a second challenge of any

antigen. Because the data in figures 1 and 2 revealed that MTSA

induced the full maturation of DC subsets, we, therefore, in-

vestigated whether MTSA-matured DC subsets still retained the

ability to respond to a challenge with various antigens, includ-

ing the CE of M. tuberculosis. Furthermore, it has been reported

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 921

Table 2. Cytokine profiles of Mycobacterium tuberculosis secretory antigen(MTSA)–stimulated dendritic cell (DC) subsets.

Group

Cytokine

TNF-a IFN-g IL-12p40 IL-10

BMDCs 192.0 � 15.2 ND ND 35 � 1.2

BMDCs � MTSAa 1766 � 165 145.5 � 20 55.2 � 2.1 59.6 � 6.5

CD8a� DCs 21.8 � 2.5 ND ND 38 � 2.5

CD8a�� MTSAa 43.7 � 3.5 71.75 � 5.2 43.8 � 3.5 43.5 � 2.4

CD8a� DCs 72.9 � 7.2 30.7 � 3.2 ND 32 � 2.8

CD8a� � MTSAa 140.9 � 15 1305.8 � 95 225.75 � 25 ND

NOTE. Data are mean � SD results (pg/mL/106 cells) of 1 of 3 experiments. Culture supernatantsfrom MTSA-stimulated DC subsets were analyzed for the presence of the cytokines, as described inMaterials and Methods. BMDCs, bone marrow–derived DCs; IFN, interferon; IL, interleukin; ND, notdetected; TNF, tumor necrosis factor.

a DC subsets were stimulated with 25 mg/mL MTSA for 24 h, and supernatants were analyzed forthe indicated cytokines.

elsewhere that the cell-wall skeleton of M. bovis BCG induces

the maturation of human DCs [35].

As expected, MTSA-specific T cells cocultured with MTSA-

matured BMDCs secreted high levels of IFN-g in culture su-

pernatants, compared with IL-10 levels (figure 4A). Of interest,

when MTSA-matured DCs were pulsed with various antigens,

the IL-2 and IFN-g T cell responses of the challenging antigens

were attenuated by up to 2–3-fold, compared with their respective

controls (figure 4A), which indicates that MTSA-matured DCs

are less proficient at processing a second challenge with any other

antigen. The reduction of IFN-g levels of CE-specific T cells,

after MTSA-matured DCs are pulsed with CE, is of particular

significance, because the CE components may well constitute

parts of whole bacteria that might be released from infected

macrophages at sites of infection. Similar observations were ob-

tained when MTSA-matured splenic CD8a� and CD8a� DCs

were pulsed with different antigens. Again, the proliferative and

proinflammatory responses of CE-specific T cells (as reflected

by IL-2 and IFN-g levels) and other antigens were down-regu-

lated by 2-fold, compared with their respective controls (figure

4B and 4C). The IFN-g responses of MTSA-specific T cells in

cocultures of MTSA-matured BMDCs pulsed with CE were also

marginally down-regulated, but not with those cocultured with

similarly treated splenic DCs (data not shown). However, no

effect of ovalbumin or hen egg lysozyme on MTSA-specific T

cell responses was observed (data not shown).

MTSA-matured DCs down-regulate T cell responses to CE

antigens in vivo. In an attempt to mimic the early events

that would occur after an infection by mycobacteria, whereby

their release from macrophages would follow that of secretory

proteins from the phagosomal complex, we separately trans-

ferred MTSA-matured DC subsets that were pulsed with CE

into naive mice. This was followed by a challenge with either

MTSA- or CE-specific enriched T cells. Lymph nodes were later

cultured, and cytokines levels in their supernatants were esti-

mated. As shown in figure 5, MTSA-specific T cell responses

induced by all DC subsets were as expected, with IFN-g levels

dominantly expressed over IL-10. However, a challenge with

CE-specific T cells in mice that had received MTSA-matured

BMDCs led to a significant down-regulation of IL-2 and IFN-

g levels by 4- and 10-fold, respectively, compared with results

obtained when CE-matured DCs were used (figure 5A). A sim-

ilar trend was evident with splenic DC subsets, insofar as the

down-regulation of IFN-g levels were concerned after a chal-

lenge with CE-specific T cells, when a 2-fold reduction in IFN-

g levels was observed (figure 5B and 5C). IL-10 levels also

showed an increase by 3-fold in CD8a� DCs, compared with

CE-matured DCs, which indicates that there was down-regu-

lation of Th1 responses, together with an increase in regulatory

responses, at least in this DC subset. These results indicate that

proliferative and proinflammatory T cell responses to M. tu-

berculosis CE are down-regulated at sites where DC-APCs ex-

pressing or loaded with MTSA predominate.

MTSA-matured DCs retain phagocytic ability. It is known

that matured or antigen-activated DCs show reduced phago-

cytosis of particulate matter [16, 17]. The results presented

above demonstrate that MTSA-matured DCs do not respond

to a second challenge with antigen. To investigate whether

MTSA-matured DCs display phagocytosis, we incubated these

cells with 0.5-mm fluorescent carboxylate–modified latex beads

and examined their internalization by flow cytometry. As shown

in figure 6A, immature BMDCs readily internalized the beads.

Internalization of the beads was, however, only marginally re-

duced in MTSA-matured BMDCs (figure 6B). As expected,

LPS-matured DCs showed a significant reduction (12-fold) in

their phagocytic ability (figure 6C), whereas activation of DCs

with MSP-119 kDa had no effect on the phagocytic ability of DCs

(figure 6D). Similar results were obtained with splenic DCs,

where no significant differences were observed between un-

stimulated and MTSA-matured DCs (data not shown). These

922 • JID 2003:187 (15 March) • Natarajan et al.

Figure 3. T cells responses induced by Mycobacterium tuberculosis secretory antigen (MTSA)–matured dendritic cell (DC) subsets are CD4� restrictedEither bone marrow–derived DCs (BMDCs; A) or CD8a� (B) or CD8a� (C) DC subsets were matured with MTSA and cocultured with either unfractionatedor CD4� or CD8� MTSA-specific T cells for 48 h, and cytokine levels were estimated in the culture supernatants at end of incubation period (seeMaterials and Methods). Groups 2–4 in all the panels represent cocultures of MTSA-matured DC subsets with unfractionated, CD4�, and CD8� Tcells, respectively. Group 1 shows the cytokine levels of unfractionated T cells cocultured with unstimulated DC subsets. The differences in levels ofcytokines between CD4� and CD8� T cell groups were statistically significant at . Data from 1 of 3 experiments are shown. IFN, interferon;P ! .05IL, interleukin.

results are in agreement with previous studies that report re-

duced, but not abolished, phagocytic ability of DCs when in-

fected with live mycobacteria [36].

DCs activated by M. tuberculosis CFPs down-regulate pro-

inflammatory responses to CE antigens. The results shown in

figures 4 and 5 indicate that maturation of DC subsets by MTSA

leads to down-regulation of proliferative and proinflammatory

responses to CE antigens, as reflected by the decrease in the levels

of IL-2 and IFN-g of the interacting T cells, thereby suggesting

a possible role of secretory antigens in governing immune re-

sponses during an M. tuberculosis infection. However, because

the above observations were made with a single antigen, it was

prudent to carry out similar studies with various other secretory

antigens to add support to the above proposition. To this end,

we stimulated various DC subsets with the CFPs of M. tuber-

culosis, because they would represent an array of antigens that

are likely to be released from the phagosomes of infected mac-

rophages at sites of infection. We first depleted MTSA from CFPs

by incubation with antibody to MTSA, followed by immuno-

precipitation with protein G–conjugated agarose beads, to rule

out any effects of MTSA that might be present in the CFP. CFP-

activated DCs were pulsed with CE and cocultured with either

CE- or CFP-specific T cells for 48 h, and IFN-g and IL-10 levels

were then scored in supernatants. As shown in figure 7, like

MTSA, CFP-activated DC subsets were also unable to stimulate

the proliferation of CE-specific T cells, as indicated by the re-

duced levels of IL-2 and IFN-g in these cells, suggesting that

MTSAs induce maturation of DCs at sites of infection that might

lead to an attenuation of proinflammatory responses to CE or

possibly whole bacteria once they are released from cytolyzed or

infected macrophages.

DISCUSSION

The recognition that immunization with M. bovis BCG has a

variable impact on the transmission of M. tuberculosis has

renewed interest in developing more effective vaccines for

tuberculosis [4–7]. However, a prerequisite for achieving this

goal is a thorough understanding of the pathogenesis of M.

tuberculosis, with respect to the kind and degree of immune

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 923

Figure 4. Mycobacterium tuberculosis secretory antigen (MTSA)–matured dendritic cells (DCs) are nonresponsive to secondary antigenic challenge.MTSA-matured bone marrow–derived DCs (BMDCs; A), CD8a� (B), and CD8a� (C) DC subsets were pulsed with 10 mg/mL of various antigens for 24h and cocultured for 48 h with enriched T cells primed with the challenging antigen. Culture supernatants were then screened for cytokine levels.“CONTROL” depicts average values of control groups of either unstimulated DCs or T cells only. Differences in levels of interleukin (IL)–2 and interferon(IFN)–g in all the panels between second antigen–challenged MTSA-matured DCs and their respective controls were statistically significant at P !

. Data from 1 of 4 independent experiments are shown. CE-T, cell extract–specific T cells; HE-T, hen egg lysozyme (HEL)–specific T cells; M-T,.05MTSA-specific T cells; OVA-T, ovalbumin (OVA)–specific T cells.

responses generated early in the course of infection. It is well

known that, after infection, cell types recruited foremost at sites

of infection are the various DC subsets and their precursors,

which are at various stages of their differentiation and matu-

ration [16, 19]. Consequently, interactions of DCs with my-

cobacteria or parts thereof likely play a determinant role in

regulating immune responses that are generated early in the

infection and that would essentially permit a framework for

the host to tailor its defense strategies for clearing the pathogen.

A number of studies have been conducted on the interaction

of mycobacteria with DCs. For example, Henderson et al. [36]

showed that infection of DCs with mycobacteria causes their

activation, as reflected by increased surface densities of various

costimulatory and MHC molecules. In addition, infected DCs

secreted elevated levels of inflammatory cytokines, including

TNF-a, IL-1, and IL-12. DCs were further shown to phago-

cytose mycobacteria. Bodnar et al. [37] further showed that

mycobacteria could replicate inside murine BMDCs and that,

although DCs were able to restrict their growth, they were

nevertheless less efficient than infected macrophages at elimi-

nating the infection. These results further suggested the im-

portance of DCs in priming immune responses to mycobac-

teria. Furthermore, stimulation of M. tuberculosis–infected DCs

via CD40 increased the ability of DCs to mount T cell responses;

this was later shown to be primarily attributed to increased

expression of costimulatory and MHC molecules on their cell

surface [38]. DCs also have been shown to induce protective

immunity against M. tuberculosis in a murine model and also

against aerosol-mediated infection [39]. A number of microbi-

al lipopeptides and proteins also have been shown to activate

and mature DCs [40]. Although a number of studies have been

conducted on secretory antigens, most have focused on CFPs

924 • JID 2003:187 (15 March) • Natarajan et al.

Figure 5. Mycobacterium tuberculosis secretory antigen (MTSA)–matured dendritic cell (DC) subsets down-regulate T cell inflammatory responsesto cell extract (CE) in vivo. MTSA-matured bone marrow–derived DCs (BMDCs; ; A), CD8a� (B), or CD8a� (C) DC subsets were pulsed with62 � 10CE for 24 h and injected (subcutaneously [sc] at the base of tail) into naive mice, followed by a challenge with an equal number of either MTSA- orCE-specific T cells 24 h later (sc at the base of tail; see Materials and Methods). Three days later, lymph nodes were cultured for 48 h, and cytokineswere measured in the culture supernatants. “CONTROL” depicts average values of groups that received either unstimulated or unstimulated DCsfollowed by antigen-specific T cells. Differences in levels of interleukin (IL)–2 and interferon (IFN)–g in all the panels between CE-pulsed, MTSA-matured and CE-matured DCs were statistically significant at . Data from 1 of 3 independent experiments are shown.P ! .05

and their potential role as vaccine candidates. For example,

early secreted antigen target (ESAT)–6 has been designated as

an important T cell antigen recognized by protective T cells in

animal models of infection with M. tuberculosis [41]. Further-

more, MPT64 and ESAT-6 have been shown to have potential

in the diagnosis of M. tuberculosis because they are recognized

by T cells in animal models [42, 43] of M. tuberculosis. Like

MTSA, both antigens have been found primarily in M. tuber-

culosis but not in most environmental mycobacteria or BCG

[44, 45]. MPT64 has been evaluated as a skin test reagent in

guinea pig models of tuberculosis [46] and in humans [47].

These antigens also have been shown to elicit delayed-type

hypersensitivity responses in guinea pig models of tuberculosis.

ESAT-6 also has been considered as a potential candidate for

subunit-based vaccines [48]. Ag85b, a member of the antigen

85 complex (a family of fibronectin-binding proteins involved

in mycobacterial cell-wall biosynthesis) [49] and other secretory

antigens have been used as potential DNA vaccine candidates

[50, 51]. However, despite these studies, information regarding

their actual roles at sites of infection in influencing the early

immune responses to this pathogen is still lacking. Only very

recently has their in vivo presence been demonstrated [11]. In

addition, an intriguing question that emerges is the physiolog-

ical relevance of these secretory antigens, more so in the light

of the fact that mycobacteria have been highly successful in

devising strategies for immune evasion [10, 52]. To identify

possible interactions of secretory antigens with DCs, we have

recently shown that mycobacterial secretory antigens, including

MTSA, induce the differentiation of DCs from BM [26]. These

DCs expressed various cell-surface markers characteristic of

DCs, such as CD11c and CD11b; costimulatory molecules

CD80, CD86, CD40, and CD54; and MHC class I and class II

molecules and also displayed DC-like morphology. Although

immature in nature, MTSA-differentiated DCs were equally

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 925

Figure 6. Mycobacterium tuberculosis secretory antigen (MTSA)–matured dendritic cells (DCs) display phagocytosis. Bone marrow–derivedDCs (BMDCs) were stimulated with either 25 mg/mL of MTSA or merozoitesurface protein (MSP)–119 kDa or 20 mg/mL of lipopolysaccharide (LPS) for24 h. Cells were washed and resuspended in buffer containing fluorescentcarboxylated latex beads (see Material and Methods) for 4 h at 37�C.Cells were thoroughly washed and analyzed by flow cytometry. A–D,Unstimulated, MTSA-matured, LPS-matured, and MSP-119 kDa–stimulatedBMDCs, respectively. Values within the marker (M1) represent percentageof phagocytic cells. Data from 1 of 4 independent experiments are shown.

proficient at stimulating allogeneic T cell responses, compared

with GM-CSF–differentiated DCs. To investigate whether

MTSA would also induce the maturation of various DC subsets

and whether MTSA-matured DCs would in any manner alter

the nature of subsequent T cell responses to mycobacterial

antigens, the present study was done. We demonstrated that

incubation of various DC subsets with MTSA induces their

maturation. Consistent with various parameters considered to

be the hallmark of DC maturation by various agents, such as

soluble proteins like gp96 [53] or CpG-containing DNA motifs

[54], increases in the levels of various markers is one aspect of

DC maturation, and the addition of MTSA to various DC

subsets indeed up-regulated the levels of CD80, CD86, CD40,

CD54, and MHC class I and class II molecules. MTSA also

increased the expression of the DC maturation marker F4/80

antigen. MTSA-matured DCs were also found to boost allo-

geneic T cell responses, compared with those of immature

DCs—another feature that is attributable to DC maturation

[16, 17] and is considered to be a direct translation of up-

regulated levels of costimulatory and MHC molecules [53, 54].

The addition of various terminal maturation–inducing stimuli

had no effect on either the surface levels of various markers or

the extent of allogeneic T cell stimulation, which indicates that

MTSA induced the full maturation of DCs.

That the observed effects were caused by MTSA and not any

contaminant in the recombinant expressed protein was ascer-

tained in the various control experiments that showed the ab-

sence of any detectable or significant levels of endotoxins, such

as LPS, or other low-molecular-weight compounds that are

known to copurify with proteins expressed in Escherichia coli (ta-

ble 1).

Infection of DCs by mycobacteria has been shown to in-

duce differential cytokine production, and the profiles were also

shown to differ from those induced by infection of macro-

phages [55]. Although infected DCs secreted IL-12, IFN-a, and

TNF-a, infected macrophages secreted IFN-g, IL-6, and IL-18.

Low levels of IL-12 observed in macrophages were attributed

to higher levels of IL-10, a known inhibitor of IL-12 secretion

[56]. Infection of human DCs by M. tuberculosis has further

been shown to lead to the secretion of type 1 IFN genes [56].

Recently, it was also shown that differential cytokine secretion

by M. tuberculosis–infected DCs and macrophages resulted in

differential effects on naive T cell polarization that were ex-

plained on the basis of differential levels of IL-12 secretion that

was predominantly expressed by DCs, compared with macro-

phages [57]. In fact, incubation of mycobacteria infected DCs

with IL-10 converted them into macrophages with increased

antibacterial activity [58]. It was thus of interest to examine

the cytokine profiles of various DC subsets after incubation

with MTSA. It has been proposed recently that the nature of

T cell responses generated after cognate interactions with APCs

is also influenced by the profiles of cytokine milieu, both before

and during the interaction [59]. For example, a predominance

of IL-12 secreted by DCs skews the response toward Th1,

whereas low levels of IL-12 and enhanced levels of IL-10 induce

a Th2/Th0 response [60]. Further characterization revealed that

MTSA-stimulated DC subsets secreted varying levels of IFN-g

and IL-12p40. CD8a� DCs, which are known to skew responses

toward Th1 [18], secreted high levels of proinflammatory cy-

tokines TNF-a, IL-12p40, and IFN-g, whereas CD8a� DCs

known to trigger Th2 responses displayed low levels of Il-12p40

and moderate levels of IFN-g after MTSA stimulation. Because

IL-12p40 (or IL-12p70) directly regulates IFN-g production

[60], the low levels of IFN-g in BMDCs and CD8a�DCs could

probably result from low levels of IL-12p40 induction by MTSA

in these DCs, whereas the higher levels of IFN-g in the CD8a�

DCs could result from enhanced secretion of IL-12p40 by this

subset, indicating that MTSA induced the differential secretion

of various cytokines from different DC subsets. That MTSA

induced the dominant secretion of proinflammatory cytokines

is consistent with related studies that report a similar profile

of cytokine secretion after infection of DCs by live mycobacteria

[61, 62].

To investigate whether maturation of DCs by MTSA also re-

sults in its internalization, processing, and presentation and to

926 • JID 2003:187 (15 March) • Natarajan et al.

Figure 7. Mycobacterium tuberculosis culture filtrate protein (CFP)–activated dendritic cells (DCs) down-regulate interferon (IFN)–g responses ofcell extract (CE) antigens. CFP-stimulated DC subsets were pulsed with CE for 24 h and cocultured with either CFP-specific T cells (CFP-T) or CE-specific T cells (CE-T) for 48 h. Culture supernatants were scored for the levels of various cytokines at the end of the incubation period. “CONTROL”depicts average values of unstimulated DCs cocultured with T cells. Differences in levels of interleukin (IL)–2 and IFN-g between CE-pulsed, M.tuberculosis secretory antigen (MTSA)–matured DCs and CE-matured DCs were statistically significant at . Data from 1 of 4 experiments areP ! .05shown. BMDCs, bone marrow–derived DCs.

investigate whether the differences in cytokine secretory profiles

would result in differential T cell responses, we next characterized

the antigen-specific T cell responses mediated by MTSA-matured

DC subsets. Our results showed that MTSA was indeed presented

on MHC molecules during maturation and that matured DCs

readily stimulated antigen-specific T cells to secrete IL-2 and IFN-

g. However, regardless of the differences in cytokine-secretion

profiles from various MTSA-matured DC subsets, the overall

differences in the relative levels of IFN-g and IL-10 from MTSA-

specific T cells were more or less similar. These results, therefore,

indicate that factors other than cytokine secretory profiles of DCs

may also influence the nature of T helper responses and may

possibly be antigen dependent. Furthermore, these results are in

agreement with those of Pulendran et al. [63], who showed that

antigen-pulsed CD8a� and CD8a� DCs were equally efficient in

inducing T cells into secreting IFN-g and IL-2, despite the fact

that CD8a� DCs secreted more IL-12, compared with CD8a�

DCs. A more detailed analysis showed that the major contri-

bution to IFN-g secretion came from the CD4� T cell subset,

which indicates that MTSA was essentially MHC class II re-

stricted. Although MTSA was added exogenously would normally

be processed and presented on MHC class II molecules, DCs are

often known to cross-present antigens. For example, the outer

membrane protein A (ompA) from Klebsiella pneumoniae was

shown to induce the maturation of DCs via Toll-like receptor 2

and was, indeed, MHC class I restricted [32]. Our results, how-

ever, showed that MTSA was largely MHC class II restricted and,

in a way, exemplified the importance of CD4� T cells in me-

diating immune responses to mycobacterial infection [10]. The

role of CD8� T cells in governing immune responses to myco-

bacteria recently has been highlighted with the identification of

a number of CD8� T cell clones mediating immune responses

to mycobacterial antigens [64] and by studies showing greater

susceptibility of b-2 microglobulin knockout mice over wild-type

control mice [65].

Because fully mature DCs are less efficient at processing a

second challenge of antigen [16, 17], pulsing of MTSA-matured

DC subsets with various antigens did not result in stimulation

of T cells specific to the challenging antigen, which indicates

that MTSA-matured DCs were attenuated in their capacity to

present secondary antigens. The components of the cell en-

velope of mycobacteria (which would constitute part of CE that

was used in the current study) are some of the early antigens

that would be recognized by APCs and have been shown to

induce the maturation of DCs [35]. We, therefore, characterized

the effects of CE-pulsed, MTSA-matured DCs in regulating

immune responses of CE-specific T cells. Consistent with the

results obtained with other challenging antigens, addition of

CE to these DCs also resulted in a decrease in the capacity of

CE-specific T cells to secrete IL-2 and IFN-g, which indicates

that these T cells were nonproliferative. Furthermore, the de-

crease in the levels of IFN-g was also accompanied by modest

changes in the levels of IL-10 in some DC subsets, such as those

observed in the case of CD8a� DCs, which are known to induce

Th2 responses [18]. The fact that the IL-10 levels were, by and

large, not affected and the decrease in IFN-g levels point to a

change in the overall T helper responses at sites of infection,

whereby a decrease in the proinflammatory responses could

contribute to down-regulation of immune responses to my-

cobacterial antigens. These results may thus indicate a possible

putative role for secretory antigens in modulating immune re-

sponses during a mycobacterial infection. This is further sup-

ported by the results obtained with CFPs, where a similar trend

was obtained with respect to the attenuation of IL-2 and IFN-

g responses from CE-specific T cells when cocultured with CE-

pulsed, CFP-matured DCs.

Maturation of Dendritic Cells by MTSA • JID 2003:187 (15 March) • 927

Maturation of DC precursors by secretory antigens may have

important bearings in the spread of infection, as noted in a

recent report by Bodnar et al. [37], which examined the use

of DCs as transport vehicles to migrate to secondary lymphoid

organs. Our results on the retention of phagocytic ability of

MTSA-matured DCs suggest that this might constitute one of

the mechanisms that the bacterium may exploit to achieve this

objective. Although maturation also results in changes in the

chemokine receptor profiling that would now also change their

receptiveness to various chemokines and would allow the DCs

to migrate, it is nevertheless possible and probable that these

DCs may still be able to phagocytose mycobacteria that are

released from macrophages, because development of a che-

mokine gradient is essential for proper migration of DCs [66].

However, more-detailed experiments are required to support

the current hypothesis. In light of the above, it is tempting to

speculate that maturation of DCs by MTSA and other secretory

antigens might provide additional frequency of such transport

vehicles for the bacterium to migrate to secondary organs.

Acknowledgements

We thank J. T. Belisle (Colorado State University, Fort Col-

lins), for the kind gift of whole-cell lysate and culture filtrate

proteins (provided through National Institutes of Health, Na-

tional Institutes of Allergy and Infectious Diseases grant AI-

75320), and Pawan Malhotra (International Center For Genetic

Engineering and Biotechnology, Aruna Asaf Ali Marg, New

Delhi, India), for the kind gift of Plasmodium falciparum mer-

ozoite surface protein–119 kDa.

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