characterization of a mycobacterium tuberculosis peptide that is recognized by human cd4+ and cd8+ t...

8/6/2019 Characterization of a Mycobacterium tuberculosis Peptide That Is Recognized by Human CD4+ and CD8+ T Cells in

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of July 7, 2011This information is current as

2004;173;1966-1977J ImmunolAndersen and Peter F. BarnesKatie Ewer, Gerald T. Nepom, David M. Lewinsohn, PeterLalvani, Patrick K. Moonan, Hassan Safi, Benjamin Wizel,Homayoun Shams, Peter Klucar, Steven E. Weis, AjitAlleles

T Cells in the Context of Multiple HLA+CD8and+Peptide That Is Recognized by Human CD4

Mycobacterium tuberculosisCharacterization of a

References

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Characterization of a Mycobacterium tuberculosis Peptide That

Is Recognized by Human CD4 and CD8 T Cells in the

Context of Multiple HLA Alleles1

Homayoun Shams,2* Peter Klucar,* Steven E. Weis, Ajit Lalvani, Patrick K. Moonan,

Hassan Safi,* Benjamin Wizel,* Katie Ewer, Gerald T. Nepom, David M. Lewinsohn,**

Peter Andersen, and Peter F. Barnes*

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high

percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by

identifying and characterizing a 15-mer peptide, CFP107185, that elicited IFN-production and CTL activity by both CD4 and

CD8 T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP107185 contained at least

two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4 cells in the context of

DRB1*04, DR5*0101, and DQB1*03, and by CD8 cells of A2 donors. T6 elicited responses by CD4 cells in the context of

DRB1*04 and DQB1*03, and by CD8

cells of B35

donors. Deleting a single amino acid from the amino or carboxy terminusof either peptide markedly reduced IFN- production, suggesting that they are minimal epitopes for both CD4 and CD8 cells.

As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell sub-

populations. The capacity of CFP107185 to stimulate IFN-production and CTL activity by CD4 and CD8 cells from persons

expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antitu-

berculosis vaccine. The Journal of Immunology, 2004, 173: 19661977.

Eight million new cases and 1.8 million deaths annually

worldwide are attributed to Mycobacterium tuberculosis,

one of the leading causes of death from a single infectious

agent (1). The burgeoning epidemic of HIV infection in regions

where tuberculosis is common has created a growing population of

persons that are highly susceptible to M. tuberculosis. In addition,

the continued spread of multidrug-resistant tuberculosis threatens

to overwhelm the public health capacity of many jurisdictions (2,

3). These unfavorable factors will cause tuberculosis to remain a

major health problem in the coming decades, and increase the ur-

gency for development of an effective vaccine.

The only available antituberculosis vaccine is bacillus Calmette-

Guerin (BCG),3 a live attenuated Mycobacterium bovis that was

created in 1921. Vaccination with M. bovis BCG reduces the se-

verity of tuberculosis in children, but does not protect against de-

velopment of tuberculosis. Furthermore, vaccination can cause

life-threatening disease in immunocompromised patients, such as

those with HIV infection (4).

T cells play a pivotal role in protection against tuberculosis, and

many studies have shown that CD4

T cells are essential for im-munity (5). A growing body of evidence in animals and in humans

suggests that CD8 cells also contribute significantly to immune

defenses against tuberculosis through lysis of infected cells, pro-

duction of IFN-, and direct microbicidal activity (612). There-

fore, the most effective vaccine is likely to be one that elicits re-

sponses by both CD4 and CD8 T cells (12).

Most published evidence indicates that secreted M. tuberculosis

Ags stimulate protective immunity (13). Two important secreted

proteins are 6-kDa early secretory antigenic target (ESAT-6) and

10-kDa culture filtrate protein (CFP10), which form a tightly

bound 1:1 heterodimeric complex (14). The encoding genes are

cotranscribed (15) and are part of the RD1 region of the M. tu-

berculosis genome, which is deleted from M. bovis BCG. Resto-ration of RD1 enhanced the capacity of BCG vaccination to protect

mice against subsequent infection with M. tuberculosis (16).

ESAT-6 and CFP10 stimulate T cells to produce IFN- and

exhibit CTL activity in animal models and in humans infected with

M. tuberculosis, making them excellent candidates for inclusion in

an antituberculosis subunit vaccine (1720). T cells from a high

percentage of persons with latent tuberculosis infection recognize

ESAT-6 and CFP10 (20, 21), suggesting that they either contain

multiple epitopes that are restricted by different MHC molecules,

or epitopes that are promiscuously recognized in the context of

multiple MHC molecules. Several epitopes for CD4 and CD8 T

cells, restricted by different MHC molecules, have been identified

*Center for Pulmonary and Infectious Disease Control, and Departments of Micro-biology, Immunology, and Medicine, University of Texas Health Center, Tyler, TX75708; Department of Internal Medicine, University of North Texas Health ScienceCenter, Fort Worth, TX 76107; Nuffield Department of Clinical Medicine, Univer-sity of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; Benaroya Re-search Institute, Seattle, WA 98101; **Division of Pulmonary and Critical Care Med-icine, Oregon Health and Science University/Portland Veterans Affairs Medical

Center, Portland, OR 97207; and Statens Seruminstitut, Copenhagen, Denmark

Received for publication December 16, 2003. Accepted for publication May 24, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health(AI44935), the Cain Foundation for Infectious Disease Research, the Wellcome Trust,and the Center for Pulmonary and Infectious Disease Control. P.F.B. holds the Mar-garet E. Byers Cain Chair for Tuberculosis Research. A.L. is a Wellcome SeniorResearch Fellow in Clinical Science.

2 Address correspondence and reprint requests Dr. Homayoun Shams, Center for Pul-monary and Infections Disease Control, University of Texas Health Center, 11937 USHighway 271, Tyler, TX 75708. E-mail address: [email protected]

3 Abbreviations used in this paper: BCG, bacillus Calmette-Guerin; BLS, bare lym-phocyte syndrome; CFP, culture filtrate protein; ESAT, early secretory antigenictarget.

The Journal of Immunology

Copyright 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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in ESAT-6, providing an explanation for its widespread recogni-

tion (18, 22, 23). In contrast, only two CD8 epitopes for CFP10

have been identified (24). In this study, we wished to determine the

molecular basis for the recognition of CFP10 by most individuals

with latent tuberculosis infection. We identified and characterized

a 15-mer peptide of CFP10 that elicited IFN- production and

CTL activity by both CD4 and CD8 T cells from the majority

of persons with latent tuberculosis infection, including those ex-

pressing several different MHC class I and class II molecules.

Materials and MethodsStudy subjects

This study was approved by the Institutional Review Boards of the Uni-versity of North Texas Health Science Center (Fort Worth, TX) and the

University of Texas Health Center (Tyler, TX). Blood was obtained from10 healthy tuberculin-negative donors without prior contact with tubercu-losis patients, and from 132 donors who were recent contacts of patients

with pulmonary tuberculosis. Seventy-six (58%) donors were Hispanic, 29(22%) were white non-Hispanic, 21 (16%) were African American, and 6

(5%) were Asian. All donors had no symptoms of tuberculosis with normalchest radiographs. Donors were classified as having latent tuberculosis in-fection if they had a tuberculin skin test showing at least a 5-mm diameter

of induration and their PBMC produced IFN- in response to CFP10 or

ESAT-6, based on the ELISPOT assay.

Peptides

We selected 15-mer peptides that overlapped by 10 aa and spanned the

CFP10 protein. Truncated peptides were also synthesized, as outlined inthe results. Peptides were synthesized by the Molecular Genetics Instru-mentation Facility at the University of Georgia (Athens, GA) and by In-

vitrogen Life Technologies (Carlsbad, CA), using Fmoc chemistry. Peptidepurity was70%, as assayed by HPLC, and their composition was verifiedby mass spectrometry. Lyophilized peptides were dissolved at 25 mg/ml in

DMSO, aliquoted, and stored at 4C.

Antibodies

We used Abs to framework MHC class I (ATCC clone W6/32; AmericanType Culture Collection (ATCC), Manassas, VA) and MHC class II

(ATCC clone 9.3F10; ATCC).

Isolation of PBMC and cell subpopulations

PBMC were obtained by centrifugation over Ficoll-Paque (Pharmacia,

Uppsala, Sweden) and cultured in RPMI 1640 (Invitrogen Life Technol-ogies, Gaithersburg, MD), supplemented with 10% heat-inactivated human

AB serum (Atlanta Biologicals, Norcross, GA). In some experiments,

FIGURE 1. Capacity of CFP10 peptides to stimulate IFN- production

by PBMC from persons with latent tuberculosis infection. PBMC from 49

CFP10-responsive persons were cultured overnight on IFN- ELISPOT

plates with 15-mer overlapping peptides spanning the CFP10 protein. The

values shown are the percent of CFP10 responders that produced

7IFN- cells per 2.5 105 cells to each individual peptide. Unstimulated

PBMC showed 0 2 IFN- cells per 2.5 105 cells.

FIGURE 2. CFP107185 and CFP107690 stimulate production of IFN-

by PBMC. PBMC from eight CFP10-responsive individuals were stimu-

lated with peptides CFP107185 and CFP107690 for 48 h. PBMC and pos-

itively selected CD4 or CD8 cells were then cultured overnight on

IFN-ELISPOT plates. Values shown are the means and SEM of triplicate

wells. Unstimulated PBMC showed 0 2 IFN- cells per 2.5 105 cells.

A, B, and C show results for PBMC, CD4 cells, and CD8 cells,

respectively.

1967The Journal of Immunology

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FIGURE 3. CTL activity of short-term T cell lines

stimulated with CFP107185 and CFP107690. PBMC

from four donors were stimulated with peptides

CFP107185 and CFP107690 for 1314 days. These

short-term lines were used as effectors. Positively se-

lected CD4 or CD8 cells from these lines were also

used as effectors. Targets were autologous dendritic

cells, either unpulsed or pulsed with relevant pep-

tides. A, B, and C show results, using peptide-stimu-

lated PBMC, CD4 cells, and CD8 cells as effec-

tors, respectively. Values shown are the means and

SEM of triplicate wells.

1968 A MYCOBACTERIAL PEPTIDE RECOGNIZED BY CD4 and CD8 T cells

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CD4, CD8, or CD14 cells were isolated from PBMC by positive se-lection with magnetic beads conjugated to the appropriate Abs (Miltenyi

Biotech, Auburn, CA). Positively selected cells were 95% pure, as de-termined by flow cytometry.

Measurement of the frequency of IFN--producing cells

To measure the frequency of cells in PBMC that produced IFN- in re-sponse to mycobacterial Ags or peptides, 2 105 cells per well were

cultured in RPMI 1640 and 10% heat-inactivated human AB serum, withpurified protein derivative (1 g/ml; Statens Seruminstitut, Copenhagen,Denmark), CFP10 (10 g/ml; Lionex, Braunschweig, Germany), ESAT-6

(10 g/ml; Statens Seruminstitut) or CFP peptides (10 g/ml) for 16 20 hin 96-well plates that were precoated with 15 g/ml anti-human IFN-mAb (1-DlK; Mabtech, Nacka, Sweden).

To measure the frequency of IFN--producing CD4 or CD8 cells,PBMC were cultured in T-25 flasks at 1.5 106 cells/ml, in medium alone,or with peptide (10 g/ml), or purified protein derivative (1 g/ml) for48 72 h. Preliminary studies showed that this period of stimulation yieldedthe maximum number of IFN- cells. After 48 72 h, cells were washedthree times, and one aliquot was placed on an anti-IFN--precoated ELIS-POT plate for 16 20 h. From two other aliquots, CD4 and CD8 cellswere positively selected and placed on an ELISPOT plate for 16 20 h.

ELISPOT plates were washed with PBS plus 0.05% Tween 20, andanti-human IFN- mAb 7-B6-1 conjugated to alkaline phosphatase

(Mabtech) was added as the detection Ab. After 90 min, the plates were

washed and 5-bromo-4-chloro-3-indolyl phosphate/NBT substrate (Moss,Pasadena, MD) was added for 25 min or until spots appeared. The spotsin air-dried plates were counted using a stereomicroscope. Responses wereconsidered positive if the Ag-stimulated well contained a mean of at least

five more spot-forming cells than the mean of the negative control wells,and the Ag-stimulated value was at least twice the mean of the negativecontrol value (20).

In some experiments, freshly isolated CD4 cells (25,000 cells/well) orCD4 clones (100 cells/well) were cultured with transfected bare lympho-

cyte syndrome (BLS) cells (25,000 cells/well), expressing a single HLAmolecule (25) as APCs on an ELISPOT plate for 16 20 h. The number ofIFN--producing cells was determined, as outlined above.

Expansion of peptide-specific CTLs

PBMC were washed, resuspended in RPMI 1640 containing 10% humanAB serum, 20 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1

mM nonessential amino acids (all from Invitrogen Life Technologies), 50U penicillin (Sigma-Aldrich, St. Louis, MO), and 10 g/ml peptide, and

seeded in 24-well plates (BD Biosciences, San Jose, CA) at 3 106 cells/well. After 3 days, 100 U/ml recombinant human IL-2 (Proleukin; Chiron,Emeryville, CA) was added to each well. After 7 days, 3 106 peptide-

pulsed irradiated (3300 rad) autologous PBMC and 100 U/ml IL-2 wereadded to each well. Six days later, effector cells were tested for CTL ac-tivity in a 51Cr release assay.

In some cases, positively selected CD4 and CD8 effectors were iso-lated from peptide-expanded short-term lines, using immunomagnetic

beads. Purity of these cells was 9599%, as assessed by flow cytometry.

Generation of peptide-specific T cell clones

Clones were generated by previously described methods (26, 27). Briefly,autologous dendritic cells were generated as described below (preparation

of target cells), pulsed with peptide T1, irradiated, and cultured at 104

cellsper well in 96-well round-bottom plates with 300 CD4 T cells in each

well. Wells that showed visible growth were tested for reactivity with

peptide T1 by ELISPOT. IFN- T cell clones were expanded with anti-CD3 mAb (OKT3; Ortho Biotech, Bridgewater, NJ), irradiated allogeneic

PBMC, and an EBV-transformed B cell line lymphoblastoid cell lines(LCL).

Assessment of CTL activity

Target cells were autologous dendritic cells, generated by incubating pos-itively selected CD14 macrophages with IL-4 (10 ng/ml; R&D Systems,

Minneapolis, MN) and GM-CSF (10 ng/ml; R&D Systems) for 5 day.Dendritic cells were either unstimulated, infected with M. tuberculosisH37Rv for 48 h, or pulsed with a peptide overnight.

Targets were labeled overnight with 100 Ci of Na251CrO

4(Amersham

Life Science, Arlington Heights, IL) at 37C. After extensive washing, theywere suspended in complete medium containing 10% FBS, and 104 cells/

well were added in triplicate to round-bottom 96-well plates, each wellcontaining 6 105 effector cells, an E:T ratio of 60:1. Plates were centri-

fuged at 500 g for 2 min, then incubated for 5 h at 37C. Supernatantswere collected (Skatron, Sterling, VA), and 51Cr release was expressed asthe mean percent specific lysis, calculated as: 100 ([experimental re-lease spontaneous release]/[maximum release spontaneous release]).Net specific lysis was calculated by subtracting the percent speci fic lysis ofunpulsed target cells from the percent specific lysis of peptide-pulsed or M.tuberculosis-infected target cells. Maximum and spontaneous release weredetermined in wells containing target cells only, with or without 2% Triton

X-100, respectively. Spontaneous release was always 15% of maximum

release.

MHC typing

DNA was extracted from PBMC, using Wizard Genomic (Promega, Mad-ison, WI). Low resolution HLA typing was performed, using PCR with

sequence-specific primers (Combi Tray; GenoVision, West Chester, PA).Briefly, DNA samples (30 ng/l) were mixed with a master mix containingTaq polymerase (GenoVision), added into the plates containing sequence-

specific primers and amplified by PCR. PCR products (10 l) were elec-trophoresed on a 2% agarose gel containing ethidium bromide. MHC al-leles were identified with the GenoVision version of HELMBERG-SCOREVirtual Sequencing software.

Table I. HLA typing of donors whose T cells recognized CFP107185a

Donor CD8IFN- Cells HLA-A HLA-B HLA-C CD4 IFN- Cells HLA-DQ HLA-DR

T225 15 2 A*01, *24 B*07, *53 Cw*04, *07 78 7 B1*05, *06 B1*1001, *15T262 32 4 A*24, *31 B*40, *51 Cw*03, *15 85 2 B1*03, *04 B1*04, *08T221 314 26 A*02, *02 B*39, *40 Cw*03, *07 535 58 B1*03, *03 B1*04, *04T193 700 52 A*02, *68 B*35, *51 Cw*04, *08 633 56 B1*03, *03 B1*04, *04T040 70 12 A*26, *68 B*40, *44 Cw*03, *05 223 25 B1*03, *03 B1*04, *04T198 223 4 A*11, *11 B*15, *3525 Cw*07, *08 39 3 B1*03, *03 B1*11, *12

T021 15 3 A*02, *03 B*40, 58 Cw*03, *06 78 2 B1*03, *05 B1*1001, *11a PBMC were cultured with or without CFP107185 for 48 h, and positively selected CD4

and CD8 cells were placed on an ELISPOT plate for 16 20 h to detect IFN-

cells. The values shown (mean SEM of triplicate wells) are the number of peptide-stimulated IFN- cells per 2.5 105 cells. Unstimulated wells contained 0 2 positivecells per 2.5 105 cells.

Table II. Truncated peptides derived from CFP107185a

CFP107185 EISTNIRQAGVQYSR

CFP107690 IRQAGVQYSRADEEQ

T110 aa IRQAGVQYSRT211 aa NIRQAGVQYSRT312 aa TNIRQAGVQYSRT413 aa STNIRQAGVQYSRT514 aa ISTNIRQAGVQYSRT69 aa EISTNIRQAT79 aa NIRQAGVQYT810 aa TNIRQAGVQYT910 aa RQAGVQYSRA

a A ladder of 10 14 aa peptides was made from CFP107185 (T1-T5). Four otherpeptides (T6-T9) were predicted to bind to 3 8 MHC class I alleles with high affinity,

using a scoring system that allocates values to every amino acid in a peptide, basedon the frequency of the respective amino acid in natural ligands, T cell epitopes, or

binding peptides (28).


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ResultsTwo CFP10 peptides elicit IFN- production by T cells from

most persons with latent tuberculosis infection

PBMC from 132 close contacts of patients with pulmonary tuber-

culosis were stimulated with the M. tuberculosis-specific proteins,

CFP10 and ESAT-6, which have previously been used to identify

persons with latent tuberculosis infection (20, 21, 23). The ELIS-

POT assay was used to identify IFN--producing cells. Fifty-three

subjects were classified as having latent tuberculosis infection,based on having positive tuberculin skin tests and IFN--produc-

ing PBMC in response to either ESAT-6 or CFP10. Of these 53, 49

responded to CFP10.

To identify the regions of CFP10 that induced IFN- produc-

tion, we tested overlapping 15-aa peptides that spanned CFP10

(20). CFP107185 and CFP107690 were the most potent, and were

recognized by 83 and 91% of responders, respectively (Fig. 1).

CFP107185 and CFP107690 did not elicit IFN- production by

PBMC from 10 tuberculin-negative persons who had no history of

contact with tuberculosis patients.

CFP107185

and CFP107690

elicit IFN- production and CTL

activity by CD4 and CD8 T cells

We stimulated PBMC from eight donors with CFP107185 and

CFP107690. After 48 h, we obtained CD4

and CD8 cells by

positive immunomagnetic selection, and cultured them on ELIS-

POT plates overnight. CFP107185 induced IFN- production by

PBMC from eight donors (mean 300 124 (SE) IFN- cells per

2.5 105 cells; Fig. 2A), by CD4 cells from seven donors (mean

205 86 IFN- cells per 2.5 105 cells; Fig. 2B) and by CD8

cells from eight donors (mean 173 85 IFN- cells per 2.5

105 cells; Fig. 2C). CFP107690 was recognized by PBMC from

six donors (mean 320 176 IFN- cells per 2.5 105 cells; Fig.

2A), by CD4 cells from seven donors (mean 146 64 IFN-

cells per 2.5 105 cells; Fig. 2B), and by CD8 cells from five

donors (mean 121

83 IFN-

cells per 2.5

10

5

cells; Fig. 2C).To determine whether CFP107185 and CFP107690 elicited

CTL activity, PBMC were cultured with either peptide for 7 days,

then restimulated for another 6 7 days. This brief period reduced

the likelihood of artifactual induction of peptide-responsive CTL.

For four donors, PBMC showed net specific lysis ranging from 6

to 45% against peptide-pulsed autologous dendritic cells (Fig. 3A).

CD4 T cells isolated from restimulated PBMC showed similar

levels of net specific lysis as PBMC (6 46%; Fig. 3B) and CD8

T cells showed lower levels of lysis (127%; Fig. 3C). Recent

studies show that M. tuberculosis-responsive CD8 T cell clones

are more potent CTL than CD4 T cell clones, using a low E:T

ratio and suboptimal peptide concentrations (28). The different re-

sults in our experimental system may be due to our use of short-

term T cell lines as effectors, a high E:T ratio and high peptide

concentrations. The precursor frequency of CD4 CTL may also

be higher than that of CD8 CTL in peptide-stimulated PBMC.

CFP107185

is recognized by donors expressing different HLA

alleles

CFP107185 elicited IFN- production by both CD8

and CD4

cells from most donors with latent tuberculosis infection, suggest-

ing that it is recognized in the context of multiple MHC alleles. To

evaluate these possibilities, we performed MHC typing of seven

donors whose CD4 and CD8 cells produced IFN- in response

to CFP107185 (Table I). No single MHC class I or class II allele

was shared between all donors. However, of these seven individ-

uals, six expressed DQB1*03 and four expressed DRB1*04. This

suggests that CFP107185 contains a single epitope that is recog-

nized promiscuously in the context of multiple MHC molecules, or

that it contains two or more epitopes, each restricted by differentMHC molecules.

Identification of minimal T cell epitopes within CFP107185

To identify the epitopes in CFP107185, we first truncated 15 aa

from the N terminus (T1-T5, Table II). We also used a motif-based

algorithm (29) to identify additional sequences in CFP107185 that

were predicted to bind with high affinity (score, 9) to 4 8 MHC

class I alleles (T6-T8, Table II). Finally, we identi fied a peptide

that was predicted to bind with high affinity to three MHC class I

alleles (T9, Table II).

PBMC from six CFP107185-responsive donors were stimulated

with peptides T1-T9, and the frequency of IFN- cells was mea-

sured by ELISPOT (Table III). The 9-mer T6 and CFP107185elicited comparable numbers of IFN- cells for all donors. The

10-mer T1 yielded similar numbers of IFN- cells as CFP107185

for four donors, and 20 60% fewer IFN- cells for two donors.

To determine whether peptides T1 and T6 contain minimal

epitopes, we deleted 12 amino acids from the N or C terminus of

these peptides. Removal of a single amino acid from either end

of both peptides markedly reduced IFN- production by PBMC

(Table IV).

Table III. Capacity of truncated peptides to stimulate IFN- production by PBMCa

Donor CFP10 Response T1 (10aa) T2 (11 aa) T3 (12 aa) T4 (13 aa) T5 (14 aa) T6 (9 aa) T7 (9 aa) T8 (10 aa) T9 (10 aa) CFP107185 (15 aa)

T221 571 671 603 606 590 578 346 384 19 569

T225 103 190 284 284 240 213 4 6 4 246T262 313 325 378 315 381 394 163 228 16 309T267 121 119 121 138 146 165 69 82 6 153

T283 255 266 263 265 305 296 130 171 23 293T294 178 141 111 149 179 156 29 62 1 189

T296 0 0 0 0 3 0 0 0 0 0T298 2 1 0 1 0 2 2 3 1 1

T299 0 0 0 0 0 1 0 1 0 0T300 0 0 0 0 0 0 0 0 0 0T301 0 0 0 0 0 0 0 0 0 0

T303 0 0 0 0 0 0 0 0 0 0T302 1 6 1 5 7 0 0 0 1 1

T297 7 3 6 5 4 6 3 2 2 2

a Freshly isolated PBMC from donors who responded to CFP107185 were stimulated overnight on IFN- ELISPOT plates with peptide (10 g/ml). For each donor, 23experiments were performed. Values shown are the mean number of IFN- cells per 2.5 105 cells, based on duplicate wells in a representative experiment. Unstimulated

wells contained 0 5 positive cells per 2.5 105 cells.


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Peptides T1 and T6 are presented by MHC class I and II

molecules

To identify the restriction elements for peptides T1 and T6, we

treated PBMC with anti-MHC class I, anti-MHC class II, or con-

trol Ab for 4 h before addition of peptide on the IFN-ELISPOT

plate. Neutralization of MHC class I reduced the mean number of

T1- and T6-responsive IFN- cells by 50% (T1, mean 119 18

vs 51 15 cells per 2.5 105 cells, p 0.02; T6, mean 139

26 vs 65 14 cells per 2.5 105

cells, p 0.04; Table V).Anti-MHC class II reduced the number of IFN- cells by 90%

(T1, mean 119 18 vs 9 5 cells per 2.5 105 cells, p 0.0001;

T6, mean 139 26 vs 8 2 cells per 2.5 105 cells, p 0.0005;

Table V). Isotype control Abs had no effect on the number of

IFN- cells (data not shown).

Peptides T1 and T6 elicit IFN- production and CTL activity by

CD4 and CD8 T cells

PBMC from four donors were stimulated with peptides T1 and T6.

Forty-eight to 72 h later, positively selected CD4 and CD8 cells

were placed on an IFN- ELISPOT plate. The frequency of pep-

tide-responsive IFN- cells was similar in CD4 cells and

PBMC (Fig. 4, A and B). The number of IFN-

CD8

cells waslower than that of CD4 cells, but higher than corresponding val-

ues for unstimulated cells in three donors for peptide T1 and four

donors for peptide T6 (Fig. 4C).

We next evaluated the ability of peptides T1 and T6 to elicit

CTL activity. PBMC from five donors were cultured with peptides

T1 and T6 for 7 days and restimulated with peptide for 6 7 more

days. PBMC effectors lysed autologous peptide-pulsed target cells

(T1, net specific lysis 8 22%; T6, net specific lysis 6 17%; Fig.

4D). Positively selected CD4 T cells showed similar results (T1

and T6, net specific lysis 8 39% and 8 20%, respectively; Fig.

4E). CD8 T cells also lysed comparable numbers of peptide-

pulsed autologous target cells. However, because CD8 cells

lysed a high percentage of unpulsed targets, net specific lysis was

relatively low (T1, 214%; T6, 4 10%; Fig. 4F).

Dendritic cells infected with M. tuberculosis express peptides T1

and T6

To determine whether peptides T1 and T6 are expressed by APCs

during M. tuberculosis infection in vivo, we cultured PBMC from

three donors with T1 and T6, and tested their capacity to lyse

autologous dendritic cells infected with H37Rv. T1-primed effec-

tor PBMC and CD4 cells showed modest lytic activity (net spe-

cific lysis 713%; Fig. 5, A and C). T6-primed effector PBMC and

CD4

cells lysed infected cells from two of three donors (netspecific lysis 0 32%; Fig. 5, B and D). CD8 effector cells pulsed

with T1 or T6 also lysed infected dendritic cells, but nonspecific

lysis was higher than for CD4 cells (net specific lysis 232%;

Fig. 5, E and F).

MHC restriction of T cells that recognize peptides T1 and T6

All nine donors whose T1- or T6-primed CD4 T cells exhibited

CTL activity or produced IFN- expressed DQB1*03, and seven

of nine also expressed DRB1*04 (Table VI). All five donors whose

T1-primed CD8 T cells showed CTL activity or IFN- produc-

tion were HLA A*02, whereas all five donors whose T6-primed

CD8 T cells showed CTL activity or IFN-production were HLA

B*35

. Three donors whose CD8

T cells responded to T1 and T6expressed both HLA A*02 and B*35 alleles.

To more definitively demonstrate that peptides T1 and T6 are

restricted by DRB1*04 and DQB1*03, we used transfected BLS

cells expressing a single HLA allele as target cells. Freshly isolated

CD4 T cells were obtained from five persons who responded to

CFP10 and were infected with M. tuberculosis, and from three

uninfected persons who did not respond to CFP10. CD4 cells

from the CFP10-responsive donors produced IFN- specifically in

response to DRB1*0401 or DQB1*0302 targets pulsed with

peptide T1 or T6, but not to unpulsed targets. In contrast, CD4

cells from CFP10-negative donors did not produce IFN- in re-

sponse to peptide-pulsed targets (Table VII).

The results above suggest that peptides T1 and T6 are recog-

nized in the context of more than one MHC class II allele. To

Table IV. Effect of truncation on the capacity of peptides T1 and T6 to elicit IFN- production by PBMCa

T1

(IRQAGVQYSR)

T11(RQAGVQYSR)

T12(IRQAGVQYS)

T13(IRQAGVQY)

T6

(EISTNIRQA)

T6 1(ISTNIRQA)

T6 2(EISTNIRQ)

CFP107185(EISTNIRQAGVQYSR)

T225 149 4 3 0 151 0 3 216T262 163 4 18 18 185 0 1 204T267 153 6 18 9 149 4 3 152

T283 191 0 63 1 178 1 0 252T294 228 0 60 14 147 1 1 208

a Peptides T1 and T6 were truncated from the amino or carboxy terminus, and the effect of truncation was assessed in the ELISPOT assay. Freshly isolated PBMC from

CFP107185-responsive donors were stimulated overnight with 10 g/ml peptide on IFN-ELISPOT plates. Values shown are the mean number of IFN- cells per 2.5 105

cells, based on duplicate wells. Unstimulated PBMC showed 0 4 IFN- cells per 2.5 105 cells.

Table V. Effect of Abs to MHC class I and MHC class II on peptide-induced IFN- production by PBMCa

Peptide T1

Peptide T1

Anti-MHC I

Peptide T1

Anti-MHC II Peptide T6

Peptide T6

Anti-MHC I

Peptide T6

Anti-MHC II

T221 100 48 0 139 63 4T225 87 18 4 94 81 5T262 170 108 10 179 94 8

T267 123 ND 31 189 ND 18T343 65 35 0 38 15 10

T375 168 45 8 195 73 5

a Freshly isolated PBMC from donors responsive to peptides T1 and T6 were incubated with Abs for 4 h, then incubatedovernight with peptide on IFN-ELISPOT plates. Values shown are the mean number of IFN- cells per 2.5 105 cells, based

on duplicate wells. Unstimulated PBMC showed 0 4 IFN- cells per 2.5 105 cells.


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confirm these findings at the level of the single cell, we used two

T1-specific CD4 T cell clones as effector cells. BLS cells lacking

endogenous MHC class II expression (BLS-1) and six BLS cell

lines, each expressing a single transfected HLA class II allele,

were used as APCs. Significant numbers of IFN- cells were

only observed in the presence of peptide, and BLS cells alone

elicited IFN- production by 10% of the clones (Fig. 6). The

frequency of IFN- cells in both clones was 3- to 6-fold higher

when BLS cells expressing DRB5*0101 or DRB1*0401 were used,

and BLS cells expressing DQB1*0602 yielded a 2-fold higher

FIGURE 4. Peptides T1 and T6 stimulate production of IFN-and CTL activity. PBMC from four donors were stimulated with peptides T1 and T6 for

48 h, and then PBMC (A) and purified CD4 (B) and CD8 T cells (C) were cultured overnight on IFN-ELISPOT plates. Values shown are the means

and SEM of triplicate wells. Unstimulated cells showed 0 2 cells per 2.5 105 PBMC. PBMC from five donors were cultured with peptides T1 or T6

for 1314 days. These short-term lines (D), or positively selected CD4 (E) or CD8 cells (F) from these lines, were used as effectors. Targets were

autologous dendritic cells, either unpulsed or pulsed with relevant peptides. Values shown are the means and SEM of triplicate wells.


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response by clone B9. Cells expressing DRB1*0401 induced the

strongest response in both clones.

DiscussionBased on data using purified CD4 and CD8 primary T cells, T

cell lines, and T cell clones, we demonstrated that a 15-mer peptide

in the secreted mycobacterial protein CFP10 elicits IFN-produc-

tion and CTL activity by both CD4 and CD8 T cells from a high

proportion of persons with latent tuberculosis infection.

CFP107185 was recognized by CD4

and CD8 T cells from

persons expressing multiple MHC class II and class I molecules,

respectively (Table I), and contains at least two epitopes, one of 10

aa (peptide T1) and another of 9 aa (peptide T6). T1 was recog-

nized by CD4 cells in the context of at least DRB1*0401,

DRB5*0101, and DQB1*0302, and by CD8 cells of A2 donors

(Tables VI and VII, and Fig. 6). T6 elicited responses by CD4

FIGURE 5. CTL activity of effector cells stimulated with peptides T1 and T6 against M. tuberculosis-infected target cells. PBMC from three donors were

cultured with peptides T1 (A, C, and E) or T6 (B, D, and F) for 14 days. These short-term lines, and positively selected CD4 or CD8 cells from these

lines, were used as effectors. Targets were autologous dendritic cells, either uninfected or infected with H37Rv. Means and SEM of triplicate wells are

shown. Effectors used in A and D are peptide-stimulated PBMC, effectors in B and E are CD4 cells, and those in C and F are CD8 cells.


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cells in the context ofDRB1*0401 and DQB1*0302, and by CD8

cells ofB35 donors (Tables VI and VII). Deleting a single amino

acid from the amino or carboxy terminus of either peptide mark-

edly reduced IFN- production, suggesting that they are minimal

epitopes for both CD4 and CD8 cells. As far as we are aware,

these are the shortest microbial peptides that are known to stimu-

late responses by both T cell subpopulations. The capacity of

CFP107185 to stimulate IFN- production and CTL activity by

CD4 and CD8 cells from persons expressing a spectrum of

MHC molecules suggests that this peptide is an excellent candidate

for inclusion in an antituberculosis vaccine.

CD4

and CD8

T cells play complementary roles in protectiveimmunity to many intracellular pathogens, including M. tubercu-

losis. CD4 cells are the major source of the macrophage-activat-

ing factor IFN-, whereas CD8 cells predominate in lysing in-

fected cells (28). CD4 cells also enhance the CD8 cell response

to Ag through interactions between CD40L on the surface of

CD4 cells and CD40 on APCs and on CD8 cells (30 32), and

we have recently shown that the CD40/CD40L pathway con

tributes significantly to the human CD8 T cell response to M.

tuberculosis (33). To maximize the protective immune response, it

is theoretically appealing to vaccinate with peptides that contain

epitopes for both CD4 and CD8 T cells. Such peptides can be

presented by the same APC to both T cell subpopulations, and their

close physical proximity may favor CD40/CD40L interactions andcytokine effects that enhance CD8 cell effector function. Admin-

istration of a peptide containing a CTL epitope of HIV fused to a

Th epitope yielded increased CTL responses (34), and vaccination

with a 35-mer peptide containing both a CTL and a Th epitope of

human papillomavirus completely eradicated papilloma virus-ex-

pressing tumors in a murine model (35).

Although epitopes for CD4 cells and those for CD8 cells can

be fused to create chimeric peptides, naturally occurring peptides

recognized by CD4 and CD8 cells may elicit more effective

immunity because they are more likely to undergo appropriate Ag

processing. Fused peptides can also create junctional epitopes that

inhibit the immune response to the desired epitopes (36). An

epitope comprising 15 aa capable of binding to both MHC class I

and class II molecules in a murine model has been identified for

HIV (37) and CD8 epitopes within CD4 epitopes are present in

Plasmodium falciparum (38). CD8 epitopes of P. falciparum that

were nested within CD4 epitopes were more antigenic for humans

than other CD8 epitopes, supporting the enhanced immunogenicity

of peptides that stimulate both classes of T cells.

Peptides within the M. tuberculosis proteins Ag 85B, ESAT-6,

mce2, and the 16-kDa proteins MPB70 and -crystallin are rec-

ognized by T cells from persons expressing more than one MHC

class II haplotype (39 44). In most of these studies, peptides of

16 25 aa were studied, minimal epitopes were not delineated by

peptide truncation, or responses of purified CD4 cells were not

tested (39 43). Therefore, these peptides may contain more than

one CD4 epitope, or a CD4 and CD8 epitope, rather than a singlepromiscuous CD4 epitope. Valle and colleagues identified a 12-aa

peptide of Ag85 that elicited proliferation by PBMC from 89% of

healthy tuberculin reactors (44). However, because a proliferative

response was defined as only 2-fold that of background levels, and

MHC typing of the donors was not performed, it is uncertain

whether this peptide is truly promiscuous. The current study pro-

vides the most definitive evidence to date that M. tuberculosis

peptides of only 9 10 aa can be recognized by persons expressing

multiple MHC class II alleles. These peptides are shorter than the

1316 aa peptides that have generally been found to bind MHC

class II molecules.

Anti-MHC class I reduced the number of peptide T1- and T6-

responsive IFN-

cells by

50%, whereas anti-MHC class IIalmost completely abrogated the response (Table V). These results

suggest that MHC class I-restricted CD8 T cells contribute sig-

nificantly to IFN- production induced by M. tuberculosis pep-

tides. However, this response depends on the presence of CD4

cells. These findings extend the results of prior studies indicating

that the capacity of CD8 T cells to produce IFN- in response to

heat-killed M. tuberculosis requires CD4 cells, probably through

CD40/CD40L interactions (33, 45).

CFP10 is recognized by T cells from the majority of persons

with latent tuberculosis infection and by persons with active tu-

berculosis, including patients with HIV infection (20, 46, 47). The

carboxy end of the molecule is highly immunogenic, and peptides

7190 elicit responses by 30 50% of PBMC from CFP10-respon-

sive persons in Zambia and India (20, 46). The current results

confirm and extend these findings, demonstrating that CFP107185

Table VI. HLA typing results and responses of CD4 and CD8 T cells to peptide T1 and peptide T6a

Donor CD4 CTL/IFN- DRB1 DQB1 CD8 CTL/IFN- HLA-A HLA-B HLA-C

Peptide T1T294 /ND DRB1*04, *15 DQB1*03, *06 /ND A*24, *3108 B*15, *4406 Cw*01, *05

T283 /ND DRB1*04, *08 DQB1*03, *04 /ND A*24, *68 B*15, *40 Cw*01, *03T295 /ND DRB1*04,*04 DQB1*03,*03 /ND A*01,*02 B*40, *57 Cw*03, *06

T013 / DRB1*04, *08 DQB1*03, *04 / A*02, *24 B*15, *35 Cw*01, *04T215 / DRB1*04,*04 DQB1*03,*03 /ND A*02, *11 B*35, *40 Cw*03, *04

T221 / DRB1*04,*04 DQB1*03,*03 / A*02, *02 B*39, *40 Cw*03, *07T193 ND/ DRB1*04,*04 DQB1*03,*03 ND/ A*02, *68 B*35, *51 Cw*04, *08T234 ND/ DRB1*03, *13 DQB1*02,*03 ND/ A*24, *74 B*35, *41 Cw*04, *17T235 / DRB1*03, *13 DQB1*02,*03 / A*24, *74 B*35, *41 Cw*04, *17

Peptide T6

T294 /ND DRB1*04, *15 DQB1*03, *06 /ND A*24, *3108 B*15, *4406 Cw*01, *05T283 /ND DRB1*04, *08 DQB1*03, *04 /ND A*24, *68 B*15, *40 Cw*01, *03T295 /ND DRB1*04,*04 DQB1*03,*03 /ND A*01, *02 B*40, *57 Cw*03, *06

T013 / DRB1*04, *08 DQB1*03, *04 / A*02, *24 B*15,*35 Cw*01, *04T215 / DRB1*04,*04 DQB1*03,*03 /ND A*02, *11 B*35, *40 Cw*03, *04

T221 / DRB1*04,*04 DQB1*03,*03 / A*02, *02 B*39, *40 Cw*03, *07T193 ND/ DRB1*04,*04 DQB1*03,*03 ND/ A*02, *68 B*35, *51 Cw*04, *08T234 ND/ DRB1*03, *13 DQB1*02,*03 ND/ A*24, *74 B*35, *41 Cw*04, *17

T235 / DRB1*03, *13 DQB1*02,*03 / A*24, *74 B*35, *41 Cw*04, *17

a Boxes show HLA class I and class II alleles that appeared to be associated with either CTL activity or IFN- production by CD8 and CD4 cells, respectively.


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contains at least two epitopes for CD4 T cells, and is recognized

in the context of DRB1*0401, DRB5*0101, and DQB1*0302 (Ta-

bles VI and VII, and Fig. 6). The responsiveness of CD4 T cells

from subject T225 to the CFP10 peptides in the absence of

DRB1*04 or DQB1*03 (Table I) may be due to the expression of

DRB5*0101, which is linked to the DRB1*1501 allele in HLA-

DR2 subjects. Peptide T1 contains isoleucine at position 1, ala-

nine at position 4, and valine at position 6, conforming to the motif

predicting strong binding to HLAB1*0401, which is the most com-

mon subtype of HLAB1*04 in the United States (48). In contrast,

peptide T6 shows no features of this motif. Because some donorswhose CD4 T cells produced IFN- in response to CFP10

7185

expressed other MHC class II alleles (Table I, and data not shown),

peptides T1 and T6, or other epitopes on CFP107185, are likely to

be presented by additional class II molecules. Our findings are

consistent with previous work demonstrating that certain peptides

can bind to at least seven common DR types, including

DRB1*0401 (49).

Previous work has shown that CFP108594 and CFP10211 are

HLA-B14- and HLA-B44-restricted epitopes, respectively, for hu-

man CD8 T cell clones (24). We found that CFP107185 contains

at least two epitopes for CD8 T cells, one recognized by persons

expressing HLA-A*02 and the other by persons expressing HLA-

B*35 (Table VI). CD8

T cells from persons expressing otherMHC class I alleles may also recognize these epitopes, as formal

restriction analysis with cells expressing a single allele was not

performed. HLA-A*02 is part of the HLA-A2 supertype, which is

expressed by 39 46% of Caucasians, North American Blacks,

Hispanics, and Asians (50). HLA-B*35 is part of the HLA-B7

supertype, which is expressed by 4357% of these ethnic groups.

Therefore, CFP107185 is likely to be recognized by CD8

T cells

from the majority of people in different populations throughout the

world.

The capacity of CFP107185 to elicit IFN-production and CTL

activity by CD4 and CD8 T cells from persons bearing multiple

MHC class I and class II alleles makes it an intriguing candidate

for inclusion in an antituberculosis vaccine. DNA vaccines encod-

ing short peptides or peptide-based vaccines are attractive because

they are substantially easier to produce than vaccines based on

whole proteins. In addition, epitopes in proteins that elicit sup-

pressive or immunopathogenic responses can be avoided. Peptides

such as CFP107185, perhaps in combination with other immuno-

dominant M. tuberculosis peptides, may also be useful to develop

a diagnostic test for latent tuberculosis infection, based on an

ELISPOT assay that detects IFN--producing cells. However, a

vaccine that includes CFP107185 would limit the clinical utility of

CFP107185-based diagnostic tests in the vaccinated population.

These potentially contrasting roles will need to be reconciled in the

future.

FIGURE 6. Peptide T1 is recognized by T cell clones in the context of

different HLA class II alleles. T1-responsive clones B9 and F10 were gen-

erated from a donor whose MHC class II alleles were DQB1*03,

DQB1*02, DRB1*04, and DRB1*07. Thirty-five percent and 58% of B9

and F10 cells produced IFN- in response to T1-pulsed autologous mac-

rophages, respectively. Clones were incubated with BLS cells lacking en-

dogenous MHC class II alleles, and with BLS cells expressing single HLA

class II alleles, in the presence or absence of peptide at concentrations of

10, 1, and 0.1 g/ml. Clones without BLS cells were also used as controls.

A total of 25,000 BLS cells and 100 clones were placed into each well in

duplicate, and the number of IFN--producing cells was enumerated after

16 h. Values shown are the mean of duplicate results obtained with a

peptide concentration of 1 g/ml. A total of 10 g/ml peptide yieldedessentially identical results. The number of IFN- cells was reduced by

90% when the peptide concentration was 0.1 g/ml.

Table VII. Presentation of peptides T1 and T6 by APC expressing a single HLA allelea

Donors

CFP10

response

Donors HLA

Type Peptide T1 Peptide T6

DR DQ

DRB1*0401

peptide

CD4

DRB1*0401

CD4

DRB1*0401

peptide

DQB1*0302

peptide

CD4

DQB1*0302

CD4

DQB1*0302

peptide

DRB1*0401

peptide

CD4

DRB1*0401

CD4

DRB1*0401

peptide

DQB1*0302

peptide

CD4

DQB1*0302

CD4

DQB1*0302

peptide

T040 B1*04 B1*03

80 8 3 45 5 0 113 20 3 110 8 0B1*04 B1*03

T262 B1*04 B1*03

78 5 3 70 8 0 85 23 3 80 5 0 B1*08 B1*04

T283 B1*04 B1*03

58 18 0 40 0 0 70 13 0 58 8 0 B1*08 B1*04

T294 B1*04 B1*03

68 10 3 65 10 0 120 10 3 55 5 0 B1*15 B1*06

T030 B1*04 B1*03

7 8 0 2 3 0 5 8 0 8 5 0 B1*13 B1*06

T062 B1*01 B1*05

19 14 0 4 7 0 22 16 0 5 5 0 B1*13 B1*06

T185 B1*03 B1*02

3 5 0 2 7 0 4 1 0 5 6 0 B1*04 B1*03

T281 B1*01 B1*05

12 18 0 2 4 0 18 12 0 2 4 0 B1*13 B1*05

a BLS cells expressing DRB1*0401 or DQB1*0302 were used as target cells, either unpulsed or pulsed with peptides T1 or T6. Purified CD4 T cells from freshly isolated

PBMC from four donors were added to target cells, and the number of IFN-

cells was measured by ELISPOT. Values shown are the mean number of IFN-

cells per 2.5

105 cells, based on duplicate wells.


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AcknowledgmentsWe are grateful to Mabtech and Staffan Paulie for providing us with pre-

coated IFN-ELISPOT plates, and to Ortho Biotech for provision of anti-

CD3 mAb. We thank Sharon Kochik for excellent technical assistance in

handling BLS cells and transfectants.

References1. Corbett, E. L., C. J. Watt, N. Walker, D. Maher, B. G. Williams,

M. C. Raviglione, and C. Dye. 2003. The growing burden of tuberculosis: global

trends and interactions with the HIV epidemic. Arch. Intern. Med. 163:1009.

2. Toungoussova, O. S., P. Sandven, A. O. Mariandyshev, N. I. Nizovtseva,

G. Bjune, and D. A. Caugant. 2002. Spread of drug-resistant Mycobacterium

tuberculosis strains of the Beijing genotype in the Archangel oblast, Russia, in

1999. J. Clin. Microbiol. 40:1930.

3. Tracevska, T., I. Jansone, V. Baumanis, O. Marga, and T. Lillebaek. 2003. Prev-

alence of Beijing genotype in Latvian multidrug-resistant Mycobacterium tuber-

culosis isolates. Int. J. Tuberc. Lung Dis. 7:1097.

4. Sterling, T. R., W. T. Brehm, R. D. Moore, and R. E. Chaisson. 1999. Tubercu-

losis vaccination versus isoniazid preventive therapy: a decision analysis to de-

termine the preferred strategy of tuberculosis prevention in HIV-infected adults

in the developing world. Int. J. Tuberc. Lung Dis. 3:248.

5. Kaufmann, S. H. E. 2001. How can immunology contribute to the control of

tuberculosis? Nat. Rev. Immunol. 1:20.

6. Tan, J. S., D. H. Canaday, W. H. Boom, K. N. Balaji, S. K. Schwander, and

E. A. Rich. 1997. Human alveolar T lymphocyte responses to Mycobacteriumtuberculosis antigens. J. Immunol. 159:290.

7. Stenger, S., D. A. Hanson, R. Teitelbaum, P. Dewan, K. R. Niazi, C. J. Froelich,

T. Ganz, S. Thoma-Uszynski, A. Melian, C. Bogdan, et al. 1998. An antimicro-

bial activity of cytolytic T cells mediated by granulysin. Science 282:121.

8. Smith, S. M., 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. 2000. Human CD8 CTL specificfor the mycobacterial major secreted antigen 85A. J. Immunol. 165:7088.

9. Geluk, A., K. E. van Meijgaarden, K. L. M. C. Franken, J. W. Drijfhout, S.

DSouza, A. Necker, K. Huygen, and T. H. M. Ottenhoff. 2000. Identification ofmajor epitopes of Mycobacterium tuberculosis AG85B that are recognized by

HLA-A*0201-restricted CD8 T cells in HLA-transgenic mice and humans.

J. Immunol. 165:6463.

10. Canaday, D. H., R. J. Wilkinson, Q. Li, C. V. Harding, R. F. Silver, and

W. H. Boom. 2001. CD4 and CD8 T cells kill intracellular Mycobacterium

tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J. Immu-

nol. 167:2734.

11. Heinzel, A.S., J. E. Grotzke, R. A. Lines, D. A. Lewinsohn, A. L. McNabb,D. N. Streblow, V. M. Braud, H. J. Grieser, J. T. Belisle, and D. M. Lewinsohn.

HLA-E-dependent presentation of Mtb-derived antigen to human CD8 T cells.

J. Exp. Med. 196:1473.

12. Lazarevic, V., and J. Flynn. 2002. CD8 T cells in tuberculosis. Am. J. Respir.

Crit. Care Med. 166:1116.

13. Orme, I. M. 1988. Characteristics and specificity of acquired immunologic mem-ory to Mycobacterium tuberculosis infection. J. Immunol. 140:3589.

14. Renshaw, P. S., P. Panagiotidou, A. Whelan, S. V. Gordon, R. G. Hewinson,

R. A. Williamson, and M. D. Carr. 2002. Conclusive evidence that the major

T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10

form a tight, 1:1 complex and characterization of the structural properties of

ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex: implications for pathogen-

esis and virulence. J. Biol. Chem. 277:21598.

15. Berthet, F. X., P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel.

1998. A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-

molecular-mass culture filtrate protein (CFP-10). Microbiology 144:3195.

16. Pym, A. S., P. Brodin, R. Brosch, M. Huerre, and S. T. Cole. 2002. Loss of RD1contributed to the attenuation of the live tuberculosis vaccines Mycobacterium

bovis BCG and Mycobacterium microti. Mol. Microbiol. 46:709.

17. Arend, S. M., A. Geluk, K. E. van Meijgaarden, J. T. van Dissel, M. Theisen,

P. Andersen, and T. H. Ottenhoff. 2000. Antigenic equivalence of human T-cell

responses to Mycobacterium tuberculosis-specific RD1-encoded protein antigensESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides.

Infect. Immun. 68:3314.

18. Lalvani, A., R. Brookes, R. Wilkinson, A. Malin, A. Pathan, P. Andersen,

H. Dockrell, G. Pasvol, and A. Hill. 1998. Human cytolytic and interferon-se-

creting CD8 T lymphocytes specific for Mycobacterium tuberculosis. Proc.Natl. Acad. Sci. USA 95:270.

19. Brandt, L., T. Oettinger, A. Holm, A. B. Andersen, and P. Andersen. 1996. Key

epitopes on the ESAT-6 antigen recognized in mice during the recall of protective

immunity to Mycobacterium tuberculosis. J. Immunol. 157:3527.

20. Lalvani, A., P. Nagvenkar, Z. Udwadia, A. A. Pathan, K. A. Wilkinson,

J. S. Shastri, K. Ewer, A. V. Hill, A. Mehta, and C. Rodrigues. 2001. Enumer-

ation of T cells specific for RD1-encoded antigens suggests a high prevalence oflatent Mycobacterium tuberculosis infection in healthy urban Indians. J. Infect.

Dis. 183:469

.

21. Lalvani, A., A. A. Pathan, H. McShane, R. J. Wilkinson, M. Latif, C. P. Conlon,

G. Pasvol, and A. V. Hill. 2001. Rapid detection ofMycobacterium tuberculosis

infection by enumeration of antigen-specific T cells. Am. J. Respir. Crit. Care

Med. 163:824.

22. Pathan, A. A., K. A. Wilkinson, R. J. Wilkinson, M. Latif, H. McShane,

G. Pasvol, A. V. Hill, and A. Lalvani. 2000. High frequencies of circulating

IFN--secreting CD8 cytotoxic T cells specific for a novel MHC class I-restricted

Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without

disease. Eur. J. Immunol. 30:2713.

23. Pathan, A. A., K. A. Wilkinson, P. Klenerman, H. McShane, R. N. Davidson,

G. Pasvol, A. V. S. Hill, and A. Lalvani. 2001. Direct ex vivo analysis of antigen-

specific IFN--secreting CD4 T cells in Mycobacterium tuberculosis-infected

individuals: associations with clinical disease state and effect of treatment. J. Im-

munol. 167:5217.

24. Lewinsohn, D. M., L. Zhu, V. J. Madison, D. C. Dillon, S. P. Fling, S. G. Reed,

K. H. Grabstein, and M. R. Alderson. 2001. Classically restricted human CD8

T lymphocytes derived from Mycobacterium tuberculosis-infected cells: defini-

tion of antigenic specificity. J. Immunol. 166:439.

25. Kovats, S., G. T. Nepom, M. Coleman, B. Nepom, W. W. Kwok, and J. S. Blum.

1995. Deficient antigen-presenting cell function in multiple genetic complemen-tation groups of type II bare lymphocyte syndrome. J. Clin. Invest. 96:217.

26. Lewinsohn, D. M., M. R. Alderson, A. L. Briden, S. R. Riddell, S. G. Reed, and

K. H. Grabstein. 1998. Characterization of human CD8 T cells reactive with

Mycobacterium tuberculosis-infected antigen-presenting cells. J. Exp. Med.

187:1633.

27. Lewinsohn, D. M., A. L. Briden, S. G. Reed, K. H. Grabstein, and

M. R. Alderson. 2000. Mycobacterium tuberculosis-reactive CD8

T lympho-cytes: the relative contribution of classical versus nonclassical HLA restriction.

J. Immunol. 165:925.

28. Lewinsohn, D. A., A. S. Heinzel, J. M. Gardner, L. Zhu, M. R. Alderson, and

D. M. Lewinsohn. 2003. Mycobacterium tuberculosis-specific CD8T cells pref-erentially recognize heavily infected cells. Am. J. Respir. Crit. Care Med.

168:1346.

29. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, and S. Stevanovic.

1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunoge-

netics 50:213.

30. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, and

W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40

signalling. Nature 393:478.

31. Ridge, J. P., F. DiRosa, and P. Matzinger. 1998. A conditioned dendritic cell can

be a temporal bridge between a CD4 T-helper and a T-killer cell. Nature 393:

474.

32. Bourgeois, C., B. Rocha, and C. Tanchot. 2002. A role for CD40 expression on

CD8 T cells in the generation of CD8 T cell memory. Science 297:2060.

33. Samten, B., E. K. Thomas, J.-H. Gong, and P. F. Barnes. 2000. Depressed CD40

ligand expression contributes to reduced interferon production in human tu-

berculosis. Infect. Immun. 68:3002.

34. Daftarian, P., S. Ali, R. Sharan, S. F. Lacey, C. La Rosa, J. Longmate, C. Buck,

R. F. Siliciano, and D. J. Diamond. 2003. Immunization with Th-CTL fusion

peptide and cytosine-phosphate-guanine DNA in transgenic HLA-A2 mice in-

duces recognition of HIV-infected T cells and clears vaccinia virus challenge.

J. Immunol. 171:4028.

35. Zwaveling, S., S. C. Ferreira Mota, J. Nouta, M. Johnson, G. B. Lipford,

R. Offringa, S. H. van der Burg, and C. J. Melief. 2002. Established human

papillomavirus type 16-expressing tumors are effectively eradicated following

vaccination with long peptides. J. Immunol. 169:350.

36. Wang, Y., J. A. Smith, T. Kamradt, M. L. Gefter, and D. L. Perkins. 1992.

Silencing of immunodominant epitopes by contiguous sequences in complex syn-

thetic peptides. Cell. Immunol. 143:284.

37. Takahashi, H., R. N. Germain, B. Moss, and J. A. Berzofsky. 1990. An immu-nodominant class I-restricted cytotoxic T lymphocyte determinant of human im-

munodeficiency virus type 1 induces CD4 class II-restricted help for itself. J. Exp.

Med. 171:571.

38. Wang, R., J. Epstein, F. M. Baraceros, E. J. Gorak, Y. Charoenvit, D. J. Carucci,

R. C. Hedstrom, N. Rahardjo, T. Gay, P. Hobart, et al. 2001. Induction of CD4

T cell-dependent CD8 type 1 responses in humans by a malaria DNA vaccine.

Proc. Natl. Acad. Sci. USA 98:10817.

39. Al Attiyah, R., F. A. Shaban, H. G. Wiker, F. Oftung, and A. S. Mustafa. 2003.

Synthetic peptides identify promiscuous human Th1 cell epitopes of the secreted

mycobacterial antigen MPB70. Infect. Immun. 71:1953.

40. Caccamo, N., A. Barera, C. Di Sano, S. Meraviglia, J. Ivanyi, F. Hudecz,

S. Bosze, F. Dieli, and A. Salerno. 2003. Cytokine profile, HLA restriction andTCR sequence analysis of human CD4 T clones specific for an immunodomi-

nant epitope ofMycobacterium tuberculosis 16-kDa protein. Clin. Exp. Immunol.

133:260.

41. Mustafa, A. S., F. Oftung, H. A. Amoudy, N. M. Madi, A. T. Abal, F. Shaban,

K. Rosen, I., and P. Andersen. 2000. Multiple epitopes from the Mycobacteriumtuberculosis ESAT-6 antigen are recognized by antigen-specific human T celllines. Clin. Infect. Dis. 30(Suppl. 3):S201.


y,

j

g


13/13

42. Mustafa, A. S., F. A. Shaban, R. Al Attiyah, A. T. Abal, A. M. El Shamy,P. Andersen, and F. Oftung. 2003. Human Th1 cell lines recognize the Myco-bacterium tuberculosis ESAT-6 antigen and its peptides in association with fre-

quently expressed HLA class II molecules. Scand. J. Immunol. 57:125.

43. Panigada, M., T. Sturniolo, G. Besozzi, M. G. Boccieri, F. Sinigaglia,G. G. Grassi, and F. Grassi. 2002. Identification of a promiscuous T-cell epitopein Mycobacterium tuberculosis Mce proteins. Infect. Immun. 70:79.

44. Valle, M. T., A. M. Megiovanni, A. Merlo, P. G. Li, L. Bottone, G. Angelini,L. Bracci, L. Lozzi, K. Huygen, and F. Manca. 2001. Epitope focus, clonal com-

position and Th1 phenotype of the human CD4 response to the secretory myco-

bacterial antigen Ag85. Clin. Exp. Immunol. 123:226.45. Shams, H., B. Wizel, S. E. Weis, B. Samten, and P. F. Barnes. 2001. Contribution

of CD8 T cells to interferon production in human tuberculosis. Infect. Immun.

69:3497.46. Chapman, A. L., M. Munkanta, K. A. Wilkinson, A. A. Pathan, K. Ewer,

H. Ayles, W. H. Reece, A. Mwinga, P. Godfrey-Faussett, and A. Lalvani. 2002.

Rapid detection of active and latent tuberculosis infection in HIV-positive indi-viduals by enumeration of Mycobacterium tuberculosis-specific T cells. AIDS16:2285.

47. Skjot, R. L., T. Oettinger, I. Rosenkrands, P. Ravn, I. Brock, S. Jacobsen, and

P. Andersen. 2000. Comparative evaluation of low-molecular-mass proteins from

Mycobacterium tuberculosis identifies members of the ESAT-6 family as immu-nodominant T-cell antigens. Infect. Immun. 68:214.

48. Livingston, B., C. Crimi, M. Newman, Y. Higashimoto, E. Appella, J. Sidney,and A. Sette. 2002. A rational strategy to design multiepitope immunogens based

on multiple Th lymphocyte epitopes. J. Immunol. 168:5499.

49. Southwood, S., J. Sidney, A. Kondo, M. F. del Guercio, E. Appella, S. Hoffman,R. T. Kubo, R. W. Chesnut, H. M. Grey, and A. Sette. 1998. Several common

HLA-DR types share largely overlapping peptide binding repertoires. J. Immu-

nol. 160:3363.50. Sette, A., and J. Sidney. 1999. Nine major HLA class I supertypes account for the

vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50:201.


y,

j

g

characterization of a mycobacterium tuberculosis peptide that is recognized by human cd4+ and cd8+ t...

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