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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-1
Rolf Billeskov, Carina Vingsbo-Lundberg, Peter Andersen
and Jes Dietrich
J Immunol 2007; 179:3973-3981; ;
doi: 10.4049/jimmunol.179.6.3973
http://www.jimmunol.org/content/179/6/3973
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References
The Journal of Immunology
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
I
t 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-␥ in the control of TB has been clearly demonstrated by the susceptibility to mycobacterial infections in mice
with a disrupted IFN-␥ gene and in humans with mutations in
genes involved in the IFN-␥ and IL-12 pathways (1– 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 ␤2-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
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 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.
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: [email protected]
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.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
mice (8 –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 (12–14). 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-␥ 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 (⌬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, ⌬ureC hly⫹rBCG was later shown to be more
protective than BCG against virulent M.tb infection (22–24).
Moreover, recent studies have shown that reintroduction of the
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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-␣, and IFN-␥. 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. The Journal of Immunology, 2007,
179: 3973–3981.
INDUCTION OF TB10.43-11 CD8 T CELLS
3974
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
(25–27).
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-␣ and IFN-␥, 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.
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 radiationsterilized 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⌬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⌬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 ⫻
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⌬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 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.
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-␮m 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 ⫻ 105 cells in 200 ␮l 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 ␮g/ml for ELISA and
2 ␮g/ml for flow cytometric analyses. Wells containing medium only or
Con 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/ml
monoclonal rat anti-murine IFN-␥ (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-␥ was detected with a 0.1 ␮g/ml biotinlabeled rat anti-murine Ab (clone XMG1.2; BD Pharmingen) and 0.35
␮g/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-␥ (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 ␮g/ml Ag and subsequently
incubated 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, FcRs
were blocked with 0.5 ␮g/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 ␮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. 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 ␮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 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 ␮g/ml TB10.43-11 for 1 h at
37°C and then labeled with a high concentration (40 ␮M) 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 ␮M) of
CFSE (CFSElow population). A 1:1 ratio of CFSElow- to CFSEhigh-labeled
cells (1.5 ⫻ 107 cells in total) were mixed together and adoptively transferred in 200 ␮l 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) ⫻ 100).
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Materials and Methods
Lymphocyte cultures
The Journal of Immunology
3975
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
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-␥ 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-␥
release (8713 ⫾ 1285 pg/ml IFN-␥, 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-␥ 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-␥ 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-␥-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-␥ 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).
Having identified the minimal CD8 epitope, we next analyzed
PBMCs taken from mice 6 wk after aerosol infection with M.tb by
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
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-␥ 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-␥.
The indicated percentages specify the proportion of the CD4/CD8 T cell populations producing IFN-␥ 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-␥ 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 (䡺)
and CD8 (f) T cells that stained positive for IFN-␥ 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.
3976
INDUCTION OF TB10.43-11 CD8 T CELLS
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 TB10.43-11specific 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-␥, TNF-␣, 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-␥-producing
CD8 T cells expressed CD25mid/high, CD44high, CD45RBmid/low,
CD62Llow, and CD69high (Fig. 2A). The majority of IFN-␥-producing CD8 T cells also expressed TNF-␣. In contrast, only few of
the IFN-␥-producing CD8 T cells costained for IL-2. As observed
for IFN-␥-positive cells, the majority of TNF-␣-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.
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-␥ 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-␥-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-␥-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-␥⫹ 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.
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
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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-␥ (upper diagrams) or TNF-␣ (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-␥⫹/IL-2⫺ cells, and the
proportion of TNF-␣-producing cells which
also produce IFN-␥. Cells were also stained
with anti-CD4, but the amount of CD4 IFN␥-producing cells was below 0.2%. B, Blood
cells taken 6 wk after infection were stained
with the H-2Kb/TB10.4 pentamer and antiCD44, anti-CD45RB, and anti-CD62L. In A
and B, PBMCs were pooled from five mice,
and are representative of three individual
experiments.
The Journal of Immunology
3977
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 TB10.43-11specific 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, TB10.43-11specific 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.
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-␥-producing CD8 T
cells, while the equivalent value for the M.tb group was 5.1%. In
contrast, the amount of specific IFN-␥-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-␥-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
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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-␥ 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-␥ following
TB10.41-18 stimulation. Each analysis
was performed on lymphocytes pooled
from four to six mice. D, The TB10.4specific 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.
3978
INDUCTION OF TB10.43-11 CD8 T CELLS
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, antiCD44, 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.
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-␥ following TB10.41-18 stimulation. Naive mice showed 0.1% IFN-␥-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⌬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-␥,
TNF-␣, and IL-2 that was also not significantly different from
that seen with lymphocytes from BCG-vaccinated mice (data
not shown).
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 TB10.4specific 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⌬RD1, in which the RD1
region has been deleted (29). Interestingly, even though
H37Rv⌬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
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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-␥ after stimulation with TB10.41-18.
Lymphocytes were pooled from four mice per group and results are representative of three individual experiments.
The Journal of Immunology
3979
Discussion
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-␥-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-␥. Percentages specify the proportion
of the CD4 and CD8 T cell population that produced IFN-␥ 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. ⴱⴱ, 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.
pentamer positive in H37Rv⌬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.
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-␥ and TNF-␣, 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 downregulated 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
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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⌬RD1 (solid symbols) after i.v. infection of C57BL/6 mice,
determined in the lungs at 3, 4, and 9 wk postinfection. ⴱ, 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.
INDUCTION OF TB10.43-11 CD8 T CELLS
3980
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⌬ESAT-6 was a gift from
Drs. Stewart Cole and Roland Brosch and H37Rv⌬RD1 was a gift from
Dr. William R. Jacobs.
Disclosures
The authors have no financial conflict of interest.
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