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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Tuberculosis (Edinb). 2011 Nov 13;91S1:S96–S104. doi: 10.1016/j.tube.2011.10.018

The ΔfbpA attenuated candidate vaccine from Mycobacterium tuberculosis, H37Rv primes for a stronger T-bet dependent Th1 immunity in mice

Cherie M Roche 1, Amanda Smith 1, Devin R Lindsey 1, Akshay Meher 2, Kimberly Schluns 3, Ashish Arora 2, Lisa Y Armitige 1, Chinnaswamy Jagannath 1
PMCID: PMC3248993  NIHMSID: NIHMS338608  PMID: 22082615

Summary

The ΔfbpA candidate vaccine derived from Mycobacterium tuberculosis (H37Rv) protects mice better than BCG against tuberculosis, and we investigated the hypothesis that ΔfbpA may induce a stronger Th1 immunity. Since T-bet transcription factor regulates Th1 immunity, mice infected with ΔfbpA, BCG vaccine and related mycobacteria were analyzed for T-bet positive T cells. Mouse dendritic cells (DCs) or macrophages were also pulsed with excretory-secreted antigens (ES; Antigen-85B, ESAT-6 and CFP10) and cocultured with T cells from immunized or naïve mice and tested for in vitro induction of T-bet and IFN-γ. In both models, ΔfbpA mutant induced a stronger response of T-bet+CD4 T cells, which correlated with an increased expansion of IFN-γ+CD4 T cells in vivo and in vitro. When DCs pulsed with ES antigens were allowed to stimulate T cells, ESAT-6 and CFP-10 failed to induce a recall expansion of T-bet+IFN-γ+CD4 T cells from BCG vaccinated mice. Thus, deletion of RD1 in BCG seems to reduce its ability to induce T-bet and induce stronger Th1 immunity. Finally, mice were vaccinated with ΔfbpA and BCG and challenged with virulent Mtb for evaluation of protection and T cell expansion. ΔfbpA vaccinated mice showed a rapid and stronger expansion of CD4+CXCR3+ IFN-γ+ T cells in the lungs of Mtb challenged mice, compared to those which had BCG vaccine. ΔfbpA immunized mice also showed a better decline of the Mtb bacterial counts of the lungs. Mtb derived ΔfbpA candidate vaccine therefore induces qualitatively better T-bet dependent Th1 immunity than BCG vaccine.

1. Introduction

Mycobacterium tuberculosis (Mtb) causes tuberculosis which is the leading cause of death due to infections in man today, with at least 2 million deaths reported worldwide. M.bovis BCG is the most frequently used vaccine with over a billion doses administered to date. Although it is effective against childhood tuberculosis, it has a variable efficacy and geographic variation [1]. Evidence suggests that BCG lacks some immunodominant antigens (e.g., ESAT-6 and CFP-10) encoded by the RD1 region, which is intact in the wild type MTB. Thus, efforts to improve vaccination have included, re-engineered BCG that can express these antigens, besides, DNA vaccines, sub unit vaccines and gene deletion attenuated mutants of Mtb [2] [3]. Vaccines more effective than BCG in animal models are available [4]. However, the parameters that determine protection against tuberculosis still remain unclear because existing vaccines have proved less than ideal to protect non-human primate animal models against tuberculosis [5].

The search for better vaccines for tuberculosis has necessitated an understanding of the immune mechanisms operative in protection. Extensive studies show that immunity to tuberculosis is dependent both on CD4 and CD8 T cells while a non-traditional CD1d-dependent mechanism is also evident [6]. The efficacy of T cell mediated immunity is in turn, dependent upon the ability of antigen presenting cells (APCs) to process and present antigens of pathogens [7,8]. After the initial aerosol implantation of Mtb, infected alveolar macrophages (Mφs) and dendritic cells (DCs) of the respiratory tract secrete cytokines and chemokines that recruit immune cells into the lungs[9]. The initial containment of infection or the granuloma consists of infiltrating elements including T cells, neutrophils and eosinophils [10]. Emerging evidence indicates that, during the primary immune response, DCs that contain Mtb or BCG vaccine migrate to lymph nodes where they prime naïve T cells. Mφs restrict infection at the lungs and can prime locally available immune T cells. Thus, Mφs and DCs play a key role in the development and maintenance of an anti-tuberculosis immune response [11] [12] [9] [3].

HIV-1 induced depletion of CD4 T cells in humans leads to enhanced susceptibility to tuberculosis strengthening the concept that, CD4 T cells are major mediators of protection in humans and relevant to vaccine development. Consequently, T cell-regulatory factors that orchestrate Th1 immunity appear to be important. During the initial induction of Th1 immunity, DCs not only present antigen to naive T cells but also induce T-bet, a transcription factor. T-bet drives IFN-γ synthesis and surface expression of CXCR3 among CD4 T cells, thereby affecting the ability of Th1 T cells to migrate to sites of inflammation and infection [13] [14] [15]. Likewise, antigen primed DCs can also induce either GATA or FoxP3 transcription factors, to respectively induce Th2 and T-regulatory T cells [8]. Mills and others have proposed that the nature of pathogen being phagocytosed by the DCs, secretion of distinct cytokines and stimulation of Toll-like receptors on DCs, together determine the ultimate lineage of naïve T cell differentiation [16] [8] [17,18]. In our earlier study, we found that naïve DCs infected with mycobacteria could drive Th1 differentiation and lead to the expansion of IFN-γ secreting T cells [19,20]. Using the mouse models, we also identified that newer vaccine strains derived from wild type Mtb can protect against tuberculosis. In this study, using in vitro and in vivo models, we have investigated the hypothesis that mycobacterial strains may induce different levels of T-bet transcription factor in T cells and thereby determine the efficacy of Th1 pathway. We propose an additional parameter to evaluate anti-tuberculosis vaccines.

2. Results

Mycobacterial strains induce a variable level of T-bet transcription factor in CD4 T cells of mice

T-bet determines multiple parameters of Th1 immunity including the expansion of IFN-γ+ CD4 and CD8 T cells, and surface expression of chemokine receptors that enable migration of T cells to sites of infection and inflammation [13] [21] [22]. Thus, we initially sought to determine if virulent and attenuated strains of mycobacteria induced T-bet in Th1 T cells that could affect their infection profile. Fig. 1a shows that after i.p. infection, mycobacterial strains induced variable levels of T-bet, with virulent M.tuberculosis inducing the strongest response followed by M.avium, BCG and M. smegmatis. Interestingly, all mycobacteria grew to detectable levels in the spleens by day 7 and then, either increased up to week 4 for Mtb-H37Rv and M.avium or plateaued for BCG and M.smegmatis. On day 7, when bacterial counts were comparable, virulent Mtb-H37Rv had the strongest T-bet induction suggesting that, induction of T-bet responses was not related to bacterial growth, at least over the initial 7 days of infection (Fig. 1b). Splenic T cells were also typed for IFN-γ secreting CD4 T cells. On day 14, Mtb-H37Rv induced elevated levels of CD4 IFN-γ+ T cells, compared to other mycobacteria (Fig. 1c).

Figure 1. Mycobacteria induce a variable level of Th1-regulating T-bet transcription factor in the T cells of mice after intra-peritoneal infection.

Figure 1

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Four to 6 week old C57Bl/6 mice were infected i.p. with various mycobacterial strains at 106 CFU per mouse and either at weekly intervals or on day 14, splenic T cells were stained with fluorescent antibodies for intracellular expression of T-bet and IFN-γ and analyzed using flow cytometry and Cellquest software, and expressed as percent positive T cells. Spleens were also cultured on 7H11 agar at weekly intervals for bacterial counts. A: Mtb-H37Rv induces stronger expansion of T-bet+CD4 T cells. (percent positive T cells ± 3 mice per time point). B: T-bet expression correlates with IFN-γ expressing CD4 T cells (percent positive T cells ± 3 mice on day 14) C: Virulent Mycobacterium tuberculosis (Mtb-H37Rv) and M. avium grow more than BCG or M.smegmatis in the spleens (3 mice per time point per strain; ± SD).

ΔfbpA candidate vaccine induces stronger Th1 responses in mice compared to BCG vaccine

In our previous studies, we demonstrated that an attenuated candidate vaccine derived from Mtb-H37Rv was able to protect mice against tuberculosis better than BCG vaccine [23]. We hypothesized that ΔfbpA induced a stronger Th1 response. Since T-bet is known to affect IFN-γ and CXCR3 expression of Th1 T cells, mice were infected i.p or s.c. with ΔfbpA and BCG followed by weekly T cell analysis. T cell profiles of mice following i.p. or s.c infections were comparable and thus, only i.p. profiles are presented. Flow cytometric analysis indicated that ΔfbpA induced an increased expansion of T-bet+(Fig. 2A), CXCR3+(Fig. 2B) and IFN-γ+CD4 T cells (Fig. 2C) in the spleens of mice, during the 4 weeks of infection. Interestingly, both ΔfbpA mutant and BCG vaccine grew similarly within spleens (Fig. 2D).

Figure 2. The attenuated ΔfbpA mutant derived from Mtb H37Rv induces a stronger Th1 response after intraperitoneal infection of mice compared to BCG vaccine.

Figure 2

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Mice were infected with candidate vaccine strains as in Fig. 1 and splenic T cells were immunotyped for intracellular T-bet, surface expression of CXCR3 and intracellular IFN-γ (3 mice per time point per strain ± SD). ΔfbpA vaccine induces a stronger T-bet+ (A), CXCR3 (B), and IFN-γ+ (C) (* p< 0.01 vs. BCG, t test) CD4 T cell response compared to BCG vaccine. The vaccines showed only a moderate difference in the growth within spleens (D).

Dendritic cells infected with ΔfbpA mutant drive a better recall T-bet response in T cells in vitro compared to BCG vaccine

During an ongoing infection, DCs present antigens to T cells under the regulatory influence of cytokines to recall Th1 expansion. To determine whether APCs infected with ΔfbpA and BCG vaccine strains prime T cells differently, DCs were infected in vitro with ΔfbpA or various sub-strains of BCG and overlaid with splenic T cells from mice infected with ΔfbpA or BCG. T cells were analyzed for T-bet, GATA-3 and FoxP3 and culture supernatants measured for soluble IFN-γ by ELISA. These experiments revealed that ΔfbpA infected DCs drive a stronger recall T-bet response compared to BCG (Fig. 3a), which correlated with a stronger expression of IFN-γ (Fig. 3b). Interestingly, none of the DC cocultures induced a significant GATA-3 or Fox-P3 recall response among immune T cells in vitro (Fig. 3c).

Figure 3. Dendritic cells infected with ΔfbpA vaccine drive a better recall T-bet response in T cells in vitro compared to BCG vaccine.

Figure 3

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Bone marrow derived dendritic cells (DCs) were infected for 4 hr in vitro with ΔfbpA or various sub-strains of BCG as indicated (MOI=1). After washing, DCs were overlaid with splenic T cells (5×106 T cells per 1×106 DCs per well in triplicates for per mouse; 3 mice per strain) from mice infected i.p. 2 weeks earlier with ΔfbpA or various strains of BCG. After 72 hr of coculture, T cells were stained for surface CD3, T-bet, GATA-3 and FoxP3 and culture supernatants measured for soluble IFN-γ by ELISA. A–B: ΔfbpA infected DCs drive a stronger recall T-bet response which correlates with a stronger production of IFN-γ (* p < 0.009 vs. BCG induced T-bet or cytokine; t test). C: None of the DC cocultures induced significant GATA-3 or Fox-P3 responses in T cells in vitro.

The ES antigens of ΔfbpA mutant and BCG vaccine induce T-bet response in T cells

Since mycobacteria induced different levels of T-bet in mice (Fig. 1 & 2) and T cells activated by DCs in vitro (Fig. 3), we sought to determine, if ES antigens of mycobacteria could account for differences in the ability to prime for T-bet. ES antigens are a cluster of secreted antigens capable of modulating Th1 responses to tuberculosis. DCs treated with two major ES antigens were therefore overlaid with T cells from mice immunized with different mycobacteria (H37Rv, H37Ra, BCG and ΔfbpA) and T-bet and IFN-γ expression evaluated using flow cytometry. Fig. 4A. illustrates the recall expansion of IFN-γ+CD4 T cells by DCs treated with ES antigens. Fig. 4B summarizes data from three separate experiments of T cell analysis. First, Ag85B treated DCs uniformly expanded IFN-γ secreting CD4 T cells from mycobacteria. This was anticipated, since it is present in all the mycobacteria tested. T cells from naïve mice overlaid on antigen pulsed DCs yielded <5% of IFN-γ+ T cells, indicating that there was no non-specific expansion. In contrast, DCs with ESAT-6 and CFP-10 failed to drive Th1 expansion among T cells from BCG infected mice, which in turn, correlated with a decreased T-bet induction. It is well established that Mtb-H37Rv, H37Ra and ΔfbpA have intact ESAT-6 and CFP10 while BCG lacks them. Reduced Th1 expansion by BCG infected DCs is therefore likely due to the absence of ESAT6 and CFP10 antigens in the BCG vaccine. We conclude that ES antigens can induce T-bet and differences in the content or expression of ES antigens can alter the ability of mycobacteria to induce Th1 response.

Figure 4. DCs pulsed with excretory-secreted (ES) antigens, Ag85B, ESAT-6 and CFP-10 can drive the recall T-bet and IFN-γ responses in vitro.

Figure 4

Figure 4

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DCs were pulsed for 24 hr in vitro with Ag85B, ESAT-6 or CFP-10 antigens (1 μg/106 DCs in triplicate wells for per mouse; 3 mice per strain), washed and were cocultured with T cells from spleens of mice infected i.p. 2 weeks earlier with ΔfbpA, H37Rv, H37Ra or BCG Pasteur strains (106 CFU per mouse, 3 mice per strain). After 72 hr, the cocultured T cells were stained for intracellular T-bet, IFN-γ and supernatants tested for IFN-γ by ELISA. A: Histograms illustrate that DCs pulsed with Ag85B, ESAT-6 and CFP-10 induce a better recall expansion of IFN-γ+ CD4 T cells in ΔfbpA immunized mice compared to T cells from Mtb H37Rv, H37Ra or BCG immunized mice. B: Cocultured T cells were stained for IFN-γ+ T-bet+ CD4 T cells and analyzed by flow cytometry using a BD Facscalibur and Cellquest software. Top two panels show that ES antigens drive a strong expansion Th1 expansion reflected by an increase in IFN-γ+ CD4+ T cells and secretion of IFN-γ into medium. T cells from naïve mice activated by DCs showed < 5% IFN-γ+ T cells. ESAT-6 and CFP-10 fail to drive Th1 expansion among T cells from BCG infected mice, while Ag85B alone was effective to expand BCG primed T cells. ES antigens also induced an expansion of T-bet in T cells but not in BCG primed T cells.

ΔfbpA mutant offers better protection than BCG vaccine in mice against virulent challenge with Mtb

Following aerosol infection of mice with Mtb, Th1 T cells infiltrate into lungs and help to contain infection through the secretion of IFN-γ and other cytokines. The ability to infiltrate into lungs is dependent upon the expression of chemokine receptors such as CXCR3 for Th1 T cells. We sought to determine if ΔfbpA or BCG which induce different types of Th1 differ in their ability to protect against tuberculosis. Thus, mice were immunized s.c. with ΔfbpA or BCG and challenged four weeks later with low dose aerosol (<100 CFU per mouse) using Mtb-Erdman strain. Fig. 5A illustrates that on day 28 after challenge, there were more IFN-γ secreting CD4 T cells in the lungs of mice vaccinated with ΔfbpA mutant than those with BCG vaccine or control groups. Such mice also showed a better protection against tuberculosis indicated by a decline in the bacterial counts of lungs (Fig. 5B). Fig. 5C summarizes the flow cytometric data of mouse lungs analyzed weeks after challenge. ΔfbpA vaccinated mice showed uniform increase in the number of CXCR3+IFN-γ+CD4 and CD8 T cells.

Figure 5. ΔfbpA mutant offers better protection than BCG vaccine against tuberculosis in mice which correlates with accumulation of CD4+CXCR3+IFN-γ+ T cells.

Figure 5

Figure 5

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C57Bl/6 mice (4–6 weeks) were immunized s.c. with ΔfbpA or BCG strains at 106 CFU per mouse and challenged 4 weeks later via aerosol with 100 CFU of Mtb-Erdman strain per mouse. Mice were sacrificed and lung homogenates were plated on 7H11 agar for bacterial counts or lung-derived cells analyzed for T cell phenotype. A: Lung harvested T cells were stained for intracellular IFN-γ on day 28 post challenge. Histograms show IFN-γ+CD4+T cells among naïve, naïve infected with Mtb-Erdman and vaccinated mice challenged with Mtb-Erdman. ΔfbpA vaccine induces a stronger Th1 response (Inset numbers show percent positive IFN-γ+CD4+T cells; 3 mice per group). B: ΔfbpA vaccine protects mice better than BCG (4 mice for CFU per time point, per group; *< 0.007 vs. BCG or naïve group on week 4; ANOVA, 2 experiments; SEM). C: At weekly intervals after challenge mice were sacrificed and lung cell stained for surface phenotype (CD4 or CD8 and CXCR3) in combination with intracellular IFN-γ using three colored antibodies followed by flow cytometric analysis. Increased protection by ΔfbpA vaccine correlates with a better accumulation of IFN-γ+CXCR3+ T cells in the lungs after aerosol challenge (numbers per lungs; 3 mice per group per time point; SD; one experiment shown of similar 2 experiments).

3. Discussion

Th1 immunity is known to be protective against tuberculosis and anti-tuberculosis vaccines induce Th1 signatures in animal models. Use of gene knock out strains of mice has shown that the Th1 response mediated by IFN-γ is a critical determinant of anti-tuberculosis immunity, while other cytokines like TNFα and IL-2 also play a major role [24]. It is generally held that Th1 immunity is initiated by immature DCs that upon engulfment of microbes migrate to lymphoid organs and within the cytokine rich milieu, process antigens from microbes and prime naïve T cells. The latter develop into IFN-γ secreting CD4 and CD8 Tells that also express appropriate homing receptors to enable to migrate into sites of infection and inflammation. While T-bet induction was found necessary for IFN-γ production, it also determines the efficacy with which T cells express CXCR3. Thus, efficacy of Th1 immunity seems to depend upon T-bet.

We demonstrated that mycobacteria differ in their ability induce T-bet in T cells of mice after infection. Curiously, virulent Mtb induced stronger T-bet and significant IFN-γ responses in mice. This observation can be explained as follows. First, virulent Mtb appears to have unique survival mechanisms. The ability of Mtb to remain sequestered in semi-permeable phagosomes of Mφs is well known. Mtb also has the ability to down-regulate the antigen processing mechanisms including, expression of MHC-II, CD1d, costimulatory molecules and alter the sensitivity of Mφs to IFN-γ [25] [26] [27] [28] [29]. In addition, Mtb derived lipids and lipoproteins have been reported to alter the phenotype of DCs, affect maturation and suppress or alter T cell priming function [30] [31] [32]. Since Mtb infects predominantly through aerosol route, we suggest that the minimal numbers of Mtb in respiratory tract may parasitize the Mφs and DCs and prevent efficient generation of Th1 immunity through evasion mechanisms. The significant peak of T-bet response in mice to Mtb we observed therefore seems to be due to size of the inoculum given into the peritoneal cavity, that enables rapid access of Mtb to APCs and T cells (Fig. 1). It should be noted that the growth of Mtb and M.avium continued despite Th1 response after day 7, while, BCG and M.smegmatis declined. It therefore appears that, the pathogens Mtb and M.avium have virulence factors that enable their growth while attenuated BCG and M.smegmatis are controlled by Th1 mechanisms. These observations indicate that, homologous Th1 responses fail to protect against tuberculosis while such responses effective against the infection with BCG vaccine strain. Paradoxically, BCG vaccine is only moderately effective against tuberculosis in C57Bl/6 mice, and prior vaccination yields about 1-log10 decrease in the growth of Mtb within lungs of challenged mice[5,33]. We therefore hypothesized that a better vaccine to prevent tuberculosis perhaps needs to prime for a stronger and longer lasting, Th1 immunity.

BCG vaccine is an attenuated derivative of M.bovis and known to induce significant CD4 T cell responses, with moderate CD8 T cell responses in mice. Since CD4 T cells seem to protect against acute tuberculosis infection and CD8 T cells against chronic or latent stages of tuberculosis, BCG has been modified to stimulate better CD4 and CD8 responses. Early research showed that BCG induced CD8 T cell response in mice can be enhanced through addition of listeriolysin, that seems increase release BCG antigens from phagosomes into the cytosol [34] [35,36]. Others have cloned several proteins of wild type Mtb into BCG, which led to an increase in the efficacy of the vaccine. Recombinant BCG (rBCG) expressing ESAT-6, Ag85B and LLO is a major, improved candidate vaccine under human trials. Even with these improvements, however, the immunological basis for protecting against tuberculosis remains far from clear. For example, many vaccines that protect better against mouse tuberculosis do not protect non-human primates against tuberculosis. We sought an alternate to BCG vaccine by evaluating an attenuated candidate vaccine from Mtb-H37Rv. The rationale was that the mutant would have nearly all antigens of wild type Mtb and those missing in BCG such as RD1 encoded antigens may help to confer better protection. Our previous studies showed that the ΔfbpA mutant protects mice better than BCG [23].

In this study, we observed that ΔfbpA mutant induced better Th1 responses in mice compared to BCG vaccine. First, infection studies in mice demonstrated that ΔfbpA induced enhanced levels of T-bet positive T cells that also secreted IFN-γ and expressed CXCR3 (Fig. 2 & 3). A striking observation was that the ES antigens of ΔfbpA were capable for priming for recall T-bet expression in T cells and amplify IFN-γ responses (Fig. 4). Finally, ΔfbpA mutant offered a better protection against tuberculosis in mice that correlated with a better accumulation of CXCR3+IFN-γ+CD4 and CD8 T cells in the lungs of vaccinated and Mtb challenged mice (Fig. 5).

It is apparent that the presence of ESAT-6 and CFP-10 (ES) antigens in Delta;fbpA, is likley responsible for the better immunogenicity of ΔfbpA mutant vaccine. These antigens are deleted in BCG and are known to carry major T cell epitopes and capable of boosting anti-tuberculosis immunity in mice. A second possibility for the increased efficacy of ΔfbpA candidate vaccine centers on its ability to undergo limited phagosome maturation. Using an in vitro antigen-85B presentation system to T cells, we found that APCs with BCG vaccine had a reduced efficacy in presenting Ag85B to T cells in vitro compared to those infected with ΔfbpA mutant[37]. Since DCs and process and present peptide antigens to prime the Th1 immunity [38] [7] [39] [8], it seems reasonable to propose that the ability of vaccines to undergo efficient processing in APCs can affect their immunogenicity. In summary, we have demonstrated that novel attenuated strains of Mtb can induce qualitatively and quantitatively different Th1 responses in mice that translate into better protection against tuberculosis. If safety can ensured, attenuated Mtb-derived vaccines can perhaps replace BCG vaccine.

4. Methods

DCs

C57Bl/6 mice derived bone marrow DCs were prepared by culturing bone marrow cells in IDM with 10 ng/mL mouse GM-CSF or 10% culture supernatant from NIH-3T3 fibroblasts secreting IL-4 (Kind gift of Kammertoens T, Institute of Immunology, Charite Campus, Benjamin Franklin, Berlin, Germany). After 7 days, the non-adherent cells were gently flushed and further purified using CD11c magnetic beads (Miltenyi Inc, USA). Resulting cells were more than 98% pure as analyzed by flow cytometry. The DCs were immunophenotyped with fluorophore labeled antibodies and largely found to contain CD8+ with lesser numbers of double negative DCs, all of which were CD11c positive but F4/80 negative, thus excluding contamination with macrophages.

Mycobacteria

Stock cultures from ATCC were grown in Dubos’ broth and used fresh after three washes in PBS. They were routinely > 90% viable as evaluated by fluorescein diacetate stains. Wild type Mtb-H37Rv (# 27294), and BCG (# 35734) were prepared from ATCC stock. Mtb-Erdman with acriflavine resistance gene was a kind gift of Dr. D. Kernodle from Vanderbilt University. ΔfbpA mutant (cultured in 25 μg/ml kanamycin medium) has been described by us before and were cultured in Dubos’ broth with 25 μg/mL kanamycin [40]. BCG Pasteur and other sub strains were obtained from ATCC (MD). All mycobacteria were grown in 7H9 broth to log phase and frozen stored as > 90% viable suspensions until use.

Recombinant antigens

Early secreted antigenic target (ESAT-6) and culture filtrate protein (CFP-10) were expressed and purified as described earlier [41]. They were ultra-purified to enable bio-physical studies on complex formation between ESAT-6 and CFP-10 (kind gift of Dr. Arora CDRI India).

Immune and naïve T cells

Mycobacteria primed mice

Live Mtb-H37Rv, Mtb-H37Ra, BCG or ΔfbpA were given i.p. (or s.c) at 106 CFU as one dose on day 0 to C57Bl/6 mice of 4–6 weeks age and mice were sacrificed for T cells 2 or 4 weeks later. For each immunization, 6 mice were used and CD3+ T cells from mouse spleens were cocultured with DCs. The entire experiment was repeated 3 times. Splenic T cells from immune and naïve non immunized mice were negatively isolated using a Pan-T cell kit (Miltenyi Inc, USA) and used for coculture experiments with DCs.

DC-T cell cocultures and Th1 recall expansion

The purified DCs were washed and plated into 6-well plates. DCs were either treated with antigens as indicated or were infected for 4 hrs with mycobacteria an MOI of 1:1 and washed before coculture with T cells. The splenic CD3+ T cells from immunized mice or naïve mice were cocultured with the DCs (5×106 T cells per 1×106 DCs, triplicate cultures per mouse, 3 mice per strain) [42] [19]. At the end of 72 hr coculture, dead cells were removed on Ficoll-Hypaque columns and viable cells were stained for flow cytometry using antibodies as needed. IFN-γ levels were determined using a sandwich ELISA kit from R & D.

Vaccine model

C57Bl/6 mice were immunized with one dose of either BCG or ΔfbpA mutant orleft unvaccinated (naïve). They were challenged after 4 weeks with virulent acriflavine resistant Mtb-Erdman strain implanting about 100 CFU per lungs. Mice were sacrificed weekly for colony counts of Mtb-Erdman on acriflavine containing 7H11 agar plates over the next 4 weeks. Mice were sacrificed separately for CFU counts and T cells. The lung derived cells were immunotyped for the Th1-transcription markers T-bet, CXCR3 and intracellular IFN-γ as previously described [19]. Total lung cells were counted before staining and the numbers of T cells positive for markers were enumerated using flow cytometry [19] [43] [44].

Flow Cytometry

DCs were typed for purity using CD11c (Integrin αx chain), while T cells were typed for both surface markers (CD4, CD8) as well as intracellular cytokines as follows. DCs were gated based on forward scatter and isotype control of Rat IgG2a (Caltag Laboratories, R2a04) was used. DCs were dispensed at 105 cells per pellet and added with Fc Receptor Blocker (CD16/32, Caltag Laboratories, MFCR00) and incubated for 15 min on ice. The appropriate antibody (CD11c) was added to each sample and incubated for 30 min in the dark. The cells were washed and then fixed in 2% paraformaldehyde for 30 min before acquisition using a BD FACSCAN. DCs were also stained with F4/80 to rule out contamination with Mφs. The intracellular staining for T cells was performed as follows. DC-T cell cocultures were treated on the day of harvest with Golgi stop and Golgi plug (GolgiStop 554715, BD Biosciences) for 6 h. T cells harvested on ficoll columns from cocultures were added with Fc Receptor Blocker (CD16/32; Caltag Laboratories, MFCR00) and incubated on ice for 15 min. The appropriate antibodies were added to each sample (CD4, L3T4; Caltag Laboratories MCD0401 or MCD0404), CD8α (Ly-2; Caltag Laboratories RM2201-3 or MCD0804-3) and incubated for 30 min in the dark. The cells were washed and then fixed with 2.7% paraformaldehyde for 30 min, followed by staining with anti-IFN-γ (Caltag Laboratories RM9001-3) diluted in a perm buffer containing 0.1% Triton-X 100, 0.1% Saponin, 0.001% Digitonin, and PBS for 2 h or overnight at 4°C. The samples were washed in PBS and acquired using BD FACSCAN and Cellquest software.

Acknowledgments

This study was supported by NIH AI49534.

Key words

Mtb-H37Rv

Strain of Mycobacterium tuberculosis

ΔfbpA

fibronectin binding protein A deletion mutant

BCG

M.bovis BCG Bacille Calmette-Guérin vaccine

Ag85B

Antigen 85 complex B protein

ESAT-6

Early secreted antigenic target-6

CFP-10

Culture filtrate protein-10

Footnotes

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