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The Journal of Immunology, 2007, 179, 3973 -3981
Copyright © 2007 by The 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 of a Functional Region of Difference-11

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although infection with Mycobacterium tuberculosis (M.tb) induces a robust CD8 T cell response, the role of CD8 T cells in the defense against M.tb, and the mechanisms behind the induction of CD8 T cells, is still not clear. TB10.4 is a recently described Ag that is expressed by both bacillus Calmette-Guérin (BCG) and M.tb. In the present study, we describe a novel CD8 T cell epitope 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 present throughout the infection in high numbers. TB10.43-11 CD8 T cells were recruited to the site of infection and expressed CD44, TNF-{alpha}, and IFN-{gamma}. In addition, TB10.43-11 CD8 T cells showed an up-regulation of FasL and LAMP-1/2 (CD107A/B), which correlated with a strong in vivo cytolytic activity. The induction of TB10.43-11-specific CD8 T cells was less pronounced following infection with BCG compared to infection with M.tb. By using a rBCG expressing the genetic region of difference-1 (RD1), we show that the presence of a functional RD1 region increases the induction of TB10.43-11-specific CD8 T cells as well as the bacterial virulence. Finally, as an M.tb variant lacking the genetic region RD1 also induced a significant amount of TB10.43-11-specific CD8 T cells, and exhibited increased virulence compared with BCG, our data suggest that virulence in itself is also involved in generating a robust CD8 T cell response against mycobacterial epitopes, such as TB10.43-11.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is still not clear exactly what constitutes a protective immune response to Mycobacterium tuberculosis (M.tb),3 but it has been demonstrated in both animals and humans that T cell-mediated, rather than Ab-mediated, immune responses are essential for control of tuberculosis (TB). The major mechanisms of cell-mediated immunity include CD4 Th1-cell mediated activation of macrophages to destroy intracellular bacterial pathogens; the central role of IFN-{gamma} in the control of TB has been clearly demonstrated by the susceptibility to mycobacterial infections in mice with a disrupted IFN-{gamma} gene and in humans with mutations in genes involved in the IFN-{gamma} and IL-12 pathways (1, 2, 3, 4).

Unlike CD4 T cells, the role of CD8 T cells in the defense against M.tb is still not clear. CD8 T cells are induced early in the infection (5) and previous studies indicated that cytotoxic CD8 T cell-mediated killing of infected host cells do play a role in the defense against an M.tb infection, especially in the later phases of the infection (6, 7). Furthermore, mice without functional CD8 T cells, caused by disruptions of the beta2-microglobulin or the TAP1 genes, or mice subjected to in vivo depletion of CD8 T cells, showed a decreased control of the infection compared with control mice (8, 9, 10, 11). Moreover, several studies using different vaccination approaches, such as dendritic cells pulsed with CD8 (and CD4) T cell epitopes or adenovirus-expressing mycobacterial Ags, showed a strong induction of CD8 T cells and a significant protection against infection with M.tb, again suggesting a role for CD8 T cells (12, 13, 14). However, as CD4 cells were also induced in these studies, they did not conclusively show that CD8 cells were required. In fact, two recent studies showed that induction of a CD8 response against a specific epitope from TB10.4 or ESAT-6 did not lead to protection against an acute infection with M.tb (15, 16). This is in agreement with other studies showing that depletion of CD8 T cells did not affect the bacterial load in the lungs of mice suffering from an acute infection (7). Thus, the role of the CD8 cells is still not fully known and one drawback regarding the study of CD8 T cells and their role in the defense against M.tb has been the limited number of identified M.tb CD8 peptide epitopes that are specifically recognized in infected animals. Lately, a number of CD8 epitopes have been identified in M.tb proteins, such as TB10.4 (17), CFP10, (18), MTB32A (19), Ag85A and Ag85B (14, 20), and these studies have demonstrated that although the exact role of this T cell subset during infection with M.tb still remains unclear, infection with M.tb does induce a strong CD8 response that encompass both IFN-{gamma} production and cytotoxicity.

Interestingly, concerning the role of CD8 T cells, it has been suggested that a major reason for the failure of the current TB vaccine (bacillus Calmette-Guérin (BCG)) is related to an inferior ability of BCG to induce specific CD8 T cells compared with M.tb (21, 22). Thus, a rBCG strain ({Delta}ureC hly+rBCG) was produced expressing the phagosome pore-forming protein listeriolysin from Listeria monocytogenes which should increase the escape to the cytosol, thereby increasing the amount of bacterial peptides available for the MHC class I (MHC-I) presentation pathway. Interestingly, {Delta}ureC hly+rBCG was later shown to be more protective than BCG against virulent M.tb infection (22, 23, 24). Moreover, recent studies have shown that reintroduction of the genetic region of difference-1 (RD1), thought to be the main cause of the attenuation of BCG, into BCG or Mycobacterium microti resulted in increased virulence, increased activation of CD8 T cells, and improved protection against M.tb, again indicating a role for CD8 T cells in the protection against M.tb (25, 26, 27).

In the present study, we describe a novel CD8 T cell epitope shared by the homologous proteins of the early secretory antigenic target-6 kDa (ESAT-6) family: TB10.3 and TB10.4. Detailed analysis showed that the TB10.43-11-specific CD8 T cells were recruited to the site of infection during M. tuberculosis infection and that these cells expressed both TNF-{alpha} and IFN-{gamma}, and up-regulated expression of FasL and LAMP-1/2 (CD107A/B) upon activation. In contrast, significantly less CD8 cells were induced following BCG vaccination. By detailed dissection of mycobacterial strains with and without RD1, it became clear that the induction of TB10.43-11-specific CD8 T cells was related to both a functional RD1 region as well as the virulence of the bacterial strain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal handling

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

Bacteria

M.tb H37Rv, H37Rv/KO26 (hereafter named H37Rv{Delta}RD1; Ref. 28), and Erdman were grown at 37°C on Middlebrook 7H11 (BD Pharmingen) agar or in suspension in Sauton medium (BD Pharmingen) enriched with 0.5% sodium pyruvate, 0.5% glucose, and 0.2% Tween 80. BCG Danish strain 1331 was grown at 37°C in Middlebrook 7H9 medium (BD Pharmingen). BCG::RD1 and BCG::RD1-esxAd76-95 (BCG::RD1{Delta}ESAT-6; Refs. 29 and 30) were grown at 37°C in Middlebrook 7H9 medium enriched with hygromycin. All bacteria were stored at –80°C in growth medium at ~5 x 108 CFU/ml. Bacteria were thawed, sonicated, washed, and diluted in PBS for immunizations and infections. All bacterial work was done at Statens Serum Institut by authorized personnel.

Antigens

rTB10.4 was produced in Escherichia coli BL21 (DE3) with a pDEST 17 vector containing the sequence for TB10.4 with the extension of a histidine tag. The protein was purified by gel filtration and further by application to an immobilized metal-affinity chromatography purification step. Synthetic overlapping peptides (18- and 9-mer) covering the complete primary structure of TB10.4 were synthesized by standard solid-phase methods on a SyRo peptide synthesizer (MultiSynTech) at the JPT Peptide Technologies, or at Schafer-N. Peptides were lyophilized and stored dry until reconstitution 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 infected with 105 CFU of M.tb H37Rv, H37Rv{Delta}RD1, BCG, BCG::RD1, or BCG::RD1{Delta}ESAT-6 per mouse in the lateral tail vein of the mouse. Mice were killed at indicated time points after challenge. Numbers of bacteria in the spleen or lung were determined by serial 3-fold dilutions of individual whole organ homogenates in duplicate on 7H11 medium. Colonies were counted after 2–3 wk of incubation at 37°C. Protective efficacies are expressed as log10 bacterial CFU.

Lymphocyte cultures

PBMC were purified on a density gradient of Mammal Lympholyte Cell Separation medium (Cedarlane Laboratories). Splenocyte cultures were obtained by passage of spleens through a metal mesh followed by two washing procedures using RPMI 1640. Lung lymphocytes were obtained by passage of lungs through a 100-µm nylon cell strainer (BD Pharmingen) followed by two washing procedures using RPMI 1640. Cells in each experiment were cultured in sterile microtiter wells (96-well plates; Nunc) containing 2–10 x 105 cells in 200 µl of RPMI 1640 supplemented with 1% (v/v) premixed penicillin-streptomycin solution (Invitrogen Life Technologies), 1 mM glutamine, and 5% (v/v) FCS at 37°C/5%CO2. The mycobacterial Ags were all used at a concentration of 5 µg/ml for ELISA and 2 µg/ml for flow cytometric analyses. Wells containing medium only or Con A were included in all experiments as negative and positive controls, respectively.

IFN-{gamma} ELISA

Microtiter plates (96-well; Maxisorb; Nunc) were coated with 1 µg/ml monoclonal rat anti-murine IFN-{gamma} (clone R4-6A2; BD Pharmingen). Free binding sites were blocked with 2% (w/v) milk powder in PBS. Culture supernatants were harvested from lymphocyte cultures after 72 h of incubation and tested in triplicate. IFN-{gamma} was detected with a 0.1 µg/ml biotin-labeled rat anti-murine Ab (clone XMG1.2; BD Pharmingen) and 0.35 µg/ml HRP-conjugated streptavidin (Zymed Laboratories/Invitrogen Life Technologies). The enzyme reaction was developed with 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide (TMB Plus; Kementec) and stopped with 0.2 M H2SO4. rIFN-{gamma} (BD Pharmingen) was used as a standard. Plates were read at 450 nm with an ELISA reader and analyzed with KC4 3.03 Rev 4 software.

Flow cytometric analysis

Intracellular cytokine staining procedure: cells from blood, spleen, or lungs of mice were stimulated for 1–2 h with 2 µg/ml Ag and subsequently incubated for 6 h with 10 µg/ml brefeldin A (Sigma-Aldrich) at 37°C. Thereafter, cells were stored overnight at 4°C. The following day, FcRs were blocked with 0.5 µg/ml anti-CD16/CD32 mAb (BD Pharmingen) for 10 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 indicated using 0.2 µg/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. Cells were then washed in FACS buffer, permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions, and stained intracellularly with 0.2 µg/ml anti-IFN-{gamma} (clone: XMG1.2), anti-TNF-{alpha} (clone: MP6-XT22), or anti-IL-2 (clone: JES6-5H4) mAbs. When using the CD107A/B (clone: ID4B/ABL-93) mAbs, these were added to the wells along with the Ags, according to the manufacturer’s instructions. 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 loaded with TB10.44-11 instead of TB10.43-11. After washing, cells were resuspended in formaldehyde solution 4% (w/v) pH 7.0 (Bie and Berntsen) and analyzed 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 Harvard RANKPEP website (http://bio.dfci.harvard.edu/Tools/rankpep.html; Refs. 31 and 32). Similarity is scored using position-specific scoring matrixes derived 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 split into two populations. One was pulsed with 10 µg/ml TB10.43-11 for 1 h at 37°C and then labeled with a high concentration (40 µM) of CFSE (CFSEhigh population), and the other population was incubated for 1 h at 37°C without peptide and labeled with a low concentration (4 µM) of CFSE (CFSElow population). A 1:1 ratio of CFSElow- to CFSEhigh-labeled cells (1.5 x 107 cells in total) were mixed together and adoptively transferred in 200 µl of PBS into M.tb-infected mice. Twenty hours later, recipient spleen cells were analyzed by flow cytometry. Percent lysis was determined by loss of the peptide-pulsed CFSEhigh population compared with control CFSElow population using the formula (1 – (%CFSEhigh cells/%CFSElow cells) x 100).

Statistical methods

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The immune response against TB10.4 following infection with 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 Erdman and analyzed 3 wk later. Epitope recognition of TB10.4 was assessed by using a panel of 18-mer peptides spanning the entire sequence of TB10.4 for in vitro stimulation of lymphocytes from infected mice. The amount of IFN-{gamma} released following stimulation for 72 h was then analyzed by ELISA. The results showed that only stimulation with TB10.41-18 resulted in a significant IFN-{gamma} release (8713 ± 1285 pg/ml IFN-{gamma}, Fig. 1A). We further analyzed the TB10.41-18-specific T cell phenotype by flow cytometry by staining TB10.41-18-stimulated lung lymphocytes with fluorescent anti-CD4, anti-CD8, and intracellularly with anti-IFN-{gamma} 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 by producing IFN-{gamma} after stimulation with TB10.41-18, while only 1.2% of the CD4 T cells in the lungs were specific for TB10.41-18. The corresponding amount of IFN-{gamma}-producing T cells from naive mice following TB10.41-18 stimulation was 0.5% CD4 T cells and 0.2% CD8 T cells (shown in parentheses in Fig. 1B). To precisely define the CD8 epitope within TB10.41-18, we next analyzed the sequence using the position-specific scoring matrix at Harvard’s RANKPEP website (http://bio.dfci.harvard.edu/Tools/rankpep.html; Ref. 31). The sequence QIMYNYPAM was predicted as the strongest binder of both H-2Kb and H-2Db. In agreement with this, PBMCs from infected mice stimulated in vitro with peptides spanning TB10.41-18 confirmed that the minimal epitope inducing the highest release of IFN-{gamma} was indeed TB10.43-11 (QIMYNYPAM; Fig. 1D). In addition, stimulating lymphocytes from infected mice with a panel of 12-mer peptides spanning the sequence of TB10.41-18 showed that the minimal MHC-II H-2b-restricted epitope was TB10.43-14 (data not shown).


Figure 1
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  		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 mice were stimulated for 72 h in vitro with a panel of nine overlapping 18-mer peptides covering the TB10.4 sequence, and IFN-{gamma} levels in supernatants were assessed by ELISA. Bars represent means and SEM of triplicate values. B, PBMCs were stimulated in vitro with the immune dominant epitope TB10.41-18 and the specific T cell phenotype was evaluated by flow cytometry of cells stained with anti-CD4, anti-CD8, and intracellular anti-IFN-{gamma}. The indicated percentages specify the proportion of the CD4/CD8 T cell populations producing IFN-{gamma} after peptide stimulation. Corresponding percentages in naive mice are shown in parentheses. C, A schematic overview of the overlapping 9-mer peptides spanning the N-terminal TB10.4 sequence used to map the minimal H-2Kb-restricted epitope of TB10.4. D, IFN-{gamma} production was assessed by flow cytometry of intracellular cytokine-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 ({square}) and CD8 ({blacksquare}) T cells that stained positive for IFN-{gamma} following stimulation. E, Specific CD8 T cells were identified using a H-2Kb pentamer loaded with 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 right quadrant 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 pooled from 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.

 
Having identified the minimal CD8 epitope, we next analyzed PBMCs taken from mice 6 wk after aerosol infection with M.tb by using a H-2Kb pentamer loaded with the minimal CD8 epitope (see Materials and Methods). A total of 7.6% of all CD8 T cells stained positive 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 mice were stimulated in vitro with TB10.43-11 and analyzed by flow cytometry for expression of CD25, CD44, CD45RB, CD62L, CD69, IFN-{gamma}, TNF-{alpha}, and IL-2. Cells were also stained for CD4 expression, but intracellular cytokine staining was at or below background levels following stimulation with TB10.43-11 and TB10.43-14 (data not shown), indicating that the CD4 response against TB10.4 following infection in C57BL/6 mice is a minor or transient response. The majority of the IFN-{gamma}-producing CD8 T cells expressed CD25mid/high, CD44high, CD45RBmid/low, CD62Llow, and CD69high (Fig. 2A). The majority of IFN-{gamma}-producing CD8 T cells also expressed TNF-{alpha}. In contrast, only few of the IFN-{gamma}-producing CD8 T cells costained for IL-2. As observed for IFN-{gamma}-positive cells, the majority of TNF-{alpha}-expressing cells expressed CD45RBmid/low and CD62Llow (Fig. 2A, lower diagrams). As in vitro stimulation with TB10.43-11 may alter the phenotype of the stimulated CD8 T cells, we also analyzed the TB10.43-11 CD8 cells specifically using the H-2Kb/TB10.4 pentamer. The results showed that the CD8 cells were of a CD44highCD45RBlowCD62Llow phenotype (Fig. 2B), thus resembling the effector phenotype observed following in vitro stimulation with TB10.43-11 (Fig. 2A). Naive mice had a background pentamer staining at 0–0.5% of all CD8 T cells. Thus, TB10.43-11 CD8 cells represent an effector CD8 T cell population induced shortly after infection with M.tb.


Figure 2
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  		FIGURE 2. Phenotypic analysis of TB10. 3-11-specific CD8 T cells. A, C57BL/6 mice were infected by the aerosol route with virulent M.tb Erdman, and PBMCs were purified from infected mice 6 wk after mycobacterial challenge and stimulated in vitro with TB10.43-11. Cells were stained intracellularly for IFN-{gamma} (upper diagrams) or TNF-{alpha} (lower diagrams), and for surface expression of CD8, CD25, CD44, CD45RB, CD62L, CD69, and intracellular IL-2. Percentages illustrate the proportion of cytokine-producing CD8 T cells that express CD25mid/high, CD44high, CD45RBlow/mid, CD62Llow, CD69high, single-positive IFN-{gamma}+/IL-2– cells, and the proportion of TNF-{alpha}-producing cells which also produce IFN-{gamma}. Cells were also stained with anti-CD4, but the amount of CD4 IFN-{gamma}-producing cells was below 0.2%. B, Blood cells taken 6 wk after infection were stained with the H-2Kb/TB10.4 pentamer and anti-CD44, anti-CD45RB, and anti-CD62L. In A and B, PBMCs were pooled from five mice, and are representative of three individual experiments.

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

We next examined whether TB10.43-11 CD8 T cells represented more than a transient cell population and to which degree these cells were recruited to the site of infection. Mice were infected with virulent M.tb Erdman by the aerosol route, whereafter cells from lungs, spleen and blood were isolated at weeks 0–50 postinfection. Following stimulation in vitro with TB10.41-18, the cells were analyzed for expression of CD4, CD8, and IFN-{gamma} by flow cytometry. By using the TB10.41-18 peptide, we were able to monitor both the TB10.4-specific CD4 and CD8 cells simultaneously. Only low amounts of IFN-{gamma}-producing CD4 T cells were generated throughout the infection. In contrast, TB10.43-11-specific CD8 T cells were present throughout the experiment (Fig. 3, A–C). In the spleen and blood, the kinetics was similar, although the responses were higher in the blood. Compared with blood and spleen, the response in the lungs peaked before the response in blood and spleen, indicating that TB10.43-11-specific CD8 cells were first observed in the lung (Fig. 3, A–C). The amount of TB10.4-specific IFN-{gamma}-producing T cells in all organs declined toward week 19 but was increased at week 48 postinfection. Furthermore, at later time points in particular, the TB10.43-11-specific CD8 T cells were found in higher numbers in the lung, compared with the blood (and spleen). Staining the cells with H-2Kb/TB10.4 pentamer showed that at week 6 postinfection, 7.3% of the entire CD8 T cell population in the blood was stained positive for the pentamer. This value decreased to 4.9% after 19 wk but as shown for TB10.43-11-specific CD8 IFN-{gamma}+ cells in Fig. 3, A–C, the amount of H-2Kb/TB10.4 pentamer-positive cells had increased slightly at week 48 after challenge (Fig. 3D). The phenotype of TB10.43-11-specific CD8 T cells in terms of effector markers did not change in the course of infection. Thus, 48 wk postinfection H-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 the TB10.43-11-specific CD8 T cells represented a long-lasting CD8 cell population.


Figure 3
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  		FIGURE 3. Kinetics of TB10.4-specific CD8 T cells following infection with M.tb. Lymphocytes from lung (A), spleen (B), and blood (C) were isolated at different time points after infection, and stimulated in vitro with the 18-mer peptide TB10.41-18 containing both the MHC-I and -II restricted epitopes of TB10.4 to evaluate kinetics of both the CD4 and CD8 T cells specific for TB10.4. IFN-{gamma} production was evaluated by flow cytometry and values represent the proportions of CD4 and CD8 T cells in the lymphocyte gate that stained positive with anti-IFN-{gamma} following TB10.41-18 stimulation. Each analysis was performed on lymphocytes pooled from four to six mice. D, The TB10.4-specific CD8 T cells in blood were stained with the H-2Kb/TB10.4 pentamer, and the results show the amount of CD4 and CD8 T cells that stained positive. Blood cells were pooled from four to six mice. E, Lung cells were taken 48 wk postinfection and stained with the H-2Kb/TB10.4 pentamer, CD62L, CD44, and CD45RB. The results show only H-2Kb/TB10.4 pentamer-positive cells.

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

The effector phenotype, longevity, and recruitment to the infection site of TB10.43-11-specific CD8 T cells indicated that these cells were actively involved in the immune response against M.tb. To examine the effector function of the TB10.43-11-specific T cells in vitro, lymphocytes from blood and lungs of infected mice were stimulated in vitro with TB10.43-11 and degranulation was quantified with CD107A/B labeling. In both lung and blood cells, stimulation with TB10.43-11 induced an increased expression of CD107A/B on CD44highCD8 T cells (Fig. 4A). In addition, we also observed enhanced CD95L (FasL) expression on the CD44highCD8 T cells (Fig. 4A). As these results indicated a cytotoxic potential of the TB10.43-11 cells, we next examined whether the TB10.43-11-specific T cells in infected mice were indeed capable of eliminating target cells expressing this epitope in vivo. We used the in vivo cytotoxic assay where CFSE-labeled splenocytes from naive mice, unpulsed or pulsed with TB10.43-11, were adoptively transferred into infected mice. Peptide-specific lysis of the transferred cells was then investigated by flow cytometric analysis of recipient spleens. Although some killing was observed upon transfer of target cells to naive mice, a strong increase in clearance of TB10.43-11-pulsed target cells was observed (up to 70%-specific killing of target cells), indicating that TB10.43-11-specific cells were able to kill their target cells, and that this epitope is an immunological target during natural infection with M.tb. Moreover, the cytotoxic activity of TB10.43-11-specific cells was maintained in chronically infected mice, although we did observe some decline in cytotoxicity, which however seemed to correlate 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 cells with a killing mechanism that involved degranulation as well as CD95L-induced apoptosis of target cells.


Figure 4
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  		FIGURE 4. The TB10.4-specific CD8 T cells elicit CTL responses involving degranulation and CD95L during chronic infection with M.tb. A, Degranulation of cytotoxic vesicles was evaluated by flow cytometry on T cells from infected mice stimulated in vitro with TB10.43-11 or medium alone. Lung and blood lymphocytes were stained with anti-CD8, anti-CD44, and anti-CD107A/B Abs, and blood lymphocytes were also stained with anti-CD95L. Percentages describe the proportion of CD44high CD8 T cells expressing CD107A/B and CD95L upon stimulation. B, CTL activity of TB10.43-11-specific CD8 T cells in vivo. Unloaded splenocytes (CFSElow) and TB10.43-11-loaded splenocytes (CFSEhigh) from naive mice were transferred into infected mice. The amount of splenocytes killed in vivo by cytotoxic CD8 T cells specific for TB10.43-11 is seen as the reduction in the CFSEhigh population. Percentages specify the total amount of CFSEhigh cells killed. C, The amount of the specific in vivo cytotoxic response against TB10.43-11-loaded splenocytes in infected mice measured at different time points post challenge. In A and B, analysis was performed 6 wk after infection.

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

Having showed that TB10.43-11-specific CD8 cells make up a substantial part of the total pool of CD8 T cells during the natural infection with M.tb, we were next interested in the requirement for the generation of these cells. As a decreased induction of CD8 T cells have been proposed to be partly responsible for the failure of BCG to efficiently protect against pulmonary infection in adults (21, 25), we first examined the induction of TB10.43-11-specific CD8 T cells following BCG vaccination or infection with M.tb. In vitro stimulation of blood lymphocytes from BCG-vaccinated mice with TB10.41-18 resulted in 1.4% IFN-{gamma}-producing CD8 T cells, while the equivalent value for the M.tb group was 5.1%. In contrast, the amount of specific IFN-{gamma}-producing CD4 T cells was comparable in the two groups (Fig. 5). This indicated that a genetic element present in virulent M.tb but absent in BCG influenced the CD8 T cell response against TB10.4. As the genetic RD1 region is encoded in all clinical isolates of M.tb but is deleted from all BCG substrains (25, 29), we next analyzed whether the RD1 region was indeed required for the elevated CD8 T cell response against TB10.4 in M.tb-infected mice. To examine this, we first used the BCG knockin strain, BCG::RD1, in which the genetic region RD1 has been reintroduced (29), and compared this to BCG. Mice were infected i.v. with mycobacteria for 6 wk and PBMCs were analyzed by flow cytometry for Ag-specific IFN-{gamma}-producing CD4 and CD8 T cells upon TB10.41-18 in vitro stimulation. As seen in Fig. 6, the mutant BCG::RD1 strain generated significantly higher numbers of TB10.4-specific CD8 T cells (5.8% of the entire CD8 T cell population) than BCG (1.0% of all CD8 T cells). In contrast, the CD4 T cell response against TB10.4 was not as dependent upon the RD1 region and between 0.3 and 0.4% of the CD4 T cells produced IFN-{gamma} following TB10.41-18 stimulation. Naive mice showed 0.1% IFN-{gamma}-producing CD4 T cells and 0.3% CD8 T cells (data not shown). In support of these results, in BCG::RD1-infected mice, 8.8 and 2.9% of the CD8 T cells were pentamer positive in the blood and spleen, respectively, 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 stained positive in blood and spleen. This influence of the RD1 region on the magnitude of the CD8 T cell response was dependent upon expression of ESAT-6. Thus, infection with BCG:: RD1{Delta}ESAT-6, lacking expression of ESAT-6, led to a CD8 T cell response in lungs and spleen that was not significantly different from that observed in BCG-vaccinated mice in terms of the number of TB10.43-11-specific CD8 T cells (Fig. 6E). In addition, in vitro stimulation of lymphocytes from lungs, spleen, or blood with TB10.43-11 induced a secretion of IFN-{gamma}, TNF-{alpha}, and IL-2 that was also not significantly different from that seen with lymphocytes from BCG-vaccinated mice (data not shown).


Figure 5
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  		FIGURE 5. Induction of TB10.4-specific CD8 T cells following infection with M.tb or BCG. PBMCs from C57BL/6 mice were purified 6 wk after s.c. immunization with BCG or aerosol infection with M.tb, and stimulated in vitro with TB10.41-18 containing both the MHC-I and -II-restricted epitopes of TB10.4. Induction of CD4 and CD8 T cells was assessed by flow cytometric analysis. Percentages specify the proportion of CD4 or CD8 T cells producing IFN-{gamma} after stimulation with TB10.41-18. Lymphocytes were pooled from four mice per group and results are representative of three individual experiments.

 

Figure 6
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  		FIGURE 6. Induction of CD8 T cells is increased upon mycobacterial expression of RD1-encoded proteins. A, PBMCs from C57BL/6 mice were purified 6 wk after infection of different mycobacteria as indicated. The proportions of IFN-{gamma}-producing TB10.4-specific T cells were analyzed by flow cytometry of the PBMCs stimulated in vitro with TB10.41-18. Cells were stained for surface expression of CD4, CD8, and for presence of intracellular IFN-{gamma}. Percentages specify the proportion of the CD4 and CD8 T cell population that produced IFN-{gamma} in response to TB10.41-18 stimulation (upper right quadrant). B, The proportions of H2-Kb/TB10.4 pentamer-positive CD8 T cells 6 wk after infection in the blood and spleen of mice infected with BCG or BCG::RD1. C, The bacterial burden was determined in the lungs of BCG- or BCG::RD1-infected C57BL/6 mice at the indicated time points. **, p < 0.01. D, Concurrent with the analysis of bacterial burden at 9 wk postinfection in the lungs, CD8 T cells specific for the minimal TB10.4 epitope were analyzed by flow cytometry. Bars represent means and SEM (n = 3) of the proportion of CD8 T cells that stained positive with the H-2Kb/TB10.4 pentamer. E and F, CD8 T cells specific for the minimal TB10.4 epitope in the lungs or spleen were analyzed by flow cytometry 4 wk following infection as indicated. Bars represent means and SEM (n = 4) of the proportion of CD8 T cells that stained positive with the H-2Kb/TB10.4 pentamer.

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

To examine whether other genetic regions than RD1 (absent in BCG, but present in M.tb) were involved in bacterial virulence, and in the induction of CD8 T cells, we next used the M.tb-mutant knockout strain, H37Rv{Delta}RD1, in which the RD1 region has been deleted (29). Interestingly, even though H37Rv{Delta}RD1 was significantly less virulent that H37Rv (p < 0.05), it was clearly more virulent than BCG, despite the lack of the genetic region RD1 (Fig. 7). Furthermore, as with the BCG/BCG::RD1 strains (Fig. 6), we observed a clear correlation between virulence and the number of CD8 T cells. Thus, 9 wk after infection, 15% of all the CD8 T cells in the lung were pentamer positive in H37Rv{Delta}RD1-infected mice with a bacterial count of 3.89 ± 0.55 log10 CFU, compared with 25% in H37Rv-infected mice with a bacterial count of 4.91 ± 0.41 log10 CFU. Taken together, these results indicated that virulence is also involved in generating a CD8 response against mycobacterial Ags.


Figure 7
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  		FIGURE 7. Mycobacterial virulence correlates with the amount of TB10.4-specific CD8 T cells. A, In vivo growth of H37Rv (open symbols) and H37Rv{Delta}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, Nine weeks postinfection in the lungs, CD8 T cells specific for the minimal TB10.4 epitope were analyzed by flow cytometry as indicated. Bars represent means and SEM (n = 3) of the proportion of CD8 T cells stained with the H-2Kb/TB10.4 pentamer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we describe a new MHC-I (H-2Kb)-restricted epitope shared by the homologous proteins TB10.3 and TB10.4 (33). Our results indicated that the minimal epitope mapped to amino acids 3–11 (Fig. 1). (It should be noted that these mapping studies did not include 10 mers. However, overlapping 12 mers did not induce stronger responses). TB10.43-11-specific CD8 T cells from M.tb-infected C57BL/6 mice secreted high amounts of Th1 cytokines such as IFN-{gamma} and TNF-{alpha}, in agreement with the works of Kamath et al. (34), who found that T cells specific for TB10.420-28 also produced significant amounts of these two cytokines. However, in contrast to TB10.420-28-specific CD8 cells in M.tb-infected BALB/c mice (17), TB10.43-11-specific CD8 T cells only produced small amounts of IL-2 in C57BL/6 mice challenged with virulent M.tb. This difference may be due to the use of different mice strains or the different experimental procedures used in each study.

The TB10.43-11-specific CD8 T cells expressed surface activation markers such as CD25, CD44, and CD69, but had down-regulated CD45RB or CD62L, thus exhibiting a typical effector T cell phenotype. This effector phenotype was observed upon stimulation with the TB10.43-11 peptide or after staining CD8 T cells from infected mice with the H-2Kb/TB10.4 pentamer ex vivo. The continued presence of TB10.43-11-specific CD8 T cells in the lungs (and spleen/blood) indicated that they were an active part of the immune response against both acute and chronic infection. Following stimulation with the specific Ag, TB10.43-11-specific CD8 T cells showed an increased expression of CD107A/B and CD95L (Fig. 4). CD107A/B is located on the inner membrane of cytotoxic granules, but is expressed on the outer cell membrane briefly after degranulation, while CD95L is a known inducer of target cell apoptosis. Thus, TB10.43-11-specific CD8 T cells may exhibit more than one killing mechanism. We were not able to show up-regulation of perforin expression on lung TB10.43-11-specific CD8 T cells upon stimulation with specific Ag (data not shown), which is in agreement with previous studies that indicated that perforin is not important for the control of mycobacterial infection (6, 35). In support of a cytotoxic role for TB10.43-11-specific CD8 T cells, we showed that these cells were able to kill naive splenocytes loaded with the TB10.43-11 peptide in vivo. This was also recently shown to apply for TB10.420-28-specific T cells (16, 17) and support that these T cells play an active part in the immune response against infection with M.tb.

It has recently been proposed that the amount of T cells may fluctuate dynamically throughout the infection (36). We found that the amounts of IFN-{gamma}-producing TB10.43-11-specific CD8 T cells exhibited a pattern consisting of a strong increase shortly after the onset of infection, followed by a decline in numbers to week 19, and an increase in numbers late in the chronic infection. This pattern was evident both in lungs (where the peak was first observed, although additional studies at earlier time points may help elucidate this point more), blood, and spleen, although the percentage of specific CD8 T cells was higher in the lungs and blood as compared with the spleen. The kinetic pattern therefore followed the general bacterial levels in the lung with a peak early in infection, followed by a decline once the adaptive immune response gains control over the infection (36, 37). Whether the increase in TB10.43-11-specific CD8 T cells after prolonged exposure (week 48) also correlated with increased CFU levels was not examined. Using the H-2Kb/TB10.4 pentamer, we observed the same overall kinetic pattern concerning the entire amount of TB10.4 CD8 T cells as seen for the cytokine-producing cells described above, and this also correlated with in vivo cytotoxicity toward transferred TB10.43-11-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 a major and as yet unresolved debate (21). Indeed, it has been suggested that the failure of BCG as a vaccine may be explained by its lack of ability to induce a robust CD8 response (21). Interestingly, the strong CD8 T cell response against TB10.4 was significantly reduced in BCG-vaccinated mice compared with BCG::RD1 (or M.tb) vaccinated mice, whereas the CD4 T cell response was less affected. Staining with the H-2Kb/TB10.4 pentamer confirmed that it was the number of CD8 T cells that declined in mice infected with BCG, and not merely the cytokine-producing ability of the specific CD8 T cells. The fact that we also saw an increased recognition of MTB32A (19) by CD8 T cells in BCG::RD1-infected mice compared with mice infected with BCG (data not shown) indicated that this is a general phenomenon that applies to the overall CD8 T cell population, in line with two recent studies showing that introduction of RD1 into BCG or M. microti increased the amount of Ag-specific CD8 T cells (25, 26). In addition, induction of CD8 T cells was dependent upon expression of ESAT-6 (Fig. 6), which could indicate a requirement for a specific function of ESAT-6 and/or that a functional RD1 region requires ESAT-6. However, experiments performed with the RD1 knockout mutant H37Rv{Delta}RD1 showed that induction of CD8 T cells was not strictly dependent upon the RD1 region, demonstrating that other RD regions are also involved in both virulence and the induction of CD8 T cells. Interestingly, in support of this, a recent study showed that mycobacterial Ags were indeed presented on MHC-I following infection of dendritic cells with H37Rv{Delta}RD1 (38) (Figs. 6 and 7).

Our results also showed a clear correlation between bacterial virulence and CD8 T cell induction. Although the mechanism by which the increased bacterial growth/virulence can lead to increased CD8 T cell response is not known, previous studies have indicated at least two ways by which this could occur: 1) through increased availability of bacterial Ag and 2) increased apoptosis/necrosis induction of infected APCs.

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

Regarding apoptosis, mycobacteria have been shown to induce apoptosis of infected macrophages (42). Increased release of apoptotic vesicles containing mycobacterial Ags may be taken up by bystander APCs, and via the cross-presentation pathway be presented on the cell surface on MHC-I molecules (24, 43). Interestingly, a recent study indicated a correlation between the RD1 region and apoptosis. Thus, while infection of THP-1 cells with H37Rv resulted in apoptosis, a deletion mutant that did not express the RD1-encoded protein ESAT-6 failed to induce significant apoptosis (42). Thus, the presence of functional RD1 may lead to increased apoptosis which in turn could increase the induction of CD8 T cells. This is in agreement with our results which showed a correlation between the expression of RD1 encoded ESAT-6 and the induction of TB10.43-11-specific CD8 T cells (Figs. 6 and 7). However, it should be noted that RD1 expression has also been shown to increase necrosis (44), and whether the observed increased CD8 T cell response in the presence of RD1 (Figs. 5 and 6) was due to increased apoptosis or increased necrosis was not shown in the present study.

In conclusion, we have described a novel CD8 T cell specific for TB10.43-11. In infected animals, the phenotype of TB10.43-11-specific CD8 T cells resembled that of an effector T cell and the killing mechanism may involve both degranulation and CD95L. Finally, the induction of TB10.43-11-specific CD8 T cells correlated with expression of ESAT-6 and virulence of the mycobacteria. We are presently examining how bacterial virulence affects the magnitude of CD8 T cells specific for mycobacterial Ags.


   Acknowledgments
 
The technical help of Kristine Persson, Lene Rasmussen, and Charlotte Fjordager is gratefully acknowledged. We thank Ida Rosenkrands for critical comments. BCG::RD1 and BCG::RD1{Delta}ESAT-6 was a gift from Drs. Stewart Cole and Roland Brosch and H37Rv{Delta}RD1 was a gift from Dr. William R. Jacobs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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. Back

2 Address correspondence and reprint requests to Dr. Jes Dietrich, Department of Infectious Disease Immunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. E-mail address: JDI{at}ssi.dk Back

3 Abbreviations used in this paper: M.tb, Mycobacterium tuberculosis; TB, tuberculosis; BCG, bacillus Calmette-Guérin; MHC-I, MHC class I; MHC-II, MHC class II; RD1, region of difference-1. Back

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


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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