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*
Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center Research Institute, San Francisco, CA 94115;
Division of TB Control, Department of Public Health, San Francisco, CA 94110; and
Stanford University Blood Center, Palo Alto, CA 94304
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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In keeping with findings from the murine models of TB, precursors of CD8+ CTLs that are capable of lysing autologous mycobacteria-infected monocytes are present in the peripheral blood and bronchoalveolar lavages of healthy tuberculin skin test-positive (TSP) individuals (12, 13). CD8+ class I-restricted T cells that are specific for epitopes in the M.tb early secretory antigenic target 6 have also been detected both in the peripheral blood of patients with active TB and in healthy contacts (14). Moreover, CD8+ CD1-restricted T cell lines derived from patients with active TB have been shown to lyse M.tb-infected monocytes and thereby restrict the growth of intracellular organisms (15). Surprisingly, however, no reports have yet appeared describing class I MHC-restricted M.tb-specific CTLs in humans. The current study represents an effort to identify and characterize such CTLs in M.tb-infected individuals. The CTL response to a single M.tb protein, the 19-kDa secreted lipoprotein (M.tb19) (16, 17), was studied, because M.tb19 and other secreted proteins have been implicated in immune protection against mycobacterial infection (5, 18). Moreover, M.tb19 induces CD4+ T cell responses in both mice and humans (19, 20), suggesting that it is a major target Ag of cellular immunity.
To screen for M.tb19 epitopes with the potential to serve as targets for class I MHC-restricted CTLs, we identified sequences within the protein containing the HLA-A*0201-binding motif (21). Synthetic peptides corresponding to these sequences were then tested for their ability to bind the HLA-A*0201 allele in an immunofluorescence-based peptide-binding assay (22); those peptides with the highest binding activity were studied as target Ag in CTL assays. The results indicate that CD8+ CTLs with specificity for residues 88 to 97 of M.tb19 (P88–97) are present in the circulation of M.tb-sensitized individuals, including patients with active TB. Furthermore, a synthetic peptide corresponding to this region can elicit CTLs in vitro from the T cells of uninfected persons.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A total of 11 HLA-A*0201+ individuals participated
in this study. The protocol and consent forms relating to our use of
human subjects were approved by the California Pacific Medical Center
Research Institute Administrative Panel on Human Subjects in Medical
Research. Informed consent for blood donation was obtained from all
donors. None of the subjects had evidence of infection with HIV. Two
subjects were healthy TSP individuals, four had pulmonary TB, two were
healthy tuberculin skin test-negative (TSN) individuals, and three were
normal blood donors (Table I
). Venous
blood from healthy TSP and TSN individuals and patients with TB was
collected into heparinized Vacutainer tubes (Becton Dickinson,
Rutherford, NJ). White blood cell concentrates from HLA-A2+
normal donors were obtained from the Stanford Medical School Blood
Center (Palo Alto, CA). HLA typing was performed by flow cytometry
using anti-HLA-A2 mAb (BB7.2) as the first Ab and FITC-labeled goat
anti-mouse F(ab')2 fragments as a second Ab. The HLA-A2
subtype of blood donors was determined by staining their B
lymphoblastoid cell line (B-LCL) with an HLA-A2.1/A2.2-specific mAb
(CR11–351) (23); the subtype was confirmed by the CTL response of
their CD8+ T cells to a previously identified
HLA-A*0201-restricted influenza A virus matrix peptide 58 to 66
(designated IMP) (24). The proliferative response of T cells to whole
heat-killed M.tb was assayed as described below.
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Antibodies
Our laboratory generated the following mAbs using hybridomas
that were obtained from the American Type Culture Collection (ATCC,
Manassas, VA): Leu-4 (CD3), Leu-3a (CD4), Leu-2a (CD8), Leu-11a
(CD16), w6/32 (class I MHC), L243 (HLA-DR), and BB7.2 (HLA-A2). For
blocking studies, mAbs were used as purified reagents at concentrations
of 10 µg/ml. The HLA-A2.1/A2.2-specific mAb (CR11–351) was a gift of
Dr. S. Ferrone from Columbia University (New York, NY).
FITC-conjugated mAbs directed against CD3, CD4, CD8, TCR
ß, HLA-DR,
class I MHC (w6/32), and CD14 (Leu-M3) were purchased from the Becton
Dickinson Monoclonal Center (Mountain View, CA). FITC-conjugated goat
anti-mouse IgG F(ab')2 fragments and affinity-purified
goat anti-mouse IgG (
-chain-specific) Ab were purchased from
Zymed Laboratories (San Francisco, CA).
Synthetic peptides
A panel of 54 M.tb19-derived peptides (8–10 mer) containing HLA-A*0201-binding motifs were identified using a computer scoring program (25). A total of 28 peptides with a computed score between 48 and 69 were selected for testing in a T cell-binding assay. Two previously identified HIV-1-derived peptides, an HLA-A2-restricted HIV-1 gag peptide (amino acids (aa) 71–85, designated HIV gag A2) and an HLA-B8-restricted HIV-1 gag peptide (aa 253–267, designated HIV gag B8) (26) were used as controls, as was an HLA-A*0201-restricted IMP (aa 58–66) (24). Peptides were synthesized by F-moc chemistry at either University Hospital (Leiden, The Netherlands) or the Beckman Center at the Stanford University Medical Center. Before use, lyophilized, HPLC-purified peptides (>90% pure) were reconstituted at 40 mg/ml in DMSO and diluted to 1 mg/ml with Iscove’s modified Dulbecco’s medium (IMDM) (Life Technologies, Grand Island, NY).
Peptide-binding assay
T2, an HLA-A*0201+ Ag-processing defective cell line (22) which was a gift of Dr. M. Cheever (University of Washington, Seattle, WA), was propagated in RPMI 1640 containing nonessential aa, sodium pyruvate, and 10% FBS. Before use, the cells were incubated for 6 h at 37°C in serum-free IMDM; next, cells were washed once, suspended in serum-free IMDM containing 20 µM of 2-ME and 15 µg/ml of human ß2-microglobulin (ß2m) (Calbiochem, La Jolla, CA), and pulsed with 0 to 200 µM of M.tb19 peptide. Control cells were pulsed with either HIV gag A2 or HIV gag B8 peptide. After a 24-h incubation at 37°C, T2 cells were washed once with cold PBS containing 0.5% BSA and 0.02% NaN3. They were then stained directly with FITC-conjugated w6/32 mAb or indirectly with anti-HLA-A2.1 mAb as first Ab and with goat anti-mouse FITC-labeled F(ab')2 fragments as a second-step Ab.
The percentage of FITC-positive cells as well as their staining
intensity (mean fluorescence intensity (MFI)) were determined on an
Epics Profile II (Coulter, Hialeah, FL). The 
MFI for a particular
mAb was calculated by subtracting the MFI of either the isotype-matched
control mAb or the second-step Ab from each MFI value. The fluorescence
ratio (FR) was calculated using the following formula: FR = (
MFI of peptide-treated T2 cells)/( MFI of nontreated T2
cells).
Culture medium
T cells were cultured in IMDM that had been supplemented with 10% pooled heat-inactivated human serum, 2 mM of L-glutamine, 100 µg/ml of streptomycin, 100 U/ml of penicillin, and 2.5 µg/ml of fungizone (hereafter designated complete medium (CM)). Monocytes were cultured in antibiotic-free RPMI 1640 containing 10% pooled human serum and 2 mM of L-glutamine (antibiotic-free CM).
Generation of CD8+ CTL lines from PBMCs
PBMCs from either patients with TB or healthy individuals were
suspended in CM containing 10 µg/ml ß2m and stimulated
with 6 µM of each M.tb peptide or IMP using a previously described
procedure with some modifications (27). A total of 4 x
106 cells/well were incubated at 37°C in 24-well plates
containing 2 LfU/ml of tetanus toxoid (Department of Public
Health, State of Michigan, East Lansing, MI) in 1 ml of CM. PBMCs from
individuals that did not respond by proliferation to tetanus toxoid
received 0.2 µg/ml of PHA (Wellcome Diagnostics, Research Triangle
Park, NC) at the initiation of culture. After 3 days, 1 ml of CM
supplemented with 10 U/ml of rIL-2 (Life Technologies) was added in
each well. On day 7, the cultures were restimulated with the 6 µM of
peptide in CM containing 10 µg/ml ß2m and 10 U/ml rIL-2
in the presence of 1 x 106 irradiated (3000 rad),
pooled, HLA-A2+, allogeneic PBMCs as feeder cells. On day
14, viable cells were recovered on Ficoll-Hypaque gradients, and
CD8+ T cells were isolated by positive-selection panning
using anti-CD8 mAb. After 1 day, the resulting cells (>95%
CD8+, TCRß+ by flow cytometric analysis)
were tested for CTL activity against autologous B-LCLs that had been
pulsed with the stimulating peptide (see below).
Cell separation
Populations that had been enriched for either dendritic cells
(DCs) or monocytes were separated from PBMCs on the basis of their
differential densities (28, 29). Briefly, the PBMCs that were obtained
by Ficoll-Hypaque gradient centrifugation were separated into
low-density (monocytes) and high-density Percoll fractions. Monocytes
that had been collected from the Percoll gradients (
90%
CD14+) were frozen in aliquots and thawed 1 day before use.
To separate DCs, cells from the high-density Percoll fraction suspended
in CM were incubated overnight in Teflon vessels at 37°C. Thereafter,
the cells were layered onto 15% (w/v) metrizamide and centrifuged at
650 x g for 10 min at room temperature (30). For
further enrichment, cells from the metrizamide interface (DC-enriched)
were refloated on a 14% metrizamide solution. The DC-enriched
population, which stained brightly with anti-HLA-DR mAb, was used
as APCs in the in vitro vaccination system, as described below.
CD8+ T cells were obtained from the high-density
metrizamide fraction by positive-selection panning (31) using
anti-CD8 mAb. The resulting cells were >95% CD8+ and
TCRß+ cells by flow cytometric analysis.
Priming of naive CD8+ T cells with synthetic peptides
Purified human CD8+ T cells (105 cells
suspended in 100 µl of CM) from healthy HLA-A*0201+,
M.tb-unresponsive (stimulation index (SI) < 2; Table I
) subjects were
added to 104 autologous DCs; the DCs had been pulsed with 6
µM of M.tb peptide by incubation for 2 h in 100 µl of CM
supplemented with 20 µg/ml ß2m and 1 U/ml of rIL-1 (R&D
Systems, Minneapolis, MN). The total volume per well was 200 µl, and
plates were incubated at 37°C in a humidified atmosphere containing
10% CO2. On day 3, a mixture of rIL-2 and rIL-4 (R&D
Systems) at 5 U/ml was added to each culture. On day 7, and weekly
thereafter, T cells were restimulated with 6 µM of original peptide
in the presence of irradiated (3000 rad) autologous monocytes in CM
supplemented with 5 U/ml of rIL-2 and rIL-4. After 4 to 6 wk of
expansion, the CD8+ T cells that had been recovered by
positive panning with anti-CD8 mAb were tested for their CTL
activity against autologous B-LCLs that had been pulsed with the
stimulating peptides. Cultures that displayed peptide-specific
cytolytic activity were expanded by weekly restimulation and retested
against autologous monocytes that had been infected with M.tb.
Generation of B-LCLs
To generate B-LCLs, PBMCs from each participant were transformed by EBV-containing supernatants from the marmoset line B-958 that was provided by Dr. S. K. H. Foung (Stanford University). Cells were incubated in 24-well plates (5 x 106 cells/well) in IMDM that had been supplemented with 30% heat-inactivated FBS, 2 mM of L-glutamine, 100 µg/ml of streptomycin, and 100 U/ml of penicillin. After 14 days, the transformed cells were expanded in IMDM containing 10% FBS, and an aliquot was stained with CR11–351 mAb. The percentage of HLA-A2.1/A2.2+ cells ranged between 82 and 98 by flow cytometry. Before their use as targets, B-LCLs were incubated for 18 h with 12 µM of each synthetic peptide.
Preparation of M.tb inoculum
The M.tb strain H37Ra (ATCC) was used to infect monocytes. Inocula were prepared by culturing bacilli on 7H11 agar plates. Before their addition to monocytes, colonies were resuspended in cold PBS (pH 7.2) and washed twice by centrifugation. To prevent clumping, the bacterial suspension was vortexed vigorously after each centrifugation and left standing at room temperature for 5 min before the upper fraction was withdrawn. After the last wash, clumps of mycobacteria in the suspension were dispersed by multiple passages through a syringe with a 25-gauge needle, and the concentration of bacilli was adjusted spectrophotometrically at 600 nm with reference to McFarland Equivalent Turbidity Standards (Remel, Lenex, KS); the viability of the organisms was determined by colony count (32).
M
infection
M.tb bacilli were incubated in polypropylene tubes with
monocytes that had been suspended in antibiotic-free CM at a
bacilli:monocyte ratio of 
5:1. Following 4 h of incubation at
37°C in a humidified atmosphere containing 5% CO2,
monocytes were washed thoroughly in PBS warmed to 37°C without
Ca2+ and Mg2+ by low-speed centrifugation and
incubated for 1 day in suspension in antibiotic-free CM in Teflon
vessels. Noninfected monocytes that were maintained in the suspension
culture for the same length of time served as a control. Before being
used as targets, both M.tb-infected and noninfected monocytes were
harvested and then washed in warmed PBS (37°C) by low-speed
centrifugation; cell viability was determined by the exclusion of
trypan blue. An aliquot of infected monocytes was analyzed to verify
infection by acid-fast staining and colony count. At 1 day after
infection, 31 to 53% of monocytes contained multiple bacteria. The
number of viable bacteria/cell ranged between 18 and 36 CFU. The cell
viability of both monocyte populations was >90%.
Cytotoxicity assay
Target cells (B-LCLs or monocytes) were labeled with 150 µCi
of 51Cr (ICN, Costa Mesa, CA) for 1 h at 37°C. A
total of 5000 cells were added to round-bottom microtiter wells
containing variable numbers of effector cells. In Ab-blocking
experiments, CD8+ T cells and target cells were treated for
1 h with 10 µg/ml of Leu-2a or w6/32 mAb, respectively, before
the addition of the opposing cells. All assays were performed in
triplicate at 37°C in a 10% CO2-humidified atmosphere.
51Cr release was measured at 5 h after the addition of
B-LCLs or at 18 h after the addition of monocytes. The percentage
of cytotoxicity was determined using the following formula: % specific
cytotoxicity = 100 x ([experimental release -
spontaneous release]/[maximum release - spontaneous
release]). The maximum release was determined by the lysis of
targets with 1% Triton X-100. Spontaneous release was <20% of the
maximum release in all assays. A positive CTL response was defined as
15% difference in the lysis of target cells that had been pulsed
with peptide or infected with M.tb and the corresponding untreated
target cells at an E:T of 40:1.
Proliferation assays
All proliferation assays were performed in round-bottom microtiter wells in a final volume of 200 µl of CM. For these experiments, 1 x 105 PBMCs were incubated with either 108 heat-killed M.tb or 2 LfU/ml of tetanus toxoid in quadruplicate. Control cells were incubated in medium alone. Stimulation with Ag was conducted for 6 days at 37°C in a humidified 10% CO2 atmosphere. Cellular proliferation was measured on the basis of the incorporation of [3H]thymidine that was added 6 h before harvesting. The SI was calculated using the following formula: SI = (mean cpm of cells cultured in the presence of stimulus)/(mean cpm of cells cultured in the absence of stimulus).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Using the MHC-binding motif for HLA-A*0201 (21), 28 peptides derived from M.tb19 were selected and synthesized for screening in a cell-based binding assay. Two previously described CTL epitopes, HIV gag A2 and HIV gag B8, were used as positive and negative controls, respectively. HLA-A*0201+ T2 cells, which have a defect in the assembly and transport of class I molecules (33, 34, 35), were used in these assays, because exogenously added HLA-A*0201-binding peptides can increase the number of properly folded HLA-A2 molecules on the cell surface. The increase in the surface expression of HLA-A2 molecules was measured by flow cytometry using mouse mAb to class I MHC (w6/32) and HLA-A2 (BB7.2) molecules.
A total of 5 of 28 candidate peptides stabilized HLA-A*0201 expression
on T2 cells (FR > 2.0 at 100 µM peptide) (Table II and Fig. 1). Two peptides, corresponding to
residues 14 to 22 and 88 to 97, bound to the cells with affinities that
were comparable with that of the control HIV gag A2 peptide. As
expected, HIV gag B8 had no effect on HLA-A*0201 expression by T2 cells
(FR = 1.1) (Fig. 1).
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To determine whether the selected HLA-A*0201-binding peptides were
recognized by CD8+ T cells that were primed in vivo,
PBMCs from an HLA-A*0201+ TSP subject were
stimulated separately with each peptide; after 2 to 4 wk of expansion,
CD8+ T cells that had been isolated by positive selection
were analyzed for their cytotoxic activity against peptide-pulsed
autologous B-LCLs. After 2 wk of stimulation, a CTL response (33%
specific lysis at a 40:1 E:T ratio) was detected against B-LCLs that
had been pulsed with P88–97 (Fig. 2A), and the intensity of
lytic activity increased after each round of restimulation with the
peptide, indicating a time-dependent enrichment of Ag-specific
effectors (Fig. 2, B and C). The cultured
effectors lysed P88–97 pulsed B-LCLs in a dose-dependent
manner at all three timepoints. Only weak lysis was observed for
nonpulsed B-LCLs or for target cells that had been pulsed with either
HIV gag A2 (Fig. 2) or two other 19-kDa-derived peptides,
P14–22 or P101–108 (data not shown). The
cytotoxic activity of these Ag-specific CTLs was markedly inhibited by
Abs to CD8 or class I MHC (Fig. 2D) but was not inhibited by
Abs to CD4 (Fig. 2D) or HLA-DR (data not shown). These
results were confirmed in two other experiments that tested the
responsiveness to P88–97 of PBMCs that were obtained 11 mo
later from the same donor (TSP-1) (Fig. 2E) or PBMCs from a
second TSP individual (donor TSP-2; 38% specific lysis at an E:T ratio
of 40:1). In parallel experiments, CD8+ T cells isolated
from the PBMCs of TSP-1 and TSP-2 subjects were incubated with an
IMP. The cells from both subjects exhibited 42% lysis (Fig. 2F and data not shown) of autologous targets that had been
pulsed with IMP at an E:T ratio of 40:1. By contrast,
P14–22 and P101–108 did not elicit CTL
activity when tested repeatedly on PBMCs that had been derived from the
same TSP participants (data not shown).
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). The
specificity of the response was established by the absence of cytotoxic
activity against B-LCLs that were pulsed with HIV gag A2 (Fig. 3), and
the MHC restriction was indicated by 64% inhibition of CTL-mediated
lysis by Ab to either class I MHC or CD8 molecules (data not shown). T
cells from one patient (TB-4) did not yield a strong CTL response
against peptide-pulsed autologous B-LCLs or a vigorous proliferative
response against M.tb (SI = 4) (Table I).
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, A and C,
P88–97-specific CD8+ CTLs were not recovered
when PBMCs from these TSN subjects were cultured in the presence of
M.tb P88–97. In contrast, incubating the PBMCs with IMP
resulted in an expansion of peptide-specific CTLs (Fig. 4, B
and D). These results, taken together with those detailed
above, indicate that the peptide-specific CTL precursors that expanded
in this experimental system had been primed in vivo.
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, P88–97 induced a specific
CTL response in both normal donors. These results confirm our earlier
observation that human peripheral DCs, when appropriately pulsed with
synthetic peptides, can sensitize naive CD8+ T cells and
consequently enable the generation of Ag-specific CTL lines in vitro
(36).
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We examined the ability of peptide-specific CTLs to recognize
endogenously synthesized epitopes by measuring their cytolytic activity
against autologous monocytes that were acutely infected with tubercle
bacilli. The results in Figure 6 show
that CTLs that were derived from both in vivo- and in vitro-primed
CD8+ T cells were able to recognize and lyse M.tb-infected
monocytes in a class I MHC-restricted manner. As shown, only weak lysis
was observed for uninfected monocytes. These results suggest that
P88–97 is generated by natural processing within the
infected cells.
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) and two independent
experimental systems to identify epitopes that elicit class I
MHC-restricted CD8+ T cell responses against tubercle
bacilli. One system detected peripheral blood CD8+ CTL
populations that were primed in vivo (27), while the other used
peptide-pulsed DCs as APCs to sensitize naive CD8+ T cells
in vitro (28, 36). The results establish that class I MHC-restricted
CD8+ CTL precursors with a specificity for
P88–97 are present in patients with active pulmonary TB as
well as in TSP individuals without a history of TB. Moreover,
peptide-specific CTLs that were expanded from in vivo-primed
CD8+ T cells or generated from naive CD8+ T
cells by in vitro sensitization with peptide-pulsed DCs recognized and
lysed autologous targets that were acutely infected with tubercle
bacilli in a class I MHC-restricted manner. Peripheral blood DCs that
have been pulsed with exogenous peptides are potent stimulators of
quiescent T cells (28, 37). In natural infection, DCs residing in
airway epithelium and lung parenchyma (38, 39, 40) are among the first
cells to encounter the bacillus and, presumably, to acquire and process
mycobacterial Ags for presentation to naive T cells (41, 42). A recent
report indicates that M.tb enters human DC progenitors in vitro,
resulting in the activation and maturation of DCs (43). These findings are potentially relevant for both vaccine development and adoptive immunotherapy. Epitopes that are generated by the intracellular processing of endogenously synthesized Ags are appropriate candidates for inclusion in the design of a peptide-based M.tb vaccine. The in vitro generation of M.tb-specific, biologically active effector cells potentially permits a large scale ex vivo expansion of CTLs for adoptive immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nahid Mohagheghpour, Kuzell Institute for Arthritis and Infectious Diseases, 2200 Webster Street, San Francisco, CA 94115. E-mail address:
3 Abbreviations used in this paper: M.tb, Mycobacterium tuberculosis; M.tb19, 19-kDa lipoprotein of M.tb; TB, tuberculosis; TSP, tuberculin skin test-positive; TSN, tuberculin skin test-negative; DC, dendritic cell; B-LCL, B lymphoblastoid cell line; aa, amino acid; IMP, influenza A virus matrix peptide; IMDM, Iscove’s modified Dulbecco’s medium; MFI, mean fluorescence intensity; FR, fluorescence ratio; CM, complete medium; ß2m, ß2-microglobulin; LfU, lines flocculation unit; SI, stimulation index.
Received for publication November 7, 1997. Accepted for publication April 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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interferon on human monocyte and murine peritoneal macrophages. Immunology 59:333.[Medline]
-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95:270.
T cell activation. J. Clin. Invest. 95:296.
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V. Lazarevic and J. Flynn CD8+ T Cells in Tuberculosis Am. J. Respir. Crit. Care Med., October 15, 2002; 166(8): 1116 - 1121. [Full Text] [PDF]
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A. Lalvani CD8 Cytotoxic T Cells and the Development of New Tuberculosis Vaccines Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 789 - 790. [Full Text]
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D. A. Lewinsohn, R. A. Lines, and D. M. Lewinsohn Human Dendritic Cells Presenting Adenovirally Expressed Antigen Elicit Mycobacterium tuberculosis-Specific CD8+ T Cells Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 843 - 848. [Abstract] [Full Text]
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Y. Maeda, M. Makino, D. C. Crick, S. Mahapatra, S. Srisungnam, T. Takii, Y. Kashiwabara, and P. J. Brennan Novel 33-Kilodalton Lipoprotein from Mycobacterium leprae Infect. Immun., August 1, 2002; 70(8): 4106 - 4111. [Abstract] [Full Text] [PDF]
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D. C. Flyer, V. Ramakrishna, C. Miller, H. Myers, M. McDaniel, K. Root, C. Flournoy, V. H. Engelhard, D. H. Canaday, J. A. Marto, et al. Identification by Mass Spectrometry of CD8+-T-Cell Mycobacterium tuberculosis Epitopes within the Rv0341 Gene Product Infect. Immun., June 1, 2002; 70(6): 2926 - 2932. [Abstract] [Full Text] [PDF]
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L. Grode, M. Kursar, J. Fensterle, S. H. E. Kaufmann, and J. Hess Cell-Mediated Immunity Induced by Recombinant Mycobacterium bovis Bacille Calmette-Guerin Strains Against an Intracellular Bacterial Pathogen: Importance of Antigen Secretion or Membrane-Targeted Antigen Display as Lipoprotein for Vaccine Efficacy J. Immunol., February 15, 2002; 168(4): 1869 - 1876. [Abstract] [Full Text] [PDF]
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M. Panigada, T. Sturniolo, G. Besozzi, M. G. Boccieri, F. Sinigaglia, G. G. Grassi, and F. Grassi Identification of a Promiscuous T-Cell Epitope in Mycobacterium tuberculosis Mce Proteins Infect. Immun., January 1, 2002; 70(1): 79 - 85. [Abstract] [Full Text] [PDF]
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A. A. Pathan, K. A. Wilkinson, P. Klenerman, H. McShane, R. N. Davidson, G. Pasvol, A. V. S. Hill, and A. Lalvani Direct Ex Vivo Analysis of Antigen-Specific IFN-{gamma}-Secreting CD4 T Cells in Mycobacterium tuberculosis-Infected Individuals: Associations with Clinical Disease State and Effect of Treatment J. Immunol., November 1, 2001; 167(9): 5217 - 5225. [Abstract] [Full Text] [PDF]
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N. V. Serbina and J. L. Flynn CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis Infect. Immun., July 1, 2001; 69(7): 4320 - 4328. [Abstract] [Full Text] [PDF]
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T. Chun, N. V. Serbina, D. Nolt, B. Wang, N. M. Chiu, J. L. Flynn, and C.-R. Wang Induction of M3-restricted Cytotoxic T Lymphocyte Responses by N-formylated Peptides Derived from Mycobacterium tuberculosis J. Exp. Med., May 21, 2001; 193(10): 1213 - 1220. [Abstract] [Full Text] [PDF]
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 C. G. Feng, C. Demangel, A. T. Kamath, M. Macdonald, and W. J. Britton Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8+ T cells with specificity for a novel mycobacterial epitope Int. Immunol., April 1, 2001; 13(4): 451 - 458. [Abstract] [Full Text] [PDF]
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F. A. Post, C. Manca, O. Neyrolles, B. Ryffel, D. B. Young, and G. Kaplan Mycobacterium tuberculosis 19-Kilodalton Lipoprotein Inhibits Mycobacterium smegmatis-Induced Cytokine Production by Human Macrophages In Vitro Infect. Immun., March 1, 2001; 69(3): 1433 - 1439. [Abstract] [Full Text] [PDF]
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D. M. Lewinsohn, L. Zhu, V. J. Madison, D. C. Dillon, S. P. Fling, S. G. Reed, K. H. Grabstein, and M. R. Alderson Classically Restricted Human CD8+ T Lymphocytes Derived from Mycobacterium tuberculosis-Infected Cells: Definition of Antigenic Specificity J. Immunol., January 1, 2001; 166(1): 439 - 446. [Abstract] [Full Text] [PDF]
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O. Neyrolles, K. Gould, M.-P. Gares, S. Brett, R. Janssen, P. O'Gaora, J.-L. Herrmann, M.-C. Prevost, E. Perret, J. E. R. Thole, et al. Lipoprotein Access to MHC Class I Presentation During Infection of Murine Macrophages with Live Mycobacteria J. Immunol., January 1, 2001; 166(1): 447 - 457. [Abstract] [Full Text] [PDF]
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S. M. Smith, R. Brookes, M. R. Klein, A. S. Malin, P. T. Lukey, A. S. King, G. S. Ogg, A. V. S. Hill, and H. M. Dockrell Human CD8+ CTL Specific for the Mycobacterial Major Secreted Antigen 85A J. Immunol., December 15, 2000; 165(12): 7088 - 7095. [Abstract] [Full Text] [PDF]
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S. M. Smith, M. R. Klein, A. S. Malin, J. Sillah, K. Huygen, P. Andersen, K. P. W. J. McAdam, and H. M. Dockrell Human CD8+ T Cells Specific for Mycobacterium tuberculosis Secreted Antigens in Tuberculosis Patients and Healthy BCG-Vaccinated Controls in The Gambia Infect. Immun., December 1, 2000; 68(12): 7144 - 7148. [Abstract] [Full Text] [PDF]
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A. Geluk, K. E. van Meijgaarden, K. L. M. C. Franken, J. W. Drijfhout, S. D'Souza, A. Necker, K. Huygen, and T. H. M. Ottenhoff Identification of Major Epitopes of Mycobacterium tuberculosis AG85B That Are Recognized by HLA-A*0201-Restricted CD8+ T Cells in HLA-Transgenic Mice and Humans J. Immunol., December 1, 2000; 165(11): 6463 - 6471. [Abstract] [Full Text] [PDF]
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 S. Cho, V. Mehra, S. Thoma-Uszynski, S. Stenger, N. Serbina, R. J. Mazzaccaro, J. L. Flynn, P. F. Barnes, S. Southwood, E. Celis, et al. Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis PNAS, October 12, 2000; (2000) 210391497. [Abstract] [Full Text]
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N. Mohagheghpour, A. van Vollenhoven, J. Goodman, and L. E. Bermudez Interaction of Mycobacterium avium with Human Monocyte-Derived Dendritic Cells Infect. Immun., October 1, 2000; 68(10): 5824 - 5829. [Abstract] [Full Text] [PDF]
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 J. W. Hodge, A. N. Rad, D. W. Grosenbach, H. Sabzevari, A. G. Yafal, L. Gritz, and J. Schlom Enhanced Activation of T Cells by Dendritic Cells Engineered to Hyperexpress a Triad of Costimulatory Molecules J Natl Cancer Inst, August 2, 2000; 92(15): 1228 - 1239. [Abstract] [Full Text] [PDF]
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D. M. Lewinsohn, A. L. Briden, S. G. Reed, K. H. Grabstein, and M. R. Alderson Mycobacterium tuberculosis-Reactive CD8+ T Lymphocytes: The Relative Contribution of Classical Versus Nonclassical HLA Restriction J. Immunol., July 15, 2000; 165(2): 925 - 930. [Abstract] [Full Text] [PDF]
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N. V. Serbina, C.-C. Liu, C. A. Scanga, and J. L. Flynn CD8+ CTL from Lungs of Mycobacterium tuberculosis-Infected Mice Express Perforin In Vivo and Lyse Infected Macrophages J. Immunol., July 1, 2000; 165(1): 353 - 363. [Abstract] [Full Text] [PDF]
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S. M. Smith, A. S. Malin, Pauline T., Lukey, S. E. Atkinson, J. Content, K. Huygen, and H. M. Dockrell Characterization of Human Mycobacterium bovis Bacille Calmette-Guerin-Reactive CD8+ T Cells Infect. Immun., October 1, 1999; 67(10): 5223 - 5230. [Abstract] [Full Text] [PDF]
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C. G. Feng, A. G. D. Bean, H. Hooi, H. Briscoe, and W. J. Britton Increase in Gamma Interferon-Secreting CD8+, as Well as CD4+, T Cells in Lungs following Aerosol Infection with Mycobacterium tuberculosis Infect. Immun., July 1, 1999; 67(7): 3242 - 3247. [Abstract] [Full Text] [PDF]
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S. Cho, V. Mehra, S. Thoma-Uszynski, S. Stenger, N. Serbina, R. J. Mazzaccaro, J. L. Flynn, P. F. Barnes, S. Southwood, E. Celis, et al. Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis PNAS, October 24, 2000; 97(22): 12210 - 12215. [Abstract] [Full Text] [PDF]
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