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The Journal of Immunology, 2006, 177: 437-442.
Copyright © 2006 by The American Association of Immunologists

Secreted Proteins from Mycobacterium tuberculosis Gain Access to the Cytosolic MHC Class-I Antigen-Processing Pathway1

David M. Lewinsohn2,*,{dagger}, Jeff E. Grotzke{dagger}, Amy S. Heinzel*, LiQing Zhu{ddagger}, Pamela J. Ovendale{ddagger}, Mark Johnson3,{ddagger} and Mark R. Alderson{ddagger}

* Division of Pulmonary and Critical Care Medicine, Oregon Health Sciences University/Portland Veterans Affairs (VA) Medical Center, Portland, OR 97239; {dagger} Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97239; and {ddagger} Corixa, Seattle, WA 98104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8+ T cells play an important role in the host response to infection with Mycobacterium tuberculosis (Mtb). Mtb resides in an arrested phagosome that is phenotypically similar to an early endosome. The mechanisms by which Mtb-derived Ags gain access to the HLA-I-processing pathway are incompletely characterized. Studies with CD8+ T cell lines have suggested that Mtb Ags gain access to the HLA-I pathway in an alternate vacuolar pathway that is both brefeldin A (BFA) and TAP independent. To define the requirements of entry of Ag into the HLA-I pathway, we have used human CD8+ T cell clones specific for the secreted Mtb Ag CFP10. Human monocyte-derived dendritic cells were pulsed with CFP10 expressed in a recombinant adenovirus, surface adsorbed to microspheres, or in its native form by Mtb. When delivered by adenovirus, processing and presentation of CFP10 were blocked by both BFA and the proteasomal blocker lactacystin. In contrast, processing of CFP10 adsorbed to the surface of microspheres was not affected by either of these Ag-processing inhibitors. BFA, lactacystin, and TAP inhibition blocked the recognition of Mtb-infected dendritic cells, suggesting that processing was via a cytosolic pathway for this secreted protein Ag. We conclude that secreted proteins from Mtb can be processed in a BFA- and proteasome-dependent manner, consistent with egress of Ag into the cytosol and subsequent loading of proteasomally derived peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis is a leading cause of infectious disease mortality worldwide. Control of the intracellular growth of Mycobacterium tuberculosis (Mtb)4 depends upon the acquisition of a robust adaptive cellular immune response. In both animal models and human tuberculosis, there is abundant evidence to support complementary roles for both CD4+ and CD8+ T cell-mediated immunity (1).

Mtb is a facultative intracellular pathogen that, following phagocytosis by a macrophage or dendritic cell (DC), resides in an arrested phagosome (2, 3). Although this compartment has been traditionally thought to be a part of the vesicular, MHC-class II processing pathway, both human and mouse Mtb-specific CD8+ T cells are capable of recognizing Mtb-infected cells.

Phagocytosis of particulate Ags and apoptotic cells by DCs and macrophages leads to MHC class-I (MHC-I) Ag processing and presentation through a process termed cross-presentation. Several models have been proposed to account for presentation of these Ags in a MHC-I-dependent manner. In the vacuolar model, peptides are generated in a vesicular acidic compartment and then loaded onto recycled MHC (4, 5). Presentation of these Ags does not require endoplasmic reticulum (ER)-Golgi transport brefeldin A (BFA) independent, and is independent of TAP. In this regard, polylactide-coglycolide (PLG)-associated Ag can be delivered to the vacuolar pathway in a cathepsin-S-dependent and TAP-independent manner (6). Conversely, particulate Ags have also been shown to be presented via a cytosolic pathway that is TAP dependent and requires proteasomal processing (7). In these examples, the identical Ag (OVA) could be targeted to distinct processing pathways by varying the nature of the particle.

The mechanisms by which Mtb-derived Ag gain access to the MHC-I pathway are incompletely understood. Infection with live, but not heat-killed, Mtb facilitates the MHC-I-dependent recognition of soluble OVA in a TAP-dependent manner (8). Similarly, macrophage phagosomes containing live bacillus Calmette-Guerin (BCG) appear to be porous to molecules of <70 kDa (9). Mtb lipids and lipoproteins, actively trafficked in phagosomally derived vesicles (10), are cross-presented by bystander APC in a TAP-independent and proteosome-independent manner (11, 12, 13). We have reported previously that presentation to HLA-E-restricted, Mtb-specific, human CD8+ T cells requires proteasomal Ag processing and is not blocked by BFA (14). Similarly, Canaday et al. (15) developed Mtb-specific CD8+ T cell lines that were insensitive to BFA blockade.

To delineate the processing pathway used by Mtb-infected cells to present to CD8+ T cells, we developed a model system to test whether Mtb-infected cells preferentially use the cytosolic or vacuolar pathways. In this study, Mtb Ag was delivered to the cytosolic or vacuolar pathways by adenovirus or PLG microspheres, respectively, and human CD8+ T cell clones (16) were used to monitor Ag presentation in the presence of Ag-processing inhibitors. Using this approach, we find that entry of Mtb-derived secreted proteins does not depend upon the vacuolar pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human subjects

Subjects were recruited from employees at Harborview Medical Center, Fred Hutchinson Cancer Research Center, Corixa, and Oregon Health and Science University. Protocols for venipuncture and apheresis were Institutional Review Board approved.

Cell lines and T cell clones

Clone 1-1B is an HLA-B44-restricted CD8+ T cell clone specific for Mtb Ag, CFP102–11, and recognizes the minimal epitope, AEMKTDAATL (16). Clone 38.1-1 is a CD4+ T cell clone specific for CFP1073–87 and recognizes the epitope STNIRQAGVQYSRAD (17). Clone A3 is an HLA-B3514-restricted CD8+ T cell clone specific for the Mtb-Ag Mtb8.433–41 and recognizes the epitope ASPVAQSYL (our unpublished observation).

mAbs and reagents

Culture medium consisted of RPMI 1640 supplemented with 10% FBS (BioWhittaker), 50 µg/ml gentamicin sulfate (BioWhittaker), and 2 mM glutamine (Invitrogen Life Technologies). Human T cell assays were performed with RPMI 1640 supplemented with 10% human serum (HS), as described previously (14). Mtb strain H37Rv was obtained from American Type Culture Collection and grown in modified Middlebrook 7H9 medium (Difco). Recombinant (18) and CFP10/Mtb11 were prepared, as described previously (19). Adenovirus-expressing ICP47 was provided by D. Johnson (Oregon Health and Science University, Portland, OR) (20).

Assays for T cell function

IFN-{gamma} ELISPOT was performed, as described (16). Human and murine IFN-{gamma} ELISAs and proliferation assays were performed, as described (14, 18).

Microsphere preparation

PLG RG502H was purchased from Boehringer Ingelheim. Polyvinyl alcohol (87–89% hydrolyzed; 13–23,000 kDa) was purchased from Sigma-Aldrich. PLG polymer was dissolved in dichloromethane and emulsified using a Silverson homogenizer. Microspheres were hardened for 2 h with mild stirring, washed twice via centrifugation, and lyophilized after the addition of mannitol. Protein was adsorbed to the microspheres at the time of use. The lyophilized microspheres were dispersed in aqueous buffer, to which the protein solution was added and gently mixed. Microspheres were collected by centrifugation with the unadsorbed protein solution removed. Protein adsorption to the microspheres was quantified by HPLC analysis of the supernatant before and after adsorption and confirmed for select lots by amino acid analysis on the microsphere formulation.

Expansion of T cell clones

To expand the CD8+ T cell clones, a rapid expansion protocol using anti-CD3 mAb stimulation was used (21). T cell clones were cultured in the presence of irradiated allogeneic PBMC (25 x 106), irradiated allogeneic lymphoblastoid cell line (5 x 106), and anti-CD3 mAb (30 ng/ml; Chiron) in RPMI 1640 medium with 10% HS in a T-25 upright flask in a total volume of 30 ml. The cultures were supplemented with IL-2 (1 ng/ml; Chiron) on days +1, +4, +7, and +10 of culture. The cell cultures were washed on day +4 to remove remaining soluble anti-CD3 mAb.

Generation of peripheral blood DCs and macrophages

Monocyte-derived DC were prepared according to the method of Romani et al. (22). Briefly, PBMC were isolated from heparinized blood by centrifugation over Ficoll-Hypaque (Sigma-Aldrich) and washed three times with culture medium. Alternatively, PBMC were obtained via leukapharesis. Cells were resuspended in 2% HS medium (BioWhittaker) and allowed to adhere to a T-75 (Costar) flask at 37°C for 1 h in the presence of 30 µg/ml DNase (Amgen). After gentle rocking, nonadherent cells were removed, and 30 ml of 10% HS medium containing 10 ng/ml IL-4 (Amgen) and 10 ng/ml GM-CSF (Amgen) was added. After 5–7 days, cells were harvested with cell-dissociation medium (Sigma-Aldrich). To generate Mtb-infected cells, 1 x 106 phagocytes were cultured overnight in the presence of Mtb (H37Rv; multiplicity of infection (MOI) 25–50) in low-adherence 16-mm wells (Costar no. 3473). After 18 h, the cells were harvested and resuspended in RPMI 1640/10% HS. Infection with these MOIs typically leads to at least one bacteria per cell, but does not result in cellular apoptosis (14, 23).

Metabolic inhibition of Ag presentation

One hour before the addition of Mtb to DC, lactacystin (40 µM; EJ Corey-Harvard Biolabs), N-acetyl-Leu-Leu-norleucinal (LLnL; Calbiochem), or BFA (Sigma-Aldrich) was added to the culture medium. After 18 h of coincubation with Mtb, cells were harvested and fixed in either 1% paraformaldehyde (Sigma-Aldrich) or 0.1% glutaraldahyde (grade I, 25% aqueous; Sigma-Aldrich). After vigorous washing, fixed DC were used as stimulators for CD8+ T cells, as described above.

Construction of adenoviral vectors

To construct the adenoviral vector expressing CFP10, the open reading frame of CFP10 was subcloned from pcDNA3/IL-1/Mtb39 and pEGFP-N1, respectively, into the multiple cloning site of the adenoviral shuttle vector, pAD1. Shuttle vectors were then cotransfected into human 293 cells with plasmid, pBGHE3 (Microbix Biosystems). Virus was purified over two CsCl gradients and titered before use.

Adenovirus infection of APC

To generate adenovirally infected APC, DC were harvested after 18 h of culture, and 5 x 105 DC were seeded in Optimem (125 ml; Invitrogen Life Technologies) into low-adherence 16-mm wells (Costar). Adenoviral liposomes were prepared by coincubating the adenovirus and lipofectamine (6 µg/ml; Invitrogen Life Technologies) for 15 min and then added to the DC cultures. After 4–6 h, fresh medium (1 ml) containing GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) was added (24).

Intracellular cytokine staining

A total of 105 monocyte-derived DC in 20 µl of Opti-Mem medium was seeded in a 96-well flat-bottom plate. After a 20-min incubation of adenovirus with Lipofectamine 2000 (Invitrogen Life Technologies), 20 µl of adenovirus liposomes was added to the DC and incubated for 2 h at 37°C. Medium containing GM-CSF and IL-4 was added, and DC were incubated for an additional 28–30 h. DC were then infected with H37Rv at varying MOIs or pulsed with peptide or protein Ag for 18 h. After washing, 1–1.5 x 105 T cells were added and incubated for 6 h, with the addition of BFA (10 µg/ml) for the final 2 h. After harvesting, cells were fixed, permeabilized, stained with anti-CD3 (UCHT1; BD Pharmingen) and anti-IFN-{gamma} (4S.B3; eBioscience), and analyzed on a FACSCalibur.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Presentation of adenovirally expressed CFP10 is blocked by both BFA and the proteasomal blocker LLnL

Viral Ags are expressed in the cytosol, and then processed for presentation by MHC-I via a TAP-dependent and BFA-sensitive pathway. To validate that BFA distinguishes between the vacuolar and cytosolic pathways in our in vitro system, we used a recombinant adenovirus expressing the Mtb protein CFP10. HLA-B44-positive DC were infected with CFP10 adenovirus using lipofectamine. As a control, we used a 10-mer peptide previously shown to be the minimal epitope within CFP10 recognized by the CD8+ T cell clone, 1-1B (CFP102–11) (16). Both the CFP102–11 peptide-pulsed DC and the CFP10 adenovirus-infected DC induced a strong IFN-{gamma} response by the 1-1B T cells (Fig. 1A). However, while the addition of BFA had no effect on the recognition of peptide-pulsed APC, it resulted in complete abrogation of Ag presentation by adenovirus-infected DC. Furthermore, as is demonstrated in Fig. 1A, treatment with proteasomal blocker LLnL abrogated recognition by the 1-1B T cell clone. In contrast, identical treatment had no effect on recognition of peptide-pulsed APC.


Figure 1
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  		FIGURE 1. Cytosolic and vacuolar Ag presentation can be distinguished by sensitivity to BFA and proteasomal blockade. A, DC were pretreated with LLnL (30 µM) or BFA (5 µg/ml) and infected with adenoviral CFP10 overnight (MOI 100), before fixing in 0.2% paraformaldehyde. APC were then coincubated with 1-1B T cells, and IFN-{gamma} was assessed by ELISA after 48 h of incubation. Each bar represents the mean of triplicate cultures, and the data are representative of two separate experiments. B, Recombinant CFP10, PLG CFP10, and the cognate peptide CFP102–11 were tested for their ability to stimulate the CFP10-specific CD8+ T cell clone 1-1B. DC (20,000) were coincubated with 1-1B T cells (20,000) in the presence of Ag at the concentrations indicated. Plates were cultured for 48 h, after which 50 µl was removed for assessment of IFN-{gamma} by ELISA. The plates were then pulsed with [3H]thymidine (1 µCi/well). After culture for an additional 18 h, tritium uptake was determined. Each point reflects the mean of triplicate cultures, and data are representative of three separate experiments. C, DC were pretreated with titrating concentrations of BFA for 30 min and then infected with CFP10-expressing adenovirus or cocultured with CFP10 PLG microspheres. After overnight culture, DC were harvested, fixed in 0.2% paraformaldehyde, and used to stimulate 1-1B T cells. Each point reflects the mean of triplicate cultures, and data are representative of three separate experiments.

 
PLG microsphere delivery of Ag is both BFA and TAP independent

Initially, we assessed whether DC pulsed with PLG-CFP10 microspheres showed enhanced Ag presentation compared with DC pulsed with soluble CFP10. DC pulsed with PLG-CFP10 microspheres, when compared on a microgram per microgram basis, are ~300-fold more efficient at delivering CFP10 protein to 1-1B T cells compared with soluble CFP10 (Fig. 1B). As is demonstrated in Fig. 1C, treatment with BFA, even at the lowest concentration of BFA tested (0.16 µg/ml), completely abrogated recognition of adenovirally presented CFP10, but had no effect on recognition of microsphere-presented Ag or on targets loaded with cognate peptide CFP102–11.

Ag presentation by Mtb-infected DC is blocked by both BFA and lactacystin

We next sought to determine which pathway was used for presentation of CFP10 to 1-1B T cells during the course of infection of DC with Mtb. Treatment of Mtb-infected monocyte-derived DC with either BFA or the proteasomal blocker lactacystin abrogated recognition of these DC over a wide range of MOIs (Fig. 2), suggesting that the cytosolic pathway is preferentially used by Mtb-infected cells presenting Ag to HLA-I-restricted CD8+ T cells.


Figure 2
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  		FIGURE 2. Ag presentation by Mtb-infected DC to 1-1B T cells is blocked by both BFA and lactacystin. DC were infected with strain H37Rv Mtb in the presence of either BFA (2 µg/ml) or lactacystin (20 µM). Following overnight incubation, DC were fixed in 0.2% paraformaldehyde and used as APC to stimulate 1-1B T cells, as described. Each point represents the mean of triplicate cultures, and data are representative of two separate experiments.

 
Ag presentation by Mtb-infected DC is blocked by the TAP inhibitor ICP47

To determine the TAP dependence of Mtb-derived Ag, the TAP inhibitor ICP47 was expressed in an adenoviral vector (20). Expression in human monocyte-derived DC resulted in a modest reduction in cell surface expression of HLA-I without affecting HLA-II expression levels (data not shown). Monocyte-derived DC infected with adenoviral ICP47 for 30 h were then infected with Mtb, and then assessed for their ability to present to the HLA-I-restricted CD8+ T cell clone 1-1B as well as to the HLA-II-restricted, CFP10-specific CD4+ T cell clone 38-1.1 (25). Expression of ICP47 abrogated the HLA-I-mediated recognition of Mtb-derived CFP10 (Fig. 3A), but had no effect on the recognition of APC pulsed with cognate peptide (Fig. 3B). Conversely, expression of ICP47 had no effect on the HLA-II-dependent recognition of Mtb-infected DC (Fig. 3D) or on the recognition of recombinant Ag (Fig. 3E). Because the recognition of Mtb-infected cells was relatively inefficient, the experiment was repeated using flow cytometric analysis of Ag-dependent IFN-{gamma} release. As is demonstrated in Fig. 3, C and F, treatment with ICP47 inhibited IFN-{gamma} release for the CFP10-specific CD8+, but not CD4+ T cell clones. Furthermore, the effect was not contingent on the relatively high MOI used as it was seen at MOIs of both 25 and 50. Hence, these data demonstrate the involvement of TAP in the processing and presentation of CFP10 to HLA-I-restricted T cells.


Figure 3
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  		FIGURE 3. Ag presentation by Mtb-infected DC to 1-1B T cells is blocked by the TAP inhibitor ICP47. DC were mock infected, infected with adenovirus-expressing ICP47 or with control adenovirus (MOI 100). After 30 h, DC were harvested and infected overnight with H37Rv at a MOI of 50 (A and D) or loaded overnight with CFP102–11 peptide (B) or CFP10 protein (E). The 1-1B or 38.1-1 T cells (1000) were then added to DC (20,000), and IFN-{gamma} was assessed by ELISPOT after 18–24 h of incubation. Each bar represents the mean of triplicate wells, and the data are representative of three separate experiments. C and F, DC were mock infected or infected with adenovirus as above for 30 h and then infected with H37Rv at MOIs of 50 or 25 for 18 h. T cell clones were added for 6 h, with BFA present for the last 2 h of the stimulation. After staining, CD3+ cells were analyzed for IFN-{gamma} expression. Each bar represents the mean of duplicate wells, and the data are representative of three separate experiments.

 
Access of Ag to the HLA-I pathway does not require the region of deletion (RD1) secretory pathway

Comparative analysis of Mtb and BCG strains has shown that one region, RD1, is present in all strains of Mtb and absent from all strains of BCG (26, 27, 28). Previous reports have demonstrated that the RD1 proteins, which includes CFP10 and ESAT-6, constitute a novel secretory pathway that may have effects on membrane permeability (17, 29, 30, 31). Additionally, reports have demonstrated that abrogation of any member of the RD1 family can abolish secretion of ESAT-6 and CFP10. As a result, we postulated that the Mtb-derived heterodimer CFP10/ESAT-6 could form a phagosomal pore allowing for the Mtb-dependent egress of Ags into the cytosol. Because secretion of CFP10 depends on the presence of an intact RD1 region, we used a Mtb8.432–40-specific, HLA-B3514-restricted T cell clone, A3, derived from an actively Mtb-infected subject (D. Lewinsohn, unpublished data). Like CFP10, recognition of Mtb-derived Mtb8.4 was blocked by both BFA and lactacystin (data not shown). Mtb mutants containing deletion of the RD1 region, or deletion of or transposon insertion in its individual gene family members (CFP10 and ESAT-6), did not abrogate recognition of the Mtb Ag Mtb8.4 (Fig. 4). Similarly, the complemented strains appeared very similar to the original knockout. These data argue that cytosolic access of Mtb-derived Ags does not rely on an intact RD1 region.


Figure 4
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  		FIGURE 4. Delivery of Ag into the HLA-I pathway does not depend on RD1 gene family members. DC were pulsed overnight with cognate peptide (Mtb8.433–41; 10 µg/ml) or each of the Mtb strains listed (MOI 50). Mtb strains deficient in RD1, CFP10 (tn3874), ESAT-6 (tn3875), and the corresponding complemented strains (tn3874:MH406; tn3875:MH408) were provided by D. Sherman (University of Washington, Seattle, WA) (17 ). DC were then harvested and used as APC (20,000/well) in an IFN-{gamma} ELISPOT assay in which 1000 A3 T cells were added as effector cells. Plates were developed following overnight incubation, and spots were enumerated. Bars represent the mean percentage of the response to H37Rv for three experiments. Statistical analysis (Student’s t test) shows no significant difference between the response to H37Rv and any of the strains tested (p > 0.1).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been reported previously that Mtb-derived Ags can gain access to the HLA-I pathway via either cytosolic (8, 9) or vacuolar (15) pathways. However, each of these reports had significant limitations. Mazzaccaro et al. (8) were able to demonstrate TAP-dependent and BFA-inhibitable recognition that required viable Mtb, using OVA codelivered in soluble form with Mtb. In the report by Teitelbaum et al. (32), size-dependent colocalization of fluorescent markers with phagosomal Mtb was demonstrated following microinjection into the cytosol. In neither case, however, was processing or presentation of Mtb-derived Ag directly demonstrated. Conversely, in the report by Canaday et al. (15), recognition of a Mtb-specific CD8+ T cell line was demonstrated to be refractory to treatment with BFA, although the sensitivity to proteasomal blockade was not reported. Neither the antigenic specificity nor HLA restriction of the cell line was determined. We have demonstrated previously that HLA-E-restricted T cell clones use a pathway that is dependent on the proteasome, but refractory to BFA blockade (14).

In this study, we demonstrate that blockade with BFA can clearly distinguish the vacuolar from cytosolic pathways for the Mtb-derived Ag CFP10. Using this model, we find that recognition of Mtb Ag CFP10 is completely blocked by BFA, depends upon the proteasomal degradation, and uses TAP. Hence, these data demonstrate HLA-I-dependent presentation of Mtb-derived CFP10 does not use the vacuolar pathway. In contrast, we confirm that class I presentation of PLG-Ag microspheres is insensitive to BFA. Furthermore, we demonstrate that RD1 gene family members are not required for access of the secreted Ag Mtb8.4 into the HLA-I pathway.

It has been demonstrated recently that ER-phagosomal fusion results in a functional, MHC-I processing compartment (33, 34). This pathway has been demonstrated to be TAP dependent, and is only partially inhibited by BFA. Our data do not distinguish between phagosomal reuptake or ER loading of proteasomally derived peptides once Mtb-derived Ags access the cytosol.

Certain Ags and lipids from Mtb have been demonstrated to gain access to vesicles that can be actively exported (35), and these vesicles contain lipoproteins that can sensitize bystander APC in a TAP-independent fashion (11). Cross-presentation by these vesicles may be the result of apoptosis, and can result in cross-presentation in both human and mouse DC (12, 13). Termed the detour pathway, this process requires phagosomal acidification, but not proteasomal degradation. Although we have demonstrated previously that heavily infected cells are preferentially recognized by both HLA-E- and HLA-Ia-restricted T cells (23), under the experimental conditions described in this work, nearly all of the APC contain at least one Mtb. Hence, we cannot distinguish direct recognition of a Mtb-infected cell from cross-presented Ag. Nonetheless, the dependence on the proteasome and sensitivity to BFA blockade clearly distinguishes our results from those predicted by the detour pathway, and would be consistent with an immunosurveillance role for CD8+ T cells in the control of Mtb.

Given evidence supporting three distinct pathways of Ag processing and presentation, we hypothesize that Ags may be processed differentially based upon their physiochemical characteristics. In this study, both of the Ags tested are secreted, of low m.w., and relatively hydrophilic. Hence, it is possible that alternate pathways favor the presentation of hydrophobic or lipidated Ags. Alternately, it is possible that alternate pathways would favor Ags that are rapidly degraded by lysosomally derived proteases. Nonetheless, the availability of Mtb Ags to the cytosolic pathway would support the use of viral vectors as vaccine for Mtb, as we would predict that T cells generated in this context could recognize Mtb-infected cells.


   Acknowledgments
 
We thank Ken Rock and Ann Hill for advice and helpful discussions, as well as Deborah Lewinsohn for thoughtful and patient consideration of the manuscript. CFP10 and Mtb8.4 adenoviruses were provided by Greg Spies (Corixa, Seattle, WA). Recombinant CFP10 and Mtb8.4 were provided by Davin Dillon and Yasir Skeiky (both from Corixa, Seattle, WA). We thank David Sherman and Mark Hickey (Corixa, Seattle, WA) for provision of RD1 mutant strains of Mtb, and David Johnson for provision of adenoviral ICP-47.


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


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

1 This work was supported by American Lung Association Research and Career Investigator Awards (to D.M.L.), a Medical Research Foundation grant, National Institutes of Health Grants 1K08AI01644 and R01AI48090 (to D.M.L.), VA Merit Review (to D.M.L.), and the Portland VA Medical Center. Back

2 Address correspondence and reprint requests to Dr. David M. Lewinsohn, R&D 11, Portland VA Medical Center, 3710 U.S. Veterans Road, Portland, OR 97239. E-mail address: lewinsod{at}OHSU.edu Back

3 Current address: Discovery Laboratories, Redwood City, CA 94043. Back

4 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; BCG, bacillus Calmette-Guerin; BFA, brefeldin A; DC, dendritic cell; MHC-I, MHC class-I; ER, endoplasmic reticulum; HS, human serum; LLnL, N-acetyl-Leu-Leu-norleucinal; MOI, multiplicity of infection; PLG, polylactide-coglycolide; RD1, region of deletion 1. Back

Received for publication December 29, 2004. Accepted for publication April 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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