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*
Division of Pulmonary and Critical Care Medicine, Oregon Health Sciences University/Portland Veterans Affairs Medical Center, and
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97207; and
Corixa Corp., Seattle, WA 98104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In tuberculosis, there is abundant evidence to support an
essential role for CD4+ T cell-mediated immunity
(2, 3). However, several lines of evidence suggest that
CD8+ CTL play a unique role as well. 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 infection than their wild-type
littermates (4), as are mice deficient in TAP
(5). Silva et al. (6), found that
CD8+ CTL clones generated to the 65-kDa Mtb heat
shock protein could confer partial immunity to Mtb infection in mice
(6). Immunization of mice with plasmids expressing Mtb Ags
such as the 65-kDa Mtb heat shock protein (7), Ag85a
(8), or the 38-kDa Ag (9) have resulted in
protection from subsequent challenge with Mtb and have been associated
with the generation of Ag-specific CD8+ CTL.
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. Monocyte-derived dendritic cells (DC) pulsed with mycobacterial chloroform/methanol extract (12, 13) have been used to elicit Mtb-reactive CD1-restriced CD8+ T cells. Using Mtb-infected, peripheral blood-derived DC, we have characterized nonclassically restricted Mtb-specific CD8+ T cells that are found preferentially in those infected with Mtb and are neither group 1 CD1 Ag nor HLA-A, -B, or -C restricted (14).
Further definition of the Ags recognized in the context of classical HLA-I (HLA-A, -B, or -C and HLA-Ia) will facilitate study of the host response to Mtb and may be important in the development of improved vaccines. With regard to HLA-Ia-processed Ag, there is increasing evidence that mycobacterially derived proteins can elicit CD8+ T cell responses. Using peptides of predicted HLA binding specificity (HLA-B52 and HLA-A*0201), Lalvani et al. were able to elicit CD8+ T cells capable of recognizing ESAT-6-expressing targets. Similarly, predicted binding peptides for the 19-kDa Ag (HLA-B44) (15) have been used to elicit CD8+ T cells that are reactive with Mtb-infected DC in an HLA-Ia-restricted manner. 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 cross-reactivity with another Ag. Similarly, it remains uncertain as to whether the peptides tested reflect dominant epitopes generated during the course of natural infection. Finally, Smith et al. (16) have used vaccinia vectors to demonstrate Ag85-, 19-kDa Ag-, and 38-kDa Ag-specific responses in individuals vaccinated with bacillus Calmette-Guérin (BCG).
We have recently reported an IFN-
enzyme-linked immunospot
(ELISPOT)-based limiting dilution assay in which
CD8+ T cell clones derived in response to
Mtb-infected DC were characterized with regard to the contribution of
classical vs nonclassical HLA restriction. In a single healthy
PPD+ donor, 92% of the Mtb-specific clones were
nonclassically restricted, while four were classically restricted
(17). This was the first report of the elicitation and
cloning of human MHC class Ia-restricted cells directly from
Mtb-infected APC. One advantage of this approach is that the immune
system has been allowed to select the Ag specificity and restricting
allele, thus strengthening the argument that these responses reflect
recall responses to Mtb. From this analysis two clones, 1-1B (HLA-B44
restricted) and 1-6F (HLA-B14 restricted), grew vigorously and
maintained specificity after repeated cycles of stimulation with
anti-CD3 and thus form the basis for this report. Here we find that
both clones are specific for the secreted Ag culture filtrate protein
10 (CFP10)/Mtb11.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subjects were recruited from employees at Harborview Medical Center, The Fred Hutchinson Cancer Research Center, Corixa Corp., and Oregon Health Sciences University. Purified protein derivative (PPD) responses were determined by the employee health service at the respective institutions. Protocols for venipuncture and apheresis were approved by the institutional review board. HLA typing was performed on PBMC by the Puget Sound Blood Center. Donor 160 is a healthy respiratory therapist who became PPD+ while working at Harborview Medical Center.
Monoclonal Abs and reagents
Culture medium consisted of RPMI 1640 supplemented with 10% FBS (BioWhittaker, Walkersville, MD), 50 µg/ml gentamicin sulfate (BioWhittaker), 5 x 10-5 M 2-ME (Sigma, St. Louis, MO), and 2 mM glutamine (Life Technologies/BRL, Grand Island, NY). For the elicitation of Mtb-reactive T cell clones, RPMI was supplemented with 10% human serum. mAbs were generated from hybridoma supernatants from the anti-panHLA (W6/32), anti-HLA-B,C (B1.23.2), or anti-HLA-DR (L243) cell lines obtained from American Type Culture Collection (Manassas, VA) using the Affi-Gel protein A MAPSII kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Mycobacterial strains (H37Rv; BCG) were obtained from American Type Culture Collection and grown in modified Middlebrook 7H9 medium (Difco, Detroit, MI). After the preparation of glycerol stocks, aliquots were frozen and subsequently titrated on Middlebrook 7H10 plates (Becton Dickinson Microbiology Systems, Cockeysville, MD). H37Rv CFP were provided through National Institute of Allergy and Infectious Disease and Colorado State University. Heat inactivation was accomplished by incubating an aliquot of Mtb at 70°C for 1 h. Recombinant Mtb39 (18), Mtb9.9 (19), Mtb8.4 (20), Ra12 (21), 19-kDa protein (22), Mtb41, Ra12/HTCC-1 (a Ra12 Rv3616c fusion protein), Mtb41 (23), CFP10/Mtb11 (24, 25), 38 kDa (26), and ESAT-6 (27) proteins were prepared as previously described (18).
Generation of peripheral blood DC and macrophages
Monocyte-derived DCs were prepared according to the method of Romani et al. (28). Briefly, PBMC were isolated from heparinized blood by centrifugation over Ficoll-Hypaque (Sigma) and were washed three times with culture medium. Alternatively, PBMC were obtained via leukapheresis. Cells were resuspended in 1% human serum (HS) medium (BioWhittaker) and allowed to adhere to a T-75 (Costar, Cambridge, MA) flask at 37°C for 1 h in the presence of 10 ng/ml of GM-CSF (Immunex, Seattle, WA). After gentle rocking, nonadherent cells were removed, and 30 ml of 10% HS medium containing 10 ng/ml of IL-4 (Immunex) and 30 ng/ml of GM-CSF (Immunex) was added. After 18 h, the medium was removed and centrifuged, and the cell-conditioned medium was placed on the adherent cells. After 5–7 days, cells were harvested with cell dissociation medium (Sigma). To generate macrophages, PBMC were adhered to a T-75 flask as described above and cultured in the absence of cytokine. To generate Mtb-infected APC, 1 x 106 monocyte-derived DC or macrophages were cultured overnight in the presence of Mtb or BCG (multiplicity of infection (MOI) = 50) in low adherence 16-mm wells (Costar no. 3473). After 18 h, the cells were harvested and resuspended in RPMI/10% HS.
IFN- ELISPOT assay
Mtb-specific effectors were detected from purified
CD8+ T cells by ELISPOT, as described previously
with minor modifications (29). Briefly, 96-well
nitrocellulose-backed plates (MAHA S4510; Millipore, Bedford, MA) were
coated as recommended by the manufacturer with 10 µg/ml capture mouse
anti-IFN- (1-D1K; Mabtech, Nacka, Sweden) overnight at room
temperature. Plates were then washed six times with PBS/0.05% Tween 20
(Sigma), and blocked with RPMI/10% HS for 1 h at room
temperature. Irradiated autologous Mtb-infected DC (2 x
104) were used as APC. Autologous purified
CD8+ T cells were then added, and the plate was
incubated overnight at 37°C. After washing with PBS/0.05% Tween 20,
100 µl of 1 µg/ml biotinylated secondary anti-IFN- mAb
(7B6-1; Mabtech) was added. After 2 h of incubation at room
temperature, plates were washed six times, 100 µl avidin/biotinylated
enzyme (HRP) complex (Vectastain ABC Elite Kit; Vector, Burlingame, CA)
was added to wells, and the plates were incubated for an additional
2 h. Then, plates were washed six times, and 100 µl
3-amino-9-ethylcarbazole substrate (Vectastain AEC substrate kit;
Vector) was added. After 4–7 min, the colorimetric reaction was
stopped by washing with distilled water, and plates were air-dried.
Where possible, spots were quantitated using a Zeiss Axioplan 2
microscope with 3200 K incident illumination equipped with a Epiplan
Neofluar 5x/0.15 objective, Sony DXC 950 CCD camera,
Märzhäuser scanning stage, MCP4 control unit, Pentium PC
computer, and KS ELISPOT software (Carl Zeiss Vision, Hallbergmoos,
Germany).
Peptide synthesis
Peptides were synthesized on a Rainin/PTI (Woburn, MA) Symphony peptide synthesizer using 9-fluorenyl-methoxycarbonyl batch chemistry with 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate activation. Peptides were analyzed by reverse-phase HPLC using a Vydac C18 column. Peptide molecular masses were verified using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer.
Purification of CD4+ and CD8+ T cell subsets
CD4+ and CD8+ T cell subsets were purified using CD4+ and CD8+ microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). In brief, PBMC were first labeled with CD4+ microbeads and passed twice over the magnetic column. CD4+-depleted cells were then labeled with CD8+ microbeads and positively selected over the magnetic column. In this manner, CD4+ T lymphocytes comprised <0.2% of the CD8+ T cell subset (data not shown).
Cytotoxicity assay
Target cell membrane damage was assessed using a standard 4-h 51Cr release assay. DC or Jurkat targets were cultured in the presence of sodium 51Cr (50 mCi/ml) for 14 h. Jurkat cells displaying the CFP10/Mtb11 peptide epitope were prepared by the addition of peptide (100 µg/ml) at the time of 51Cr labeling. Target cells were washed before incubation with the effector CTL clone at various E:T cell ratios for 4–5 h at 37°C. The percent specific lysis was calculated as previously described (30).
Expansion of T cell clones
To expand the CD8+ T cell clones, a rapid expansion protocol using anti-CD3 mAb stimulation was used (31). T cell clones were cultured in the presence of irradiated allogeneic PBMC (25 x 106), irradiated allogeneic LCL (5 x 106), and anti-CD3 mAb (30 ng/ml; Chiron) in RPMI medium with 10% human serum 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two Mtb-specific CD8+ T cell clones 1-1B
and 1-6F were derived from a single donor (D160; HLA-A2, -A3, -B14, and
-B44) in response to Mtb-infected DC. Both were previously tested for
HLA restriction specificity using Mtb-infected DC from donors that
matched at a single HLA-Ia locus and were found to be restricted by
HLA-B44 and HLA-B14, respectively (17). To further
validate that these clones were HLA-Ia restricted, mAb inhibition was
employed. As shown in Fig. 1
a
(Table I), addition of either an
anti-panHLA (W6/32) or an anti-HLA-B,C (B1.23.2) Ab inhibits
IFN- production by both clones in response to autologous
Mtb-infected DC. In contrast, addition of an anti-HLA-DR (L243) or
an isotype-matched control Ab had no effect.
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Prior work by Mazzaccaro et al. had suggested that entry of
Mtb-derived peptides into the HLA-Ia pathway is dependent upon the
presence of viable mycobacteria (32). To test this
hypothesis, clones 1-1B and 1-6F were tested for their ability to
recognize autologous monocyte-derived DC pulsed with either viable or
heat-killed Mtb (strain H37Rv). As shown in Fig. 1
b (Table I), DC pulsed with viable, but not heat-killed, Mtb were strongly
recognized. Because BCG-derived proteins have also been shown to gain
access to the HLA-Ia pathway, clones were then tested for their ability
to recognize Mtb- vs BCG-infected DC. Fig. 1c (Table I)
demonstrates strong IFN- production of HLA-B44-restricted clone 1-1B
in response to Mtb-infected, but not BCG-infected, targets. These
results were extended to the HLA-B14-restricted clone 1-6F (Fig. 1e and Table I). Both clones secrete comparable IFN- in
response to Mtb-infected autologous macrophages (data not shown).
BCG has been distinguished from Mtb on the basis of several well-characterized genomic deletions (33). The first deletion characterized, RD01, contains within it an operon encoding two small secreted proteins, both of which have been shown to be potent inducers of CD4+ T cell immunity in persons latently infected with Mtb. ESAT-6 is a 6-kDa protein initially characterized from short term culture filtrate proteins of Mtb (27). Ag Mtb11 is a 10.8-kDa protein discovered by screening an Mtb genomic expression library using human serum from subjects with active tuberculosis (25). The same protein has also been recently characterized from short-term culture filtrate proteins and has been termed CFP10 (24).
We reasoned that failure to recognize either heat-killed Mtb or BCG
would be consistent with recognition of a secreted protein absent from
BCG. To test this hypothesis, autologous DC were pulsed overnight with
a panel of recombinant Mtb-derived Ags (50 µg/ml) and tested for
their ability to elicit IFN-. Fig. 1d (Table I)
demonstrates that clone 1-1B uniquely recognizes DC pulsed with
CFP10/Mtb11. This result is extended to clone 1-6F in Fig. 1e. Weak activity is observed in DC pulsed with 50 µg/ml
of either PPD or CFP from H37Rv.
Mapping of CFP10/Mtb11 epitopes
To define the peptide epitopes recognized by clones 1-1B and 1-6F,
a set of 15-mer peptides, each overlapping by 11 aa and covering the
entire protein sequence of CFP10/Mtb11, was synthesized. Autologous DC
were pulsed overnight with peptide (10 µg/ml) and assessed for
IFN- release. As shown in Fig. 2, clone 1-1B uniquely recognized the peptide
CFP10/Mtb111–15. Clone 1-6F responded to
peptides CFP10/Mtb1181–95 and
CFP10/Mtb1184–100, suggesting that the minimal
epitope lay within the 11-aa overlap.
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a, none of these 9-mer
epitopes was effective. Of the 10-mer peptides,
CFP10/Mtb112–11 efficiently sensitized DC
targets, while the 11-mer CFP10/Mtb112–12 was
less optimal. Thus, the minimal HLA-B44 epitope is
CFP10/Mtb112–11 (AEMKTDAATL).
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b, peptide 85–93 had very modest activity, whereas peptide
86–94 was comparable to the 15-mer 85–100. These data suggested that
the optimal HLA-B14 epitope is also 10 aa in length. Peptides
CFP10/Mtb1186–95 and
CFP10/Mtb1186–95 were synthesized and used to
sensitize autologous LCL targets in an IFN- ELISPOT assay. Fig. 3c demonstrates that the minimal HLA-B14 epitope is
CFP10/Mtb1185–94 (RADEEQQQAL). CFP10/Mtb11-specific CD8+ T cell effectors are present at high frequency in peripheral blood from the PPD+ individual
Given that the T cell clones 1-1B and 1-6F were derived using
autologous DC infected with Mtb, it was important to determine that
these responses represented previous exposure to Mtb in vivo and were
not the result of priming in vitro. To determine the effector cell
frequency of CFP10/Mtb11-specific CD8+ T cells
and to determine whether other HLA-Ia-restricted epitopes are present,
magnetic bead-purified CD4+ and
CD8+ T lymphocytes from the
PPD+ donor (D160) were assessed by IFN-
ELISPOT for responses to autologous DC pulsed with each of the
overlapping 15-aa peptides as well as the smaller peptides defined
above. As shown in Fig. 4, CD4+ T cell recognition was distributed among
peptide regions CFP10/Mtb111–23,
CFP10/Mtb1169–100, and, to a lesser extent,
CFP10/Mtb1141–59. In contrast,
CD8+ T cell responses were seen primarily for
peptides CFP10/Mtb111–15,
CFP10/Mtb1181–95, and
CFP10/Mtb1184–100. Effector cell frequencies
were 1:1400, 1:1600, and 1:2000, respectively. These data suggested
that the epitopes defined by clones 1-1B and 1-6F were the dominant
CFP10/Mtb11 epitopes. To further test this hypothesis, effector cell
frequencies among circulating lymphocytes were determined for the
minimal epitopes defined above. As shown in Fig. 4, the effector cell
frequency for CD8+ T cells specific for the
HLA-B44 peptide AEMKTDAATL was 1:700, while the effector cell frequency
for CD8+ T cells specific for the HLA-B14 peptide
ADEEQQQAL was 1:2100. These data suggest that the CFP10/Mtb11
CD8+ T cell response can largely be accounted for
by these two epitopes. In addition, CD8+ T cells
reacted strongly to high concentrations (100 µg/ml) of CFP10/Mtb11
but weakly to low concentrations (0.1 µg/ml; data not shown),
consistent with the inefficient access of recombinant protein to the
HLA-Ia-processing pathway. In contrast, the CD4+
T cells responded equally well to high and low doses of CFP10/Mtb11 and
did not respond to the HLA-B14 epitope.
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Because the initial clone screening and further characterization
of the clones were based upon IFN- secretion, it was important to
determine whether the CD8+ T cell clones could
lyse Mtb-infected target cells. Fig. 5a demonstrates lysis of
Mtb-infected DC targets. Fig. 5b demonstrates that clone
1-1B is able to lyse HLA-44-transfected Jurkat cells in the presence of
peptide CPF10/Mtb112–11. Because macrophages and
DC are relatively resistant to Fas-mediated apoptosis, and because the
51Cr release assay is largely reflective of the
granule exocytosis pathway (30), these data would suggest
that the clones are able to use this pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CFP10/Mtb11 is a secreted Ag. Thus, it is not surprising that recognition of DC infected with heat-killed Mtb was not observed. Whether the presence of viable Mtb is required for entry of Ag into the class I pathway as has been suggested by Mazzaccaro et al. (32) will require comparison of CD8+ T cell recognition of secreted vs nonsecreted proteins. Similarly, it has been proposed that proteins secreted early in the course of Mtb infection account for the ability of live, but not heat-killed, Mtb to confer protection in small animal systems (34). Whether the enhanced efficacy of these small secreted proteins is associated with CD8+ T cell responses is an intriguing possibility that awaits further definition.
CFP10/Mtb11 is part of an operon that is deleted from BCG. Both members of the operon, CFP10/Mtb11 and ESAT-6, are potent CD4+ T cell Ags in persons infected with Mtb (24, 25). Here, we demonstrate that CFP10/Mtb11 is also a potent CD8+ T cell Ag. As such, this Ag may be a candidate for inclusion in a tuberculosis vaccine designed to elicit both CD4+ and CD8+ T cell immunity. Importantly, because the protein is not present in BCG, it may serve to supplement or boost immunity in persons who have previously received the BCG vaccine.
Our data suggest that the CD8+ T cell response to CFP10/Mtb11 in the single PPD+ donor tested can be largely accounted for by the two epitopes defined herein. The high effector cell frequencies defined are similar to that reported in response to viruses known to elicit potent CD8+ T cell responses, such as CMV and influenza. Similarly, the tightly focused response is consistent with a model of immunodominance that has been proposed for virally induced CD8+ T cell responses in which a limited set of epitopes comprises the majority of the CD8+ T cell response for an individual to a given protein Ag (35). Our data also may explain why obtaining HLA-Ia-restricted CTL using Mtb-infected APC has been historically difficult, in that we were able to discern the minority HLA-Ia population only by using a limiting dilution analysis-based approach.
The data highlight the limitations inherent in using peptides of predicted binding specificity to define CD8+ T cell responses to pathogens. While binding to HLA-I molecules is a critical factor in the development of CD8+ T cell responses, proteasomal processing, TAP binding, and the T cell repertoire all play important roles as well. While the donor used is HLA-A*201, no HLA-A-restricted Mtb-specific CTL were detected. Moreover, using the Parker prediction algorithm (36) (http://bimas.cit.nih.gov/molbio/hla_bind/), the 10-mer peptide AEMKTDAATL has a predicted half-time of dissociation for HLA-B*4403 of 22 s and would be a reasonable candidate. In contrast, the 10-mer peptide RADEEQQQAL has a predicted half-time of dissociation for HLA-B14 of 1 s and thus would not appear to be a likely candidate. The use of peptides of predicted binding specificity is further complicated by the observation that the HLA-B44-restricted clone does not recognize a 9-aa peptide. Finally, our data support the use of nested 15-aa peptides with an 11-aa overlap as an efficient means of determining whether circulating CD8+ T lymphocytes are capable of recognizing a candidate Ag (37) (L. J. Picker, unpublished observation). The present study is limited in that we have analyzed the CD8+ T cell response of only one PPD+ donor to CFP10/Mtb11. Future studies will use the 15-aa peptide approach to analyze Ag-specific CD4+ and CD8+ T cell responses among PPD+, PPD-, and actively infected subjects.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Lewinsohn, R&D 11, Portland Veterans Affairs Medical Center, 3710 U.S. Veterans Road, Portland, OR 97207.
3 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; BCG, bacillus Calmette-Guérin; DC, dendritic cell; ELISPOT, enzyme-linked immunospot; CFP, culture filtrate protein; PPD, purified protein derivative; HS, human serum; MOI, multiplicity of infection; LCL, lymphoblastoid cell line; EGFP, enhanced green fluorescent protein.
Received for publication July 10, 2000. Accepted for publication September 27, 2000.
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References |
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interferon-secreting CD8+, as well as CD4+, T cells in lungs following aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 67:3242.
T cell pool. J. Immunol. 162:366.This article has been cited by other articles:
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V. Trajkovic, G. Singh, B. Singh, S. Singh, and P. Sharma Effect of Mycobacterium tuberculosis-Specific 10-Kilodalton Antigen on Macrophage Release of Tumor Necrosis Factor Alpha and Nitric Oxide Infect. Immun., December 1, 2002; 70(12): 6558 - 6566. [Abstract] [Full Text] [PDF]
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V. Lazarevic and J. Flynn CD8+ T Cells in Tuberculosis Am. J. Respir. Crit. Care Med., October 15, 2002; 166(8): 1116 - 1121. [Full Text] [PDF]
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A. Lalvani CD8 Cytotoxic T Cells and the Development of New Tuberculosis Vaccines Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 789 - 790. [Full Text]
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D. A. Lewinsohn, R. A. Lines, and D. M. Lewinsohn Human Dendritic Cells Presenting Adenovirally Expressed Antigen Elicit Mycobacterium tuberculosis-Specific CD8+ T Cells Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 843 - 848. [Abstract] [Full Text]
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M. R. Klein, A. S. Hammond, S. M. Smith, A. Jaye, P. T. Lukey, and K. P. W. J. McAdam HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c Infect. Immun., February 1, 2002; 70(2): 981 - 984. [Abstract] [Full Text] [PDF]
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A. A. Pathan, K. A. Wilkinson, P. Klenerman, H. McShane, R. N. Davidson, G. Pasvol, A. V. S. Hill, and A. Lalvani Direct Ex Vivo Analysis of Antigen-Specific IFN-{gamma}-Secreting CD4 T Cells in Mycobacterium tuberculosis-Infected Individuals: Associations with Clinical Disease State and Effect of Treatment J. Immunol., November 1, 2001; 167(9): 5217 - 5225. [Abstract] [Full Text] [PDF]
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J. W. Mills, L. Ryan, R. LaCourse, and R. J. North Extensive Mycobacterium bovis BCG Infection of Liver Parenchymal Cells in Immunocompromised Mice Infect. Immun., May 1, 2001; 69(5): 3175 - 3180. [Abstract] [Full Text] [PDF]
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2 ng/ml of IFN-
