Human Dendritic Cells Presenting Adenovirally Expressed Antigen ...

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Human Dendritic Cells Presenting Adenovirally Expressed Antigen Elicit Mycobacterium tuberculosis–Specific CD8 T Cells Deborah A. Lewinsohn, Rebecca A. Lines, and David M. Lewinsohn Division of Pediatric Infectious Diseases, Department of Molecular Microbiology and Immunology; and Division of Pulmonary and Critical Care Medicine, Oregon Health and Science University/Portland VA Medical Center, Portland, Oregon Previous studies in murine and human models have suggested an important role for CD8 T cells in host defense to Mycobacterium tuberculosis (Mtb). Consequently, a successful tuberculosis vaccine may require the elicitation of sustained CD4 and CD8 T cell responses. We tested the hypothesis that the potent CD4 T cell antigen Mtb39 is also a CD8 T cell antigen. A recombinant adenovirus–expressing Mtb39 (adenoMtb39) was used to infect monocyte-derived dendritic cells. Using interferon- enzyme-linked immunospot, Mtb39-specific CD8 T lymphocytes were detected in three healthy individuals with latent tuberculosis infection who also had strong anti–Mtb39-specific CD4 T cell responses. An Mtb39-specific CD8 T cell line was generated using Mtb39-expressing dendritic cells. Mtb39-specific T cell clones were obtained by limiting dilution cloning. All seven T cell clones obtained were HLA-B44 restricted. Using a panel of synthetic overlapping peptides representative of Mtb39, the peptide epitope was identified for two clones. Furthermore, all T cell clones recognized Mtb-infected dendritic cells and were cytolytic. We conclude that infection of dendritic cells with adenoviral vectors expressing Mtb proteins allows for measurement of antigen-specific CD8 T cell responses from peripheral blood mononuclear cells. The technique will be useful in defining CD8 T cell antigens and in measuring immunogenicity of tuberculosis vaccines. Keywords: CD8-positive T lymphocytes; dendritic cells; tuberculosis

It is estimated that a third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Consequently, tuberculosis is a leading cause of infectious mortality worldwide, accounting for over 8 million new cases and 2.9 million deaths annually (1). Mtb is an intracellular pathogen, and thus the control of infection relies on a coordinated cellular immune response. In the tuberculosis literature, there is abundant evidence to support an essential role for CD4 T cell–mediated immunity (2, 3). Several lines of evidence suggest, however, that CD8 cytotoxic T lymphocytes play a unique role as well. Major histocompatibility complex (MHC) class I–deficient, and thus CD8 T cell–deficient, mice in which the gene for 2-microglobulin has been disrupted are more susceptible to Mtb infec-

(Received in original form October 26, 2001; accepted in final form April 23, 2002) This study was supported by NIH 1KO8AI01645-03 (D.A.L.), NIH 1K08AI01644 (D.M.L.), an American Lung Association Research Grant (D.M.L.), a Medical Research Foundation Research Grant (D.M.L.), and Corixa Corporation. Dr. D. A. Lewinsohn was supported in part as a Junior Investigator of the Oregon Child Health Research Center by NIH NICHHD HD33703. The Portland VA Medical Center has provided laboratory space and partial salary support (D.M.L.). Correspondence and requests for reprints should be addressed to Dr. David Lewinsohn, R&D 11, Portland VA Medical Center, 3710 SW US Veterans Road, Portland, OR 97201. E-mail: [email protected] This article has an online data supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org Am J Respir Crit Care Med Vol 166. pp 843–848, 2002 DOI: 10.1164/rccm.2110094 Internet address: www.atsjournals.org

tion than their wild-type littermates (4) as are mice deficient in transporter associated with antigen processing (5). In 1994, Silva and coworkers found that CD8 cytotoxic T lymphocyte clones generated to the Mtb heat shock protein (hsp 65) could confer partial immunity to Mtb infection in mice (6). Immunization of mice with plasmids expressing Mtb antigens such as hsp 65 (7), Ag85a (8), or the 38-kD (9) antigen has resulted in protection from subsequent challenge with Mtb and has been associated with the generation of antigen-specific CD8 cytotoxic T lymphocytes. Finally, CD8 T cells have been shown to localize preferentially to the mouse lung following infection with Mtb (10, 11). In humans, there is increasing evidence to suggest that CD8 T cells are elicited in response to infection with mycobacteria. Using peptides of predicted HLA-binding specificity (HLA-B52 and HLA-A*0201), Lalvani and colleagues were able to elicit CD8 T cells capable of recognizing ESAT-6– expressing targets (12). Similarly, predicted binding peptides for the 19-kD antigen, restricted by HLA-B44 (13), have been used to elicit CD8 T cells that are reactive with Mtb-infected dendritic cells in an HLA-Ia–restricted manner. Finally, Cho and coworkers described three HLA-A*0201-restricted epitopes for which CD8 T cell lines mediated an antimycobacterial effect in vitro (14). One limitation of these peptide-based approaches is that it is difficult to ascertain whether these responses are primed by mycobacterial infection or represent low-affinity crossreactivity with another antigen. Similarly, it remains uncertain whether the peptides tested reflect dominant epitopes generated during the course of natural infection. Recently, we have used human CD8 T cell clones elicited by Mtb-infected dendritic cells to define CFP10/Mtb11 as a strong CD8 T cell antigen in a person latently infected with Mtb. However, the question as to whether or not antigens recognized by CD4 T lymphocytes are also recognized by CD8 T lymphocytes remains an important and unresolved question. In this report, we have used a replication-deficient adenovirus to introduce a potent CD4 antigen, Mtb39 (15), into the cytosol of human peripheral blood dendritic cells and to use these professional antigen-presenting cells to elicit Mtb39-specific CD8 T cell responses.

METHODS Human Subjects Subjects were recruited from employees at Harborview Medical Center, The Fred Hutchinson Cancer Research Center, Corixa Corporation, and Oregon Health and Science University as previously described (16). Human subjects protocols were approved by the institutional review boards of these institutions.

Monoclonal Antibodies and Reagents Medium for T cell culture and assays was RPMI supplemented with Hepes (25 mM), L-glutamine (4 mM), gentamicin (5 g/ml), and 11% human serum. Interleukin-2 (0.5 ng/ml; Chiron; Emeryville, CA) was added to all T cell assays. Mycobacterial strain H37Rv was obtained

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from the American Type Culture Collection (Rockville, MD) and prepared as previously described (16). Construction of adenoviral vectors and recombinant vaccinia viruses expressing Mtb39 and enhanced green fluorescent protein (EGFP) is described in the online data supplement. Synthetic peptides representative of Mtb39 were kindly provided by Corixa Corporation (Seattle, WA).

Interferon- ELISPOT and Cytotoxicity Assays The interferon- (IFN-) enzyme-linked immunospot (ELISPOT) has been previously described (16, 17). Target cell membrane damage was assessed using a standard 4-hour 51Cr-release assay. Percent specific lysis was calculated as previously described (18).

Generation and Infection of Peripheral Blood Dendritic Cells and Macrophages Monocyte-derived dendritic cells were prepared according to a modified method of Romani and coworkers (19, 20). To generate macrophages, peripheral blood mononuclear cells (PBMC) were adhered to a T-75 flask and cultured in the absence of cytokine. To generate Mtbor vaccinia-infected antigen presenting cells, monocyte-derived dendritic cells or macrophages (1  106) were cultured overnight in the presence of Mtb (multiplicity of infection [MOI]  50:1) or vaccinia recombinant (MOI  10:1), respectively, in low-adherence 16-mm wells (Costar No. 3,473). After 18 hours, the cells were harvested and resuspended in RPMI/10% HS. Adenovirus infection and the analysis of protein expression in antigen-presenting cells are described in the online data supplement.

Purification of CD4 and CD8 T Cell Subsets CD4 and CD8 T cell subsets were purified using CD4 and CD8 microbeads per the manufacturer’s instructions (Miltenyi Biotec Inc., Auburn, CA). For CD8 T cell purification, PBMC were twice depleted of CD4 T lymphocytes and then positively selected for CD8 T lymphocytes. CD4 T lymphocytes constituted less than 0.2% of the CD8 T cell subset.

Generation of Mtb39-Specific T Cell Lines Adenovirus-infected, monocyte-derived dendritic cells (1  105) were cultured with CD8 T cells (1  106). T cells were restimulated with fresh, adenovirally infected macrophages on Day 7. Two days later, cells were harvested and assessed for their ability to generate IFN- in response to lymphoblastoid cell line (LCL) targets infected with either vaccinia Mtb39 (vMtb39) or EGFP (vEGFP). Specific release of IFN- was assessed by ELISPOT.

Generation and Expansion of Mtb39-Specific CD8  T Cell Clones T cells were cloned from an Mtb39-specific CD8 T cell line by limiting dilution as described previously (20). Evaluation of the specificity of T cell clones for Mtb39 was performed as follows. LCL infected with either vMtb39 or vEGFP were prepared and seeded at 2  104 cells per well in RPMI/10% HS in ELISPOT plates coated with IFN- mAb. Aliquots of each clone (25 l) were then added and the ELISPOT assay completed after 18 hours incubation at 37C. To expand the CD8 T cell clones, a rapid expansion protocol using antiCD3 monoclonal antibody stimulation was used (21).

Figure 1. Expression of adenoviral gene products in human dendritic cells. (A) Flow cytometric analysis of EGFP expression in adenoEGFP-infected dendritic cells. The left, short curve represents adenoMtb39-infected dendritic cells, whereas the long curve represents adenoEGFP-infected dendritic cells. (B, C) Expression of Mtb39 in adenoMtb39-infected dendritic cells was determined using a Mtb39-specific CD4 T cell clone, TbH9-9. B shows proliferation of TbH9-9 (squares) in response to various concentrations of recombinant Mtb39 (positive control). C shows proliferation of TbH9-9 (squares) to increasing amounts of antigen preparation from adenoMtb39-infected dendritic cells, compared with that of EGFP-infected dendritic cells (negative control; circles).

dritic cells infected with adenoMtb39 or adenoEGFP and lysed by repeated freeze–thaw. Using a lymphoproliferative assay, these antigen preparations were compared with recombinant Mtb39 protein for the ability to stimulate proliferation of TbH9-9. As a positive control, T cell clone proliferation to recombinant Mtb39 was assessed and was linearly correlated with protein concentration (Figure 1B). TbH9-9 proliferated in the presence of antigen prepared from adenoMtb39-infected but not adenoEGFP-infected dendritic cells, demonstrating that dendritic cells infected with adenoMtb39 express immunologically relevant amounts of Mtb39 (Figure 1C). Mtb39-Specific CD8 Effector T Cells Can be Detected in Healthy Individuals with Latent Mtb Infection Who Have Primed Mtb39-Specific CD4 T Cell Responses

We defined individuals with latent Mtb infection as those with a positive tuberculin skin test (TST), without radiographic abnormalities consistent with tuberculosis and without history of bacillus Calmette-Guérin (BCG) vaccination. We hypothesized that these individuals, who had primed CD4 T cell responses

RESULTS Expression of Adenovirally Encoded Proteins in Human Peripheral Blood–derived Dendritic Cells

First, to evaluate the expression of EGFP in human peripheral blood–derived dendritic cells infected with adenovirus encoding EGFP (adenoEGFP), protein expression was assessed by flow cytometric analysis. As shown in Figure 1A, roughly 80% of dendritic cells infected with adenoEGFP at an MOI of 50:1 expressed EGFP. Second, to confirm the expression of Mtb39 in adenoMtb39-infected dendritic cells, a CD4 T cell clone specific for Mtb39 (TbH9-9; epitope Mtb39133–147; [15, 22]) was employed. In this experiment, antigen was prepared from den-

Figure 2. Healthy individuals with latent Mtb infection have CD8 T cell responses specific for Mtb39. A panel of three donors was chosen based on strong Mtb39-specific CD4 T cell responses. Using IFN- ELISPOT analysis, donor PBMC-derived CD8 T cells were assessed for reactivity against autologous dendritic cells infected with either adenoMtb39 or adenoEGFP. Each point represents the mean of duplicate determinations. Effector cell frequency was estimated using linear regression analysis. Squares, DC-EGFP; circles, DC-TbH9.

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teins, the line was restimulated with vMtb39-infected adherent macrophages. After 3 days, the line was tested for Mtb39 reactivity using IFN- ELISPOT for recognition of autologous LCL infected with either vMtb39 or negative control (vEGFP). As shown in Figure 3A, recognition of the Mtb39-expressing but not EGFP-expressing target cells was observed. Roughly 1 in 50 of the cells in this line were able to release IFN- in response to Mtb39-expressing LCL. To generate Mtb39-specific T cell clones, limiting dilution cloning using anti-CD3 mAb was employed. One hundred ninety-two wells exhibiting growth were screened for Mtb39 specificity. Cells from individual wells were tested for reactivity against either vMtb39- or control vEGFP–infected LCL using IFN- ELISPOT (Figure 3B, [16]). Nineteen Mtb39-specific clones were identified and expanded using anti-CD3 mAb. Of these, seven could be repeatedly expanded without loss of reactivity or specificity, and all seven were determined to be 100% CD8 by flow cytometric analysis (data not shown). These seven clones form the basis for the remainder of this report. All Seven Mtb39-Specific CD8 T Cell Clones Are HLA-B44 Restricted

Figure 3. Generation of Mtb39-specific CD8 T cell lines and clones. (A) PBMC-derived, purified CD8 T cells were cultured with Mtb39expressing antigen-presenting cells and after 10 days assessed for reactivity against vMtb39- or vEGFP-infected autologous LCL target cells using IFN- ELISPOT. Squares, TbH9 LCL; circles, EGFP LCL. (B) Limiting dilution cloning from an Mtb39-specific CD8 T cell line was performed using anti-CD3 mAb, and wells exhibiting growth were screened via IFN- ELISPOT against either vMtb39- or vEGFP-infected autologous LCL target cells. Positive wells are dark and negative wells are light. Mtb39 specificity is defined by reactivity against vMtb39-infected LCL (positive) but not against vEGFP-infected LCL (negative). Examples of Mtb39-specific clones are illustrated in wells d5 and h2.

directed toward Mtb39, would also have primed Mtb39-specific CD8 T cell responses. Three donors were selected who had vigorous CD4 T cell responses (15). Dendritic cells were generated from each donor and were infected with either adenoMtb39 or adenoEGFP. Expression of adenovirally encoded proteins was confirmed for each donor as described previously (data not shown). Donor PBMC-derived CD8 lymphocytes were tested for their ability to recognize adenovirusinfected autologous dendritic cells using an IFN- ELISPOT assay. All three donors had demonstrable IFN-–secreting CD8 T cells specific for Mtb39 with effector cell frequencies ranging from 1:3,000 (D131) to 1:15,000 (D160, Figure 2). In contrast, a parallel analysis performed with PBMC from three Mtb-uninfected (TST-negative; low risk) individuals demonstrated no Mtb39-specific CD8 T cells (effector cell frequency 1:50,000; data not shown). Generation of Mtb39-Specific CD8  T Cell Lines and Clones from an Individual with Latent Mtb Infection

To confirm these results, we isolated Mtb39-specific CD8 T cell lines and clones from an individual with latent Mtb infection. Donor 131 was chosen because of the vigorous response that had been observed in the peripheral blood (Figure 2). To generate an Mtb39-specific CD8 line, adenoMtb39-infected dendritic cells were used to stimulate PBMC-derived, purified CD8 T cells. To avoid repeated exposure to adenovirus pro-

To determine the HLA-restriction allele for the T cell clones, each was tested against either autologous (D131; HLA A11, B38, B44) or single-locus HLA-matched LCL that were infected with vMtb39 or negative control (vEGFP). All seven clones responded only to autologous or HLA-B44 matched LCL infected with vMtb39, indicating that all seven clones are HLA-B44 restricted. To define the peptide epitope recognized by each clone, a panel of synthetic overlapping peptides (20 amino acids in length, overlapping by 10 amino acids), representative of the entire Mtb39 protein, was used. T cell clones were tested for reactivity against autologous LCL pulsed with individual peptides (10 g/ml) using IFN- ELISPOT. Using this approach, two clones, 3-D5 and 10-E12 strongly recognized LCL pulsed with peptide 15 (Mtb39141–160) and did not recognize any other peptide-pulsed LCL. Clone 10-G4 weakly recognized LCL pulsed with peptide 35 (Mtb39341–360) and did not recognize any other peptide-pulsed LCL. The four remaining clones recognized positive control, vMtb39-infected LCL, but failed to recognize any peptide-loaded LCL. Figure 4 demonstrates the HLA restriction and broad peptide mapping for two clones, 3-D5 and 3-H2.

Figure 4. Determination of HLA restriction and broad epitope mapping of Mtb39-specific CD8 T cell clones. Mtb39-specific CD8 T cell clones, 3-D5 (solid bars) and 3-H2 (open bars), were tested in an IFN- ELISPOT assay for reactivity against a panel of LCL targets. D131Mtb39  autologous LCL infected with vMtb39. D131-control  autologous LCL infected with negative control, vEGFP. HLA-B44  LCL matched only for B44, infected with vMtb39. HLA-B38  LCL matched only for B38, infected with vMtb39. All peptide targets are autologous LCL pulsed with individual peptides (10 g/ml) from a panel of synthetic overlapping peptides (20 amino acids in length, overlapping by 10 amino acids). Both clones are restricted by HLAB44. Clone 3-D5 recognizes an epitope represented by peptide 15.

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Figure 5. Definition of the minimal peptide epitope recognized by Mtb39-specific CD8 T cell clones. (A) The amino acid sequence of peptide 15 and of the complete set of 10-mer peptides overlapping by nine amino acids for peptide 15 are shown on the right-hand side of the figure. Reactivity of clones 3-D5 and 10-E12 against autologous LCL target cells displaying individual peptides (10 g/ml), assessed by IFN- ELISPOT is shown. Clones were tested in duplicate against individual peptide-loaded LCL targets. Dark wells represent positive responses, and light wells represent negative responses. Both clones recognize only targets displaying peptide 15, YGEMWAQDAAAMFGYAAATA, and peptide 15(4), MWAQDAAAMF. The 9-mer peptide, MWAQDAAAM, is not recognized (data not shown). (B) Serial dilutions of peptides 15 (circles), 15(4) (triangles), and control peptide (squares), Mtb39133–147, AIAVNEAEYGEMWAQ, were used to sensitize autologous LCL targets for recognition by clones 3-D5 and 10-E12 in an IFN- ELISPOT assay. As little as 2  10 8 g/ml of peptide 15(4) can sensitize LCL targets for T cell recognition.

Definition of Minimal Peptide Epitopes for Three Mtb39-Specific CD8 T Cell Clones

To define the minimal peptide epitope for clones 3-D5 and 10E12, peptides nine amino acids in length, overlapping by eight amino acids, representing the 20-mer, peptide 15, were synthesized. None of these peptides were capable of sensitizing autologous LCL targets for T cell recognition. Therefore, peptides 10 amino acids in length, overlapping by 9 amino acids, were synthesized. As shown in Figure 5A, both peptide 15– specific clones recognized only autologous LCL pulsed with peptide 15(4), Mtb39144–153. To confirm that this peptide represented the minimal epitope recognized by these clones, 3-D5 and 10-E12 were tested against autologous LCL that had been incubated with serial dilutions of peptide. As shown in Figure 5B, clones 3-D5 and 10-E12 released IFN- in response to LCL targets sensitized with as little as 2  10 8 g/ml of the peptide 15(4), indicating that peptide 15(4), Mtb39144–153, represents the minimal epitope, MWAQDAAAMF, recognized by these T cell clones. To more finely map the epitope recognized by the peptide 35–specific clone, 10-G4, a panel of 10-mer peptides overlapping by nine amino acids spanning peptide 35, Mtb341–360, was synthesized. The 20-mer, peptide 35, only weakly sensitized LCL targets for T cell recognition. In contrast, the 10-mer peptide, Mtb346–355, efficiently sensitized autologous LCL targets (data not shown), mapping the epitope more finely to the region, AAERGPGQML.

peptide-loaded LCL targets. Interestingly, maximal chromium release occurred at approximately 10 g/ml of peptide, whereas maximal cytokine release required higher peptide concentrations.

Mtb39-Specific CD8 T Cell Clones Are Capable of Both Cytokine Release and Cytolysis

Because clones were screened with IFN- ELISPOT, we asked if they were capable of cytolysis as well. Autologous LCL targets displaying peptide 15, Mtb141–160, were used as antigenpresenting cells, and clones 10-E12 and 3-D5 were tested for IFN- release as assessed in an ELISPOT assay, and for cytolytic function as measured in a 4-hour chromium-release assay. As shown in Figure 6, the clones were able to both release IFN- and use the granule exocytosis pathway in response to

Figure 6. Mtb39-specific CD8 T cell clones are capable of both cytolysis and IFN- release. (A) Mtb39-specific CD8 T cell clones, 3-D5 (squares) and 10-E12 (circles), mediate cytolysis as measured by a chromium-release assay. LCL target cells were prepared by incubation with serial dilutions of peptide overnight, before addition to the assay. The E:T ratio was 10:1. (B) Mtb 39-specific CD8 T cell clones, 3-D5 and 10-E12, release IFN- as measured by ELISPOT assay. For this assay, T cell, LCL, and peptide were coincubated at the time of the assay.

Lewinsohn, Lines, and Lewinsohn: Human Mtb-Specific CD8  T Cells Mtb39-Specific CD8 T Cell Clones Recognize Mtb-infected Target Cells

Because the T cell clones were isolated using antigen-presenting cells overexpressing Mtb39, we asked whether or not the clones were able to recognize Mtb39 expressed during infection with Mtb. Figure 7A demonstrates lysis of Mtb-infected dendritic cells as measured by a chromium release assay, indicating that the epitopes recognized by the clones are processed and presented during the course of Mtb infection. Figure 7B demonstrates that the Mtb39-specific clones recognize Mtb-infected human macrophages in an ELISPOT assay.

DISCUSSION In this report, we identify Mtb39-specific CD8 T cells as part of the host immune response to Mtb and describe two distinct epitopes. These data highlight the limitations inherent in using peptides of predicted HLA Class I–binding specificity to define CD8 T cell responses to pathogens. Although binding to HLA-I molecules is a critical factor in the development of CD8 T cell responses, other determinants such as proteasomal processing, binding to the transporter associated with antigen processing, and the T cell repertoire, all play important roles as well. In this case, using the Parker prediction algorithm for peptide binding to HLA Class I molecules, ([23] http://bimas.cit.nih.gov/molbio/hla_bind/), the 10-mer peptides MWAQDAAAMF and AAERGPGQML have a predicted half time of dissociation for HLA-B*4403 of less than 1 second. Therefore, these peptides would be predicted to have a relatively low affinity for HLA-B44 and would not likely have been chosen as candidates to screen for CD8 T cell responses. Thus, this part of the Mtb39-specific CD8 T cell response would have been missed if peptides with predicted high affinity for HLA Class I molecules had been solely employed. This underscores one of the important advantages in using viral expression vectors such as recombinant adenoviruses, and pools of overlapping peptides, when attempting to fully characterize the immune response to a pathogen protein. Bothamley has suggested that HLA-B44 positive individuals may be more likely to successfully contain infection with Mtb than are HLA-B44–negative individuals (24). Although our experience is still limited, it is interesting that epitopes that we have described previously for CFP10/Mtb11 (25) and in this report are HLA-B44–restricted.

Figure 7. Mtb39-specific CD8 T cell clones recognize Mtb-infected target cells. (A) T cell clones 3-D5 and 10-E12 were tested against autologous dendritic cells either infected with Mtb (circles) or mockinfected (control; squares) in a chromium-release assay. (B) Using an IFN- ELISPOT assay, several Mtb39specific CD8 T cell clones were assessed for IFN- release in the presence of autologous macrophages either infected with Mtb (solid bars) or mock-infected (control; shaded bars).

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The search for protective T cell antigens for Mtb has largely focused on proteins that are secreted by Mtb. This focus followed from observations that immunization with secreted proteins from Mtb conferred protection from Mtb challenge in mouse and guinea pig models (26–28) and was associated with the development of a potent CD4 cellular immune response (27, 29). Furthermore, three secreted Mtb proteins (ESAT-6, Ag85, and CFP10/Mtb11) have recently been shown to be targets of the human CD8 T cell response (25, 30, 31). Furthermore, using similar methods to our own, Lalvani and colleagues have used overlapping synthetic peptides representing ESAT-6 to detect T cell responses in Mtb-infected individuals (32). However, Mtb antigens targeted by CD8 T cells are not limited to the secreted proteins, as this report identifies Mtb39, which is not a secreted protein, as a CD8 antigen. Furthermore, Mtb39 is a potent CD4 antigen as well (15). Collectively, the data argue that the Mtb39 protein might be an excellent candidate for inclusion in a subunit TB vaccine. Acknowledgment : The authors thank Mark Alderson, Davin Dillon, Greg Spies, and Sean Steen of Corixa Corporation for Mtb39 peptides, recombinant protein, and ongoing support and advice. They thank the Immunex Corporation for the provision of cytokine reagents.

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