of July 30, 2018.This information is current as

a Functional Region of Difference-1Mycobacterial Virulence and the Presence ofEpitope in TB10.4: Correlation with Induction of CD8 T Cells against a Novel

and Jes DietrichRolf Billeskov, Carina Vingsbo-Lundberg, Peter Andersen

http://www.jimmunol.org/content/179/6/3973doi: 10.4049/jimmunol.179.6.3973

2007; 179:3973-3981; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/179/6/3973.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Induction of CD8 T Cells against a Novel Epitope in TB10.4:Correlation with Mycobacterial Virulence and the Presence ofa Functional Region of Difference-11

Rolf Billeskov, Carina Vingsbo-Lundberg, Peter Andersen, and Jes Dietrich2

Although infection with Mycobacterium tuberculosis (M.tb) induces a robust CD8 T cell response, the role of CD8 T cells in thedefense against M.tb, and the mechanisms behind the induction of CD8 T cells, is still not clear. TB10.4 is a recently describedAg that is expressed by both bacillus Calmette-Guerin (BCG) and M.tb. In the present study, we describe a novel CD8 T cellepitope in TB10.4, TB10.43-11. We show that TB10.43-11-specific CD8 T cells are induced at the onset of infection and are presentthroughout the infection in high numbers. TB10.43-11 CD8 T cells were recruited to the site of infection and expressed CD44,TNF-�, and IFN-�. In addition, TB10.43-11 CD8 T cells showed an up-regulation of FasL and LAMP-1/2 (CD107A/B), whichcorrelated with a strong in vivo cytolytic activity. The induction of TB10.43-11-specific CD8 T cells was less pronounced followinginfection with BCG compared to infection with M.tb. By using a rBCG expressing the genetic region of difference-1 (RD1), weshow that the presence of a functional RD1 region increases the induction of TB10.43-11-specific CD8 T cells as well as the bacterialvirulence. Finally, as an M.tb variant lacking the genetic region RD1 also induced a significant amount of TB10.43-11-specific CD8T cells, and exhibited increased virulence compared with BCG, our data suggest that virulence in itself is also involved ingenerating a robust CD8 T cell response against mycobacterial epitopes, such as TB10.43-11. The Journal of Immunology, 2007,179: 3973–3981.

I t is still not clear exactly what constitutes a protective im-mune response to Mycobacterium tuberculosis (M.tb),3 but ithas been demonstrated in both animals and humans that T

cell-mediated, rather than Ab-mediated, immune responses are es-sential for control of tuberculosis (TB). The major mechanisms ofcell-mediated immunity include CD4 Th1-cell mediated activationof macrophages to destroy intracellular bacterial pathogens; thecentral role of IFN-� in the control of TB has been clearly dem-onstrated by the susceptibility to mycobacterial infections in micewith a disrupted IFN-� gene and in humans with mutations ingenes involved in the IFN-� and IL-12 pathways (1–4).

Unlike CD4 T cells, the role of CD8 T cells in the defenseagainst M.tb is still not clear. CD8 T cells are induced early in theinfection (5) and previous studies indicated that cytotoxic CD8 Tcell-mediated killing of infected host cells do play a role in thedefense against an M.tb infection, especially in the later phases ofthe infection (6, 7). Furthermore, mice without functional CD8 Tcells, caused by disruptions of the �2-microglobulin or the TAP1genes, or mice subjected to in vivo depletion of CD8 T cells,showed a decreased control of the infection compared with control

mice (8–11). Moreover, several studies using different vaccinationapproaches, such as dendritic cells pulsed with CD8 (and CD4) Tcell epitopes or adenovirus-expressing mycobacterial Ags, showeda strong induction of CD8 T cells and a significant protectionagainst infection with M.tb, again suggesting a role for CD8 Tcells (12–14). However, as CD4 cells were also induced in thesestudies, they did not conclusively show that CD8 cells were re-quired. In fact, two recent studies showed that induction of a CD8response against a specific epitope from TB10.4 or ESAT-6 did notlead to protection against an acute infection with M.tb (15, 16).This is in agreement with other studies showing that depletion ofCD8 T cells did not affect the bacterial load in the lungs of micesuffering from an acute infection (7). Thus, the role of the CD8cells is still not fully known and one drawback regarding the studyof CD8 T cells and their role in the defense against M.tb has beenthe limited number of identified M.tb CD8 peptide epitopes thatare specifically recognized in infected animals. Lately, a number ofCD8 epitopes have been identified in M.tb proteins, such asTB10.4 (17), CFP10, (18), MTB32A (19), Ag85A and Ag85B (14,20), and these studies have demonstrated that although the exactrole of this T cell subset during infection with M.tb still remainsunclear, infection with M.tb does induce a strong CD8 responsethat encompass both IFN-� production and cytotoxicity.

Interestingly, concerning the role of CD8 T cells, it has beensuggested that a major reason for the failure of the current TBvaccine (bacillus Calmette-Guerin (BCG)) is related to an inferiorability of BCG to induce specific CD8 T cells compared with M.tb(21, 22). Thus, a rBCG strain (�ureC hly�rBCG) was producedexpressing the phagosome pore-forming protein listeriolysin fromListeria monocytogenes which should increase the escape to thecytosol, thereby increasing the amount of bacterial peptidesavailable for the MHC class I (MHC-I) presentation pathway.Interestingly, �ureC hly�rBCG was later shown to be moreprotective than BCG against virulent M.tb infection (22–24).Moreover, recent studies have shown that reintroduction of the

Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen,Denmark

Received for publication April 4, 2007. Accepted for publication July 10, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was partially supported by Danish Research Agency, Ministry of Science,Technology and Innovation.2 Address correspondence and reprint requests to Dr. Jes Dietrich, Department ofInfectious Disease Immunology, Statens Serum Institut, Artillerivej 5, DK-2300Copenhagen S, Denmark. E-mail address: JDI@ssi.dk3 Abbreviations used in this paper: M.tb, Mycobacterium tuberculosis; TB, tubercu-losis; BCG, bacillus Calmette-Guerin; MHC-I, MHC class I; MHC-II, MHC class II;RD1, region of difference-1.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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genetic region of difference-1 (RD1), thought to be the maincause of the attenuation of BCG, into BCG or Mycobacteriummicroti resulted in increased virulence, increased activation ofCD8 T cells, and improved protection against M.tb, again in-dicating a role for CD8 T cells in the protection against M.tb(25–27).

In the present study, we describe a novel CD8 T cell epitopeshared by the homologous proteins of the early secretory antigenictarget-6 kDa (ESAT-6) family: TB10.3 and TB10.4. Detailed anal-ysis showed that the TB10.43-11-specific CD8 T cells were re-cruited to the site of infection during M. tuberculosis infection andthat these cells expressed both TNF-� and IFN-�, and up-regulatedexpression of FasL and LAMP-1/2 (CD107A/B) upon activation.In contrast, significantly less CD8 cells were induced followingBCG vaccination. By detailed dissection of mycobacterial strainswith and without RD1, it became clear that the induction ofTB10.43-11-specific CD8 T cells was related to both a functionalRD1 region as well as the virulence of the bacterial strain.

Materials and MethodsAnimal handling

Studies were performed with 7- to 9-wk-old female C57BL/6 mice fromHarlan Scandinavia. Noninfected mice were housed in cages in appropriateanimal facilities at Statens Serum Institut. Infected animals were housed incages contained within laminar flow safety enclosures (Scantainer; Scan-bur) in a separate biosafety level 3 facility. All mice were fed radiation-sterilized 2016 Global Rodent Maintenance diet (Harlan Scandinavia) andwater ad libitum. All animals were allowed a 1-wk rest period after deliv-ery before the initiation of experiments. The handling of mice was con-ducted in accordance with the regulations set forward by the Danish Min-istry of Justice and animal protection committees by Danish AnimalExperiments Inspectorate Permit 2004-561-868 (of January 7, 2004), andin compliance with European Community Directive 86/609 and the U.S.Association for Laboratory Animal Care recommendations for the care anduse of laboratory animals. All animal handling was done at Statens SerumInstitut by authorized personnel.

Bacteria

M.tb H37Rv, H37Rv/KO26 (hereafter named H37Rv�RD1; Ref. 28), andErdman were grown at 37°C on Middlebrook 7H11 (BD Pharmingen) agaror in suspension in Sauton medium (BD Pharmingen) enriched with 0.5%sodium pyruvate, 0.5% glucose, and 0.2% Tween 80. BCG Danish strain1331 was grown at 37°C in Middlebrook 7H9 medium (BD Pharmingen).BCG::RD1 and BCG::RD1-esxAd76-95 (BCG::RD1�ESAT-6; Refs. 29and 30) were grown at 37°C in Middlebrook 7H9 medium enriched withhygromycin. All bacteria were stored at �80°C in growth medium at �5 �108 CFU/ml. Bacteria were thawed, sonicated, washed, and diluted in PBSfor immunizations and infections. All bacterial work was done at StatensSerum Institut by authorized personnel.

Antigens

rTB10.4 was produced in Escherichia coli BL21 (DE3) with a pDEST 17vector containing the sequence for TB10.4 with the extension of a histidinetag. The protein was purified by gel filtration and further by application toan immobilized metal-affinity chromatography purification step. Syntheticoverlapping peptides (18- and 9-mer) covering the complete primary struc-ture of TB10.4 were synthesized by standard solid-phase methods on aSyRo peptide synthesizer (MultiSynTech) at the JPT Peptide Technologies,or at Schafer-N. Peptides were lyophilized and stored dry until reconsti-tution in PBS.

Experimental infections

When challenged by the aerosol route, the animals were infected with�50 CFU of M.tb Erdman/mouse with an inhalation exposure system(Glas-Col). When challenged by the i.v. route, the animals were in-fected with 105 CFU of M.tb H37Rv, H37Rv�RD1, BCG, BCG::RD1,or BCG::RD1�ESAT-6 per mouse in the lateral tail vein of the mouse.Mice were killed at indicated time points after challenge. Numbers ofbacteria in the spleen or lung were determined by serial 3-fold dilutionsof individual whole organ homogenates in duplicate on 7H11 medium.Colonies were counted after 2–3 wk of incubation at 37°C. Protectiveefficacies are expressed as log10 bacterial CFU.

Lymphocyte cultures

PBMC were purified on a density gradient of Mammal Lympholyte CellSeparation medium (Cedarlane Laboratories). Splenocyte cultures were ob-tained by passage of spleens through a metal mesh followed by two wash-ing procedures using RPMI 1640. Lung lymphocytes were obtained bypassage of lungs through a 100-�m nylon cell strainer (BD Pharmingen)followed by two washing procedures using RPMI 1640. Cells in each ex-periment were cultured in sterile microtiter wells (96-well plates; Nunc)containing 2–10 � 105 cells in 200 �l of RPMI 1640 supplemented with1% (v/v) premixed penicillin-streptomycin solution (Invitrogen Life Tech-nologies), 1 mM glutamine, and 5% (v/v) FCS at 37°C/5%CO2. The my-cobacterial Ags were all used at a concentration of 5 �g/ml for ELISA and2 �g/ml for flow cytometric analyses. Wells containing medium only orCon A were included in all experiments as negative and positive controls,respectively.

IFN-� ELISA

Microtiter plates (96-well; Maxisorb; Nunc) were coated with 1 �g/mlmonoclonal rat anti-murine IFN-� (clone R4-6A2; BD Pharmingen). Freebinding sites were blocked with 2% (w/v) milk powder in PBS. Culturesupernatants were harvested from lymphocyte cultures after 72 h of incu-bation and tested in triplicate. IFN-� was detected with a 0.1 �g/ml biotin-labeled rat anti-murine Ab (clone XMG1.2; BD Pharmingen) and 0.35�g/ml HRP-conjugated streptavidin (Zymed Laboratories/Invitrogen LifeTechnologies). The enzyme reaction was developed with 3,3�,5,5�-tetram-ethylbenzidine, hydrogen peroxide (TMB Plus; Kementec) and stoppedwith 0.2 M H2SO4. rIFN-� (BD Pharmingen) was used as a standard. Plateswere read at 450 nm with an ELISA reader and analyzed with KC4 3.03Rev 4 software.

Flow cytometric analysis

Intracellular cytokine staining procedure: cells from blood, spleen, or lungsof mice were stimulated for 1–2 h with 2 �g/ml Ag and subsequentlyincubated for 6 h with 10 �g/ml brefeldin A (Sigma-Aldrich) at 37°C.Thereafter, cells were stored overnight at 4°C. The following day, FcRswere blocked with 0.5 �g/ml anti-CD16/CD32 mAb (BD Pharmingen) for10 min. After the cells were washed in FACS buffer (PBS containing 0.1%sodium azide and 1% FCS), they were stained for surface markers as in-dicated using 0.2 �g/ml anti-CD4 (clone: RM4-5), anti-CD8 (53-6, 7),anti-CD25 (clone: PC61), anti-CD44 (clone: IM7), anti-CD45RB (clone:C363.16A), anti-CD62 ligand (anti-CD62L, clone: MEL-14), anti-CD69(clone: H1.2F3) or anti-CD95 ligand (CD95L, clone MFL3) mAbs. Cellswere then washed in FACS buffer, permeabilized using the Cytofix/Cyto-perm kit (BD Biosciences) according to the manufacturer’s instructions,and stained intracellularly with 0.2 �g/ml anti-IFN-� (clone: XMG1.2),anti-TNF-� (clone: MP6-XT22), or anti-IL-2 (clone: JES6-5H4) mAbs.When using the CD107A/B (clone: ID4B/ABL-93) mAbs, these wereadded to the wells along with the Ags, according to the manufacturer’sinstructions. Furthermore, a PE-conjugated Pro5 MHC-I (H-2Kb) pentamer(Proimmune) loaded with the minimal CD8 epitope of TB10.4 was used.Due to technical issues, the MHC-I molecules of the pentamer were loadedwith TB10.44-11 instead of TB10.43-11. After washing, cells were resus-pended in formaldehyde solution 4% (w/v) pH 7.0 (Bie and Berntsen) andanalyzed by flow cytometry on a six-color BD FACSCanto flow cytometer(BD Biosciences).

MHC-ligand prediction

The prediction of potential MHC-binding epitopes was done at the HarvardRANKPEP website (http://bio.dfci.harvard.edu/Tools/rankpep.html; Refs.31 and 32). Similarity is scored using position-specific scoring matrixesderived from aligned peptides known to bind to the given MHC molecule.

In vivo CTL assay

Splenocyte target cell suspensions from naive C57BL/6 were evenly splitinto two populations. One was pulsed with 10 �g/ml TB10.43-11 for 1 h at37°C and then labeled with a high concentration (40 �M) of CFSE(CFSEhigh population), and the other population was incubated for 1 h at37°C without peptide and labeled with a low concentration (4 �M) ofCFSE (CFSElow population). A 1:1 ratio of CFSElow- to CFSEhigh-labeledcells (1.5 � 107 cells in total) were mixed together and adoptively trans-ferred in 200 �l of PBS into M.tb-infected mice. Twenty hours later, re-cipient spleen cells were analyzed by flow cytometry. Percent lysis wasdetermined by loss of the peptide-pulsed CFSEhigh population comparedwith control CFSElow population using the formula (1 � (%CFSEhigh cells/%CFSElow cells) � 100).

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Statistical methods

The data obtained were tested by ANOVA. Differences between meanswere assessed for statistical significance by Tukey’s test. A p value of�0.05 was considered significant. When only comparing the means of twogroups, the Student t test was applied.

ResultsThe immune response against TB10.4 following infectionwith M.tb

To study the immune response against TB10.4 in infected mice,C57BL/6 mice were infected by the aerosol route with M.tb Erd-man and analyzed 3 wk later. Epitope recognition of TB10.4 wasassessed by using a panel of 18-mer peptides spanning the entiresequence of TB10.4 for in vitro stimulation of lymphocytes frominfected mice. The amount of IFN-� released following stimula-tion for 72 h was then analyzed by ELISA. The results showed thatonly stimulation with TB10.41-18 resulted in a significant IFN-�release (8713 � 1285 pg/ml IFN-�, Fig. 1A). We further analyzedthe TB10.41-18-specific T cell phenotype by flow cytometry bystaining TB10.41-18-stimulated lung lymphocytes with fluorescentanti-CD4, anti-CD8, and intracellularly with anti-IFN-� Abs. The

majority of TB10.41-18-specific T cells were of the CD8 phenotype(Fig. 1B). Thus, in the blood, 6.2% of CD8 T cells responded byproducing IFN-� after stimulation with TB10.41-18, while only1.2% of the CD4 T cells in the lungs were specific for TB10.41-18.The corresponding amount of IFN-�-producing T cells from naivemice following TB10.41-18 stimulation was 0.5% CD4 T cells and0.2% CD8 T cells (shown in parentheses in Fig. 1B). To preciselydefine the CD8 epitope within TB10.41-18, we next analyzed thesequence using the position-specific scoring matrix at Harvard’sRANKPEP website (http://bio.dfci.harvard.edu/Tools/rankpep.html; Ref. 31). The sequence QIMYNYPAM was predicted as thestrongest binder of both H-2Kb and H-2Db. In agreement with this,PBMCs from infected mice stimulated in vitro with peptides span-ning TB10.41-18 confirmed that the minimal epitope inducing thehighest release of IFN-� was indeed TB10.43-11 (QIMYNYPAM;Fig. 1D). In addition, stimulating lymphocytes from infected micewith a panel of 12-mer peptides spanning the sequence ofTB10.41-18 showed that the minimal MHC-II H-2b-restrictedepitope was TB10.43-14 (data not shown).

Having identified the minimal CD8 epitope, we next analyzedPBMCs taken from mice 6 wk after aerosol infection with M.tb by

FIGURE 1. Infection with virulent M.tb induce CD8 T cells specific for a novel epitope in TB10.4. A, PBMCs pooled from infected C57BL/6 micewere stimulated for 72 h in vitro with a panel of nine overlapping 18-mer peptides covering the TB10.4 sequence, and IFN-� levels in supernatantswere assessed by ELISA. Bars represent means and SEM of triplicate values. B, PBMCs were stimulated in vitro with the immune dominant epitopeTB10.41-18 and the specific T cell phenotype was evaluated by flow cytometry of cells stained with anti-CD4, anti-CD8, and intracellular anti-IFN-�.The indicated percentages specify the proportion of the CD4/CD8 T cell populations producing IFN-� after peptide stimulation. Correspondingpercentages in naive mice are shown in parentheses. C, A schematic overview of the overlapping 9-mer peptides spanning the N-terminal TB10.4sequence used to map the minimal H-2Kb-restricted epitope of TB10.4. D, IFN-� production was assessed by flow cytometry of intracellularcytokine-stained CD4/CD8 blood T cells stimulated in vitro with the five 9-mer peptides shown in C. The bars show the proportions of CD4 (�)and CD8 (f) T cells that stained positive for IFN-� following stimulation. E, Specific CD8 T cells were identified using a H-2Kb pentamer loadedwith TB10.44-11 (see Materials and Methods). Lymphocytes from mice aerosol infected for 6 wk were gated as in B and D, and the upper rightquadrant shows the proportion of CD8 T cells that stained positive with the H-2Kb/TB10.4 pentamer. In A and B, and D and E, PBMCs were pooledfrom four to six mice. Results are representative of three individual experiments. In A, B, and D, mice were infected by the aerosol route for 3 wk.

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using a H-2Kb pentamer loaded with the minimal CD8 epitope (seeMaterials and Methods). A total of 7.6% of all CD8 T cells stainedpositive with the H-2Kb/TB10.4 pentamer (Fig. 1E).

Phenotype of the TB10.43-11-specific T cells

To more precisely characterize the phenotype of the TB10.43-11-specific CD8 T cells, PBMCs from the infected C57BL/6 micewere stimulated in vitro with TB10.43-11 and analyzed by flowcytometry for expression of CD25, CD44, CD45RB, CD62L,CD69, IFN-�, TNF-�, and IL-2. Cells were also stained for CD4expression, but intracellular cytokine staining was at or belowbackground levels following stimulation with TB10.43-11 andTB10.43-14 (data not shown), indicating that the CD4 responseagainst TB10.4 following infection in C57BL/6 mice is a minoror transient response. The majority of the IFN-�-producingCD8 T cells expressed CD25mid/high, CD44high, CD45RBmid/low,CD62Llow, and CD69high (Fig. 2A). The majority of IFN-�-pro-ducing CD8 T cells also expressed TNF-�. In contrast, only few ofthe IFN-�-producing CD8 T cells costained for IL-2. As observedfor IFN-�-positive cells, the majority of TNF-�-expressing cellsexpressed CD45RBmid/low and CD62Llow (Fig. 2A, lower dia-grams). As in vitro stimulation with TB10.43-11 may alter the phe-notype of the stimulated CD8 T cells, we also analyzed theTB10.43-11 CD8 cells specifically using the H-2Kb/TB10.4 pen-tamer. The results showed that the CD8 cells were of aCD44highCD45RBlowCD62Llow phenotype (Fig. 2B), thus resem-bling the effector phenotype observed following in vitro stimula-tion with TB10.43-11 (Fig. 2A). Naive mice had a background pen-tamer staining at 0–0.5% of all CD8 T cells. Thus, TB10.43-11

CD8 cells represent an effector CD8 T cell population inducedshortly after infection with M.tb.

TB10.43-11-specific CD8 T cells are long lived and recruited tothe site of infection

We next examined whether TB10.43-11 CD8 T cells representedmore than a transient cell population and to which degree thesecells were recruited to the site of infection. Mice were infected

with virulent M.tb Erdman by the aerosol route, whereafter cellsfrom lungs, spleen and blood were isolated at weeks 0–50 postin-fection. Following stimulation in vitro with TB10.41-18, the cellswere analyzed for expression of CD4, CD8, and IFN-� by flowcytometry. By using the TB10.41-18 peptide, we were able to mon-itor both the TB10.4-specific CD4 and CD8 cells simultaneously.Only low amounts of IFN-�-producing CD4 T cells were gener-ated throughout the infection. In contrast, TB10.43-11-specific CD8T cells were present throughout the experiment (Fig. 3, A–C). Inthe spleen and blood, the kinetics was similar, although the re-sponses were higher in the blood. Compared with blood andspleen, the response in the lungs peaked before the response inblood and spleen, indicating that TB10.43-11-specific CD8 cellswere first observed in the lung (Fig. 3, A–C). The amount ofTB10.4-specific IFN-�-producing T cells in all organs declinedtoward week 19 but was increased at week 48 postinfection. Fur-thermore, at later time points in particular, the TB10.43-11-specificCD8 T cells were found in higher numbers in the lung, comparedwith the blood (and spleen). Staining the cells with H-2Kb/TB10.4pentamer showed that at week 6 postinfection, 7.3% of the entireCD8 T cell population in the blood was stained positive for thepentamer. This value decreased to 4.9% after 19 wk but as shownfor TB10.43-11-specific CD8 IFN-�� cells in Fig. 3, A–C, theamount of H-2Kb/TB10.4 pentamer-positive cells had increasedslightly at week 48 after challenge (Fig. 3D). The phenotype ofTB10.43-11-specific CD8 T cells in terms of effector markers didnot change in the course of infection. Thus, 48 wk postinfectionH-2Kb/TB10.4 pentamer-positive cells in the lung (and blood/spleen, data not shown) were still CD44high, CD45RBlow, CD62low

(Fig. 3E). Taken together, these results demonstrated that theTB10.43-11-specific CD8 T cells represented a long-lasting CD8cell population.

TB10.43-11-specific CD8 T cells are cytotoxic

The effector phenotype, longevity, and recruitment to the infectionsite of TB10.43-11-specific CD8 T cells indicated that these cellswere actively involved in the immune response against M.tb. To

FIGURE 2. Phenotypic analysis of TB10.3-11-specific CD8 T cells. A, C57BL/6mice were infected by the aerosol routewith virulent M.tb Erdman, and PBMCswere purified from infected mice 6 wk af-ter mycobacterial challenge and stimulatedin vitro with TB10.43-11. Cells werestained intracellularly for IFN-� (upper di-agrams) or TNF-� (lower diagrams), andfor surface expression of CD8, CD25,CD44, CD45RB, CD62L, CD69, and in-tracellular IL-2. Percentages illustrate theproportion of cytokine-producing CD8 Tcells that express CD25mid/high, CD44high,CD45RBlow/mid, CD62Llow, CD69high, sin-gle-positive IFN-��/IL-2� cells, and theproportion of TNF-�-producing cells whichalso produce IFN-�. Cells were also stainedwith anti-CD4, but the amount of CD4 IFN-�-producing cells was below 0.2%. B, Bloodcells taken 6 wk after infection were stainedwith the H-2Kb/TB10.4 pentamer and anti-CD44, anti-CD45RB, and anti-CD62L. In Aand B, PBMCs were pooled from five mice,and are representative of three individualexperiments.

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examine the effector function of the TB10.43-11-specific T cells invitro, lymphocytes from blood and lungs of infected mice werestimulated in vitro with TB10.43-11 and degranulation was quan-tified with CD107A/B labeling. In both lung and blood cells, stim-ulation with TB10.43-11 induced an increased expression ofCD107A/B on CD44highCD8 T cells (Fig. 4A). In addition, we alsoobserved enhanced CD95L (FasL) expression on theCD44highCD8 T cells (Fig. 4A). As these results indicated a cyto-toxic potential of the TB10.43-11 cells, we next examined whetherthe TB10.43-11-specific T cells in infected mice were indeed ca-pable of eliminating target cells expressing this epitope in vivo.We used the in vivo cytotoxic assay where CFSE-labeled spleno-cytes from naive mice, unpulsed or pulsed with TB10.43-11, wereadoptively transferred into infected mice. Peptide-specific lysis ofthe transferred cells was then investigated by flow cytometric anal-ysis of recipient spleens. Although some killing was observedupon transfer of target cells to naive mice, a strong increase inclearance of TB10.43-11-pulsed target cells was observed (up to70%-specific killing of target cells), indicating that TB10.43-11-specific cells were able to kill their target cells, and that thisepitope is an immunological target during natural infection withM.tb. Moreover, the cytotoxic activity of TB10.43-11-specific cellswas maintained in chronically infected mice, although we did ob-serve some decline in cytotoxicity, which however seemed to cor-relate with the decline in the number of TB10.43-11-specific T cells�20 wk postinfection (Fig. 4C and 3, A–D). Thus, TB10.43-11-specific cells represented a cytotoxic population of CD8 T cellswith a killing mechanism that involved degranulation as well asCD95L-induced apoptosis of target cells.

The role of the genetic RD1 and bacterial virulence in theinduction of TB10.43-11-specific CD8 T cells

Having showed that TB10.43-11-specific CD8 cells make up a sub-stantial part of the total pool of CD8 T cells during the naturalinfection with M.tb, we were next interested in the requirement forthe generation of these cells. As a decreased induction of CD8 Tcells have been proposed to be partly responsible for the failure ofBCG to efficiently protect against pulmonary infection in adults(21, 25), we first examined the induction of TB10.43-11-specificCD8 T cells following BCG vaccination or infection with M.tb. Invitro stimulation of blood lymphocytes from BCG-vaccinatedmice with TB10.41-18 resulted in 1.4% IFN-�-producing CD8 Tcells, while the equivalent value for the M.tb group was 5.1%. Incontrast, the amount of specific IFN-�-producing CD4 T cells wascomparable in the two groups (Fig. 5). This indicated that a geneticelement present in virulent M.tb but absent in BCG influenced theCD8 T cell response against TB10.4. As the genetic RD1 region isencoded in all clinical isolates of M.tb but is deleted from all BCGsubstrains (25, 29), we next analyzed whether the RD1 region wasindeed required for the elevated CD8 T cell response againstTB10.4 in M.tb-infected mice. To examine this, we first used theBCG knockin strain, BCG::RD1, in which the genetic region RD1has been reintroduced (29), and compared this to BCG. Mice wereinfected i.v. with mycobacteria for 6 wk and PBMCs were ana-lyzed by flow cytometry for Ag-specific IFN-�-producing CD4and CD8 T cells upon TB10.41-18 in vitro stimulation. As seen inFig. 6, the mutant BCG::RD1 strain generated significantly highernumbers of TB10.4-specific CD8 T cells (5.8% of the entire

FIGURE 3. Kinetics of TB10.4-spe-cific CD8 T cells following infectionwith M.tb. Lymphocytes from lung (A),spleen (B), and blood (C) were isolated atdifferent time points after infection, andstimulated in vitro with the 18-mer pep-tide TB10.41-18 containing both theMHC-I and -II restricted epitopes ofTB10.4 to evaluate kinetics of both theCD4 and CD8 T cells specific forTB10.4. IFN-� production was evaluatedby flow cytometry and values representthe proportions of CD4 and CD8 T cellsin the lymphocyte gate that stainedpositive with anti-IFN-� followingTB10.41-18 stimulation. Each analysiswas performed on lymphocytes pooledfrom four to six mice. D, The TB10.4-specific CD8 T cells in blood werestained with the H-2Kb/TB10.4 pen-tamer, and the results show the amount ofCD4 and CD8 T cells that stained posi-tive. Blood cells were pooled from fourto six mice. E, Lung cells were taken 48wk postinfection and stained with theH-2Kb/TB10.4 pentamer, CD62L, CD44,and CD45RB. The results show onlyH-2Kb/TB10.4 pentamer-positive cells.

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CD8 T cell population) than BCG (1.0% of all CD8 T cells). Incontrast, the CD4 T cell response against TB10.4 was not asdependent upon the RD1 region and between 0.3 and 0.4% ofthe CD4 T cells produced IFN-� following TB10.41-18 stimu-lation. Naive mice showed 0.1% IFN-�-producing CD4 T cellsand 0.3% CD8 T cells (data not shown). In support of theseresults, in BCG::RD1-infected mice, 8.8 and 2.9% of the CD8T cells were pentamer positive in the blood and spleen, respec-tively, whereas in BCG-vaccinated mice we observed 0.7%pentamer-positive CD8 T cells in blood and 0.3% in the spleen.In naive mice, �0.2% of the CD8 T cell population stainedpositive in blood and spleen. This influence of the RD1 regionon the magnitude of the CD8 T cell response was dependentupon expression of ESAT-6. Thus, infection with BCG::RD1�ESAT-6, lacking expression of ESAT-6, led to a CD8 Tcell response in lungs and spleen that was not significantly dif-ferent from that observed in BCG-vaccinated mice in terms ofthe number of TB10.43-11-specific CD8 T cells (Fig. 6E). Inaddition, in vitro stimulation of lymphocytes from lungs,

spleen, or blood with TB10.43-11 induced a secretion of IFN-�,TNF-�, and IL-2 that was also not significantly different fromthat seen with lymphocytes from BCG-vaccinated mice (datanot shown).

The RD1 region has been suggested to be involved in virulence,which in turn could lead to a stronger CD8 response (25). To betterunderstand how the RD1 region could influence the generation ofTB10.4-specific CD8 T cells, we therefore tested the correlationbetween the RD1 region, or bacterial growth/dissemination (forthese purposes defined as virulence), and the number of TB10.4-specific CD8 T cells. Mice were infected i.v. with BCG::RD1 orBCG, and CFU levels in the lung were measured between 3 and 9wk following infection. The results showed that BCG::RD1 wasmore virulent than BCG ( p � 0.01). Importantly, the numbers ofTB10.4-specific CD8 T cells measured at week 9 postvaccinationcorrelated with the numbers of bacteria. Thus, for both the CFUnumbers and the number of H-2Kb/TB10.4 pentamer-positive CD8T cells, we observed increased numbers in BCG::RD1-infectedmice, compared with BCG-infected mice (Fig. 6). Initially, BCGdid induce up to 10% H-2Kb/TB10.4 pentamer-positive CD8 Tcells in the lung after 4 wk of infection (Fig. 6E). However, thesenumbers declined rapidly and were always significantly below thatseen in BCG::RD1-infected mice. These results demonstrated thatthe presence of a functional RD1 region led to increased bacterialvirulence, and increased numbers of TB10.43-11-specific CD8 Tcells.

To examine whether other genetic regions than RD1 (absentin BCG, but present in M.tb) were involved in bacterial viru-lence, and in the induction of CD8 T cells, we next used theM.tb-mutant knockout strain, H37Rv�RD1, in which the RD1region has been deleted (29). Interestingly, even thoughH37Rv�RD1 was significantly less virulent that H37Rv ( p �0.05), it was clearly more virulent than BCG, despite the lack ofthe genetic region RD1 (Fig. 7). Furthermore, as with theBCG/BCG::RD1 strains (Fig. 6), we observed a clear correla-tion between virulence and the number of CD8 T cells. Thus, 9wk after infection, 15% of all the CD8 T cells in the lung were

FIGURE 4. The TB10.4-specific CD8 T cells elicit CTL responses in-volving degranulation and CD95L during chronic infection with M.tb. A,Degranulation of cytotoxic vesicles was evaluated by flow cytometry on Tcells from infected mice stimulated in vitro with TB10.43-11 or mediumalone. Lung and blood lymphocytes were stained with anti-CD8, anti-CD44, and anti-CD107A/B Abs, and blood lymphocytes were also stainedwith anti-CD95L. Percentages describe the proportion of CD44high CD8 Tcells expressing CD107A/B and CD95L upon stimulation. B, CTL ac-tivity of TB10.43-11-specific CD8 T cells in vivo. Unloaded splenocytes(CFSElow) and TB10.43-11-loaded splenocytes (CFSEhigh) from naive micewere transferred into infected mice. The amount of splenocytes killed invivo by cytotoxic CD8 T cells specific for TB10.43-11 is seen as the re-duction in the CFSEhigh population. Percentages specify the total amount ofCFSEhigh cells killed. C, The amount of the specific in vivo cytotoxicresponse against TB10.43-11-loaded splenocytes in infected mice measuredat different time points post challenge. In A and B, analysis was performed6 wk after infection.

FIGURE 5. Induction of TB10.4-specific CD8 T cells following infec-tion with M.tb or BCG. PBMCs from C57BL/6 mice were purified 6 wkafter s.c. immunization with BCG or aerosol infection with M.tb, and stim-ulated in vitro with TB10.41-18 containing both the MHC-I and -II-re-stricted epitopes of TB10.4. Induction of CD4 and CD8 T cells was as-sessed by flow cytometric analysis. Percentages specify the proportion ofCD4 or CD8 T cells producing IFN-� after stimulation with TB10.41-18.Lymphocytes were pooled from four mice per group and results are rep-resentative of three individual experiments.

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pentamer positive in H37Rv�RD1-infected mice with a bacte-rial count of 3.89 � 0.55 log10 CFU, compared with 25% inH37Rv-infected mice with a bacterial count of 4.91 � 0.41log10 CFU. Taken together, these results indicated that viru-lence is also involved in generating a CD8 response againstmycobacterial Ags.

DiscussionIn the present study, we describe a new MHC-I (H-2Kb)-restrictedepitope shared by the homologous proteins TB10.3 and TB10.4(33). Our results indicated that the minimal epitope mapped toamino acids 3–11 (Fig. 1). (It should be noted that these mappingstudies did not include 10 mers. However, overlapping 12 mers didnot induce stronger responses). TB10.43-11-specific CD8 T cellsfrom M.tb-infected C57BL/6 mice secreted high amounts of Th1cytokines such as IFN-� and TNF-�, in agreement with the worksof Kamath et al. (34), who found that T cells specific forTB10.420-28 also produced significant amounts of these two cyto-kines. However, in contrast to TB10.420-28-specific CD8 cells inM.tb-infected BALB/c mice (17), TB10.43-11-specific CD8 T cellsonly produced small amounts of IL-2 in C57BL/6 mice challengedwith virulent M.tb. This difference may be due to the use of dif-ferent mice strains or the different experimental procedures used ineach study.

The TB10.43-11-specific CD8 T cells expressed surface activa-tion markers such as CD25, CD44, and CD69, but had down-regulated CD45RB or CD62L, thus exhibiting a typical effector Tcell phenotype. This effector phenotype was observed upon stim-ulation with the TB10.43-11 peptide or after staining CD8 T cellsfrom infected mice with the H-2Kb/TB10.4 pentamer ex vivo. Thecontinued presence of TB10.43-11-specific CD8 T cells in the lungs(and spleen/blood) indicated that they were an active part of theimmune response against both acute and chronic infection. Fol-lowing stimulation with the specific Ag, TB10.43-11-specific CD8T cells showed an increased expression of CD107A/B and CD95L(Fig. 4). CD107A/B is located on the inner membrane of cytotoxicgranules, but is expressed on the outer cell membrane briefly afterdegranulation, while CD95L is a known inducer of target cell ap-optosis. Thus, TB10.43-11-specific CD8 T cells may exhibit morethan one killing mechanism. We were not able to show up-regu-lation of perforin expression on lung TB10.43-11-specific CD8 T

FIGURE 6. Induction of CD8 T cells is increased upon mycobacte-rial expression of RD1-encoded proteins. A, PBMCs from C57BL/6mice were purified 6 wk after infection of different mycobacteria asindicated. The proportions of IFN-�-producing TB10.4-specific T cellswere analyzed by flow cytometry of the PBMCs stimulated in vitro withTB10.41-18. Cells were stained for surface expression of CD4, CD8, andfor presence of intracellular IFN-�. Percentages specify the proportionof the CD4 and CD8 T cell population that produced IFN-� in responseto TB10.41-18 stimulation (upper right quadrant). B, The proportions ofH2-Kb/TB10.4 pentamer-positive CD8 T cells 6 wk after infection inthe blood and spleen of mice infected with BCG or BCG::RD1. C, Thebacterial burden was determined in the lungs of BCG- orBCG::RD1-infected C57BL/6 mice at the indicated time points. ��, p �0.01. D, Concurrent with the analysis of bacterial burden at 9 wk postin-fection in the lungs, CD8 T cells specific for the minimal TB10.4epitope were analyzed by flow cytometry. Bars represent means andSEM (n 3) of the proportion of CD8 T cells that stained positive withthe H-2Kb/TB10.4 pentamer. E and F, CD8 T cells specific for theminimal TB10.4 epitope in the lungs or spleen were analyzed by flowcytometry 4 wk following infection as indicated. Bars represent meansand SEM (n 4) of the proportion of CD8 T cells that stained positivewith the H-2Kb/TB10.4 pentamer.

FIGURE 7. Mycobacterial virulence correlates with the amount ofTB10.4-specific CD8 T cells. A, In vivo growth of H37Rv (open symbols)and H37Rv�RD1 (solid symbols) after i.v. infection of C57BL/6 mice,determined in the lungs at 3, 4, and 9 wk postinfection. �, p � 0.05. B, Nineweeks postinfection in the lungs, CD8 T cells specific for the minimalTB10.4 epitope were analyzed by flow cytometry as indicated. Bars rep-resent means and SEM (n 3) of the proportion of CD8 T cells stainedwith the H-2Kb/TB10.4 pentamer.

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cells upon stimulation with specific Ag (data not shown), which isin agreement with previous studies that indicated that perforin isnot important for the control of mycobacterial infection (6, 35). Insupport of a cytotoxic role for TB10.43-11-specific CD8 T cells, weshowed that these cells were able to kill naive splenocytes loadedwith the TB10.43-11 peptide in vivo. This was also recently shownto apply for TB10.420-28-specific T cells (16, 17) and support thatthese T cells play an active part in the immune response againstinfection with M.tb.

It has recently been proposed that the amount of T cells mayfluctuate dynamically throughout the infection (36). We found thatthe amounts of IFN-�-producing TB10.43-11-specific CD8 T cellsexhibited a pattern consisting of a strong increase shortly after theonset of infection, followed by a decline in numbers to week 19,and an increase in numbers late in the chronic infection. This pat-tern was evident both in lungs (where the peak was first observed,although additional studies at earlier time points may help eluci-date this point more), blood, and spleen, although the percentage ofspecific CD8 T cells was higher in the lungs and blood as com-pared with the spleen. The kinetic pattern therefore followed thegeneral bacterial levels in the lung with a peak early in infection,followed by a decline once the adaptive immune response gainscontrol over the infection (36, 37). Whether the increase inTB10.43-11-specific CD8 T cells after prolonged exposure (week48) also correlated with increased CFU levels was not examined.Using the H-2Kb/TB10.4 pentamer, we observed the same overallkinetic pattern concerning the entire amount of TB10.4 CD8 Tcells as seen for the cytokine-producing cells described above, andthis also correlated with in vivo cytotoxicity toward transferredTB10.43-11-loaded target cells, indicating that the TB10.43-11

CD8 T cells retain their cytotoxicity throughout the infection.The role of CD8 T cells during infection and the different ability

to induce these cells by BCG and M.tb has been a subject for amajor and as yet unresolved debate (21). Indeed, it has been sug-gested that the failure of BCG as a vaccine may be explained by itslack of ability to induce a robust CD8 response (21). Interestingly,the strong CD8 T cell response against TB10.4 was significantlyreduced in BCG-vaccinated mice compared with BCG::RD1 (orM.tb) vaccinated mice, whereas the CD4 T cell response was lessaffected. Staining with the H-2Kb/TB10.4 pentamer confirmed thatit was the number of CD8 T cells that declined in mice infectedwith BCG, and not merely the cytokine-producing ability of thespecific CD8 T cells. The fact that we also saw an increased rec-ognition of MTB32A (19) by CD8 T cells in BCG::RD1-infectedmice compared with mice infected with BCG (data not shown)indicated that this is a general phenomenon that applies to theoverall CD8 T cell population, in line with two recent studiesshowing that introduction of RD1 into BCG or M. microti in-creased the amount of Ag-specific CD8 T cells (25, 26). In addi-tion, induction of CD8 T cells was dependent upon expression ofESAT-6 (Fig. 6), which could indicate a requirement for a specificfunction of ESAT-6 and/or that a functional RD1 region requiresESAT-6. However, experiments performed with the RD1 knock-out mutant H37Rv�RD1 showed that induction of CD8 T cellswas not strictly dependent upon the RD1 region, demonstratingthat other RD regions are also involved in both virulence and theinduction of CD8 T cells. Interestingly, in support of this, a recentstudy showed that mycobacterial Ags were indeed presented onMHC-I following infection of dendritic cells with H37Rv�RD1(38) (Figs. 6 and 7).

Our results also showed a clear correlation between bacterialvirulence and CD8 T cell induction. Although the mechanism bywhich the increased bacterial growth/virulence can lead to in-creased CD8 T cell response is not known, previous studies have

indicated at least two ways by which this could occur: 1) throughincreased availability of bacterial Ag and 2) increased apoptosis/necrosis induction of infected APCs.

Concerning the increased numbers of bacteria in mice infectedwith virulent bacteria (Figs. 6 and 7), it was recently shown thatCD8 T cells are indeed more activated by heavily infected APCs,compared with APCs subjected to a low-grade infection (39), andsubjecting dendritic cells to increasing amounts of peptide loadedbeads correlated, in particularly, with the amount of cross-pre-sented CD8 epitopes (40, 41). It could therefore be speculated thatthe increased numbers of virulent bacteria lead to an increasedamount of bacterial Ag in phagocytotic cells, such as dendriticcells and macrophages, which in turn would increase the numberof Ags available for the MHC-I presentation pathway.

Regarding apoptosis, mycobacteria have been shown to induceapoptosis of infected macrophages (42). Increased release of apo-ptotic vesicles containing mycobacterial Ags may be taken up bybystander APCs, and via the cross-presentation pathway be pre-sented on the cell surface on MHC-I molecules (24, 43). Interest-ingly, a recent study indicated a correlation between the RD1 re-gion and apoptosis. Thus, while infection of THP-1 cells withH37Rv resulted in apoptosis, a deletion mutant that did not expressthe RD1-encoded protein ESAT-6 failed to induce significant ap-optosis (42). Thus, the presence of functional RD1 may lead toincreased apoptosis which in turn could increase the induction ofCD8 T cells. This is in agreement with our results which showeda correlation between the expression of RD1 encoded ESAT-6 andthe induction of TB10.43-11-specific CD8 T cells (Figs. 6 and 7).However, it should be noted that RD1 expression has also beenshown to increase necrosis (44), and whether the observed in-creased CD8 T cell response in the presence of RD1 (Figs. 5 and6) was due to increased apoptosis or increased necrosis was notshown in the present study.

In conclusion, we have described a novel CD8 T cell specific forTB10.43-11. In infected animals, the phenotype of TB10.43-11-spe-cific CD8 T cells resembled that of an effector T cell and the killingmechanism may involve both degranulation and CD95L. Finally,the induction of TB10.43-11-specific CD8 T cells correlated withexpression of ESAT-6 and virulence of the mycobacteria. We arepresently examining how bacterial virulence affects the magnitudeof CD8 T cells specific for mycobacterial Ags.

AcknowledgmentsThe technical help of Kristine Persson, Lene Rasmussen, and CharlotteFjordager is gratefully acknowledged. We thank Ida Rosenkrands forcritical comments. BCG::RD1 and BCG::RD1�ESAT-6 was a gift fromDrs. Stewart Cole and Roland Brosch and H37Rv�RD1 was a gift fromDr. William R. Jacobs.

DisclosuresThe authors have no financial conflict of interest.

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