Polyclonal activation of naïve T cells by urease deficient-recombinant BCG that produced protein complex composed of heat shock protein 70, CysO and major membrane protein-II
© Tsukamoto et al.; licensee BioMed Central Ltd. 2014
Received: 25 October 2013
Accepted: 21 March 2014
Published: 2 April 2014
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs.
Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags).
BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB.
The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation.
KeywordsTuberculosis BCG T cell activation
Tuberculosis is a chronic infectious disease, induced by intracellular infection with MTB and is responsible for around 1.4 million deaths yearly worldwide [1–3], and it is estimated that one-third of the global populations is latently infected with MTB. The emergence and worldwide spread of multidrug-resistant strains of MTB mandates the development of more effective preventive and therapeutic tools . Studies using T cell receptor transgenic mice specific for Ag85B-derived CD4+ T cells epitope which is one of the most useful animal model for understanding host defense mechanisms against MTB clearly demonstrated that the MTB-susceptible APCs including DC and macrophages need 7–10 days to initiate activating type 1 CD4+ T cells and CD8+ T cells in regional lymph nodes after aerosol MTB infection, and the stimulated T cells need 4–5 weeks to initiate inhibiting the growth of MTB in primary mice [5, 6]. However, after the adequate activation of both subsets of T cells, the number of MTB residing in the lung remains stable [5, 6]. These observations clearly demonstrate that type 1 T cells are essential elements in inhibiting the multiplication of MTB and also that lack of memory type T cells capable of reacting to MTB-infected APCs strongly and immediately allows MTB to multiply. In addition to the animal studies, in vitro studies using human APCs or T cells reveal that host defense against MTB is conducted chiefly by type 1 CD4+ T cells and CD8+ T cells [7–9]. Among them, IFN-γ produced from both subsets of T cells is considered as one of the most important element for mycobacteriocidal action , and cytotoxic T lymphocytes differentiated from the activated CD8+ T cells act chiefly at late stage of MTB infection [1, 11, 12]. The killing process of MTB-infected APCs is via a granule-dependent mechanism [13, 14]. Although BCG has been used as a vaccine against tuberculosis widely, BCG cannot prevent the manifestation of adult lung tuberculosis . The major reason why BCG is not fully functional, remains to be elucidated. BCG, as a vaccine, is not convincing in terms of T cell activation, since BCG activates naïve CD4+ T cells substantially, but poorly activates naïve CD8+ T cells [16, 17]. The reason why BCG cannot activate naïve T cells fully, may be based on the lack of the ability to induce phagosomal maturation [18–20]. Therefore, improvement of T cell-stimulating ability of BCG is strongly required. Presently various new protein vaccine candidates have been selected for clinical trials [18–20]. Actually the vaccine candidates including early secretory antigenic target-6, culture filtrate protein 10, Ag85 family and polyprotein Ag designated Mtb72F and a fusion protein ID93 [9, 21–26] are produced based on Ags that are recognized in infected individuals. However, fully reliable new vaccine has not been established yet.
Recently, we have produced recombinant BCG, termed BCG-DHTM which strongly activated human naïve CD4+ T cells and naïve CD8+ T cells, and, in mice, produced T cells responsive to H37Rv-derived cytosolic protein . In the production of BCG-DHTM, we employed two independent strategies in order to overcome the intrinsic defect of BCG, that is an ability to block phagosome maturation to inhibit processing of Ag and presentation to type 1 T cells. One of the strategies is inactivation of ureC gene of BCG, which encodes urease, from BCG [19, 20]. The urease produces ammonia from urea and inhibits the phagosomal acidification. The urease depletion facilitates the translocation of BCG to lysosome, and enhanced the ability of BCG to activate human naïve CD4+ T cells [19, 20]. The other one is the intracellular secretion of antigenic molecule. As the key antigen, we used MMP-II, since MMP-II is recognized by human T cells after infection with M. leprae or BCG, and can ligate Toll like receptor (TLR)2 and consequently activate both DC and macrophages [28–31]. Also we used HSP70, since HSP70 has a chaperon activity and can prime cytotoxic T lymphocytes. The intraphagosomal secretion of HSP70-MMP-II fusion protein induced strong activation of naïve CD4+ T cells and CD8+ T cells . Since both strategies, that is urease depletion and an intracellular secretion of HSP70-MMP-II fusion protein, worked synergistically in terms of T cell activation, the gene encoding the HSP70-MMP-II fusion protein was introduced into urease-depleted rBCG (BCG-ΔUT-11-3) for production of BCG-DHTM .
Although BCG-DHTM activated both subsets of T cells to produce IFN-γ, production of memory T cells capable of responding to MTB-derived molecules which can be induced chiefly in the activating phase of MTB growth, is needed for the tuberculosis protection. To address this point, we selected CysO (Rv1335 or CFP10A) as the target gene, since CysO also participates in cysteine biosynthesis pathway in MTB . MTB has two independent cysteine biosynthesis pathways, namely the conventional pathway and the alternative pathway. CysO is engaged in the alternative pathway. This pathway is more advantageous under the oxidative conditions. Indeed, CysO expression is induced under diamide stress, one of the oxidative stress conditions . This implies that CysO may be essential for MTB to survive within macrophages, since MTB is exposed to oxidative stress in macrophages . Furthermore, CysO expression is repressed in hypoxic condition and induced in reaeration condition, indicating that CysO may be essential for MTB in the growth phase . Moreover CysO is categorized under ubiquitin superfamily, and may direct protein towards proteasome degradation pathway, which is essential for many cellular processes . Therefore, the CysO involvement in improved cellular response against MTB within macrophages or in growth phase was considered.
In this study, we introduced CysO gene in combination with HSP70-MMP-II fusion gene into urease-deficient BCG-ΔUT-11-3, and produced new rBCG termed BCG-dHCM, and evaluated its T-cell stimulating activities.
Preparation of cells and Ags
Peripheral blood was obtained from healthy PPD-positive individuals under informed consent. The study was approved by the ethics committee of the National Institute of Infectious Diseases, Tokyo. In Japan, BCG vaccination is compulsory for children (0 ~ 1 year-old). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and cryopreserved in liquid nitrogen until use, as previously described . The viability of T cells obtained from cryopreserved PBMCs was more than 90% and no selection in terms of functionality was induced in both monocytes, a precursor of DC and macrophages, and T cells. For the preparation of peripheral monocytes, CD3+ T cells were removed from either freshly isolated heparinized blood, or cryopreserved PBMCs using immunomagnetic beads coated with anti-CD3 monoclonal antibody (mAb) (Dynabeads 450; Dynal Biotech, Oslo, Norway). The CD3- PBMC fraction was plated on tissue culture plates and the non-plastic adherent cells were removed by extensive washing. The remaining adherent cells were used as monocytes . Monocyte-derived DC were differentiated as described previously [38, 40]. Briefly, monocytes were cultured in the presence of 50 ng of rGM-CSF (Pepro Tech EC LTD, London, England) and 10 ng of rIL-4 (Pepro Tech) per ml . On day 4 of culture, immature DC were infected with rBCG at an indicated multiplicity of infection (MOI) and, on day 6 of culture, DC were used for further analyses of surface Ag and for mixed lymphocyte assays. Macrophages were differentiated as described previously [41, 42]. In brief, monocytes were cultured in the presence of 10 ng of rM-CSF (R & D Systems, Inc., Minneapolis, MN) per ml. On day 5 of culture, macrophages were infected with rBCG at an indicated MOI and, on day 7 of culture, they were used for further analyses of surface Ag and mixed lymphocyte assay. The rMMP-II protein was produced as described previously [28, 43], and the CysO protein was produced in an LPS-free condition by using M. smegmatis. Polyclonal Ab against CysO was produced by immunizing rabbit with the recombinant protein. The rHSP70 protein was purchased (Hy Test Ltd., Turku, Finland) and H37Rv-derived cytosolic protein was produced as described previously .
Vector construction and preparation of rBCG
The genomic DNAs were obtained from BCG substrain Tokyo and from MTB H37Rv strain. The oligonucleotide primers used for the amplication of hsp70 gene were F-Mb70Bal (5′-aaaTGGCCAtggctcgtgcggtcggg-3′) and R-Mb70Eco (5′-aaaGAATTCcttggcctcccggccg-3′). MMP-II sequence from MTB genomic DNA was amplified with primers: F-MMP TB Eco (aattGAATTCatgcaaggtgatcccgatgt) and R-MMP TB Sal (5′-aattGTCGACtcaggtcggtgggcgaga). CysO sequence from MTB genomic DNA was amplified with primers: F-CysO (5′-ggccgggaggccaagaacgtcaccgtatccattcc-3′) and R-CysO: (5′-atcgggatcaccttgcccaccggccacggcgggga-3′). The amplified HSP70 and MMP-II sequence were digested with appropriate restriction enzymes and cloned into parental pMV261H plasmid. For the cloning of CysO sequence into pMV261, In-Fusion HD cloning kit (Clontech laboratories, Mountain View, CA) was used. For replacing kanamycin resistance gene to hygromycin resistance cassette, the Xba I-Nhe I fragment from pYUB854  was cloned into Spe I-Nhe I fragment of the plasmid . The rBCG (BCG-ΔUT-11) of which ureC gene was disrupted, was produced as described previously . The hygromycin cassette in the BCG-ΔUT-11 was removed by using pYUB870 encoding γδ-resolvase (γδ-tnpR) . The unmarked BCG was named BCG-ΔUT-11-3. The HSP70-CysO-MMP II fusion protein expressing vector was introduced into BCG-ΔUT-11-3 by electroporation method. BCG-ΔUT-11-3 containing pMV-HSP70-CysO-MMP-II as an extrachromosomal plasmid is referred to as BCG-dHCM, and that containing pMV-261-hygromycin is referred to as BCG-261H (BCG vector control). Recombinant BCGs and MTB H37Rv strain were grown to log phase, and stored at 108 colony forming unit (CFU)/ml at -80°C. Before infection to DC and macrophages, BCGs were counted by colony assay method. There is no significant difference in the in vitro culture growth between BCG-261H and BCG-dHCM.
Analysis of cell surface Ag
The expression of cell surface Ag on DC was analyzed using FACSCalibur (BD Bioscience, San Jose, CA). Dead cells were eliminated from the analysis by staining with propidium iodide (Sigma-Aldrich, St. Louis, MO) and 1 × 104 live cells were analyzed. For the analysis of the cell surface Ag, the following mAbs were used: FITC-conjugated mAb against HLA-ABC (G46-2.6, BD Bioscience), HLA-DR (L243, BD Bioscience), CD86 (FUN-1, BD Bioscience), and CD83 (HB15a, Immunotech, Marseille, France).
APC function of DC
The ability of DC infected with BCG or pulsed with recombinant protein and BCG-infected macrophages to stimulate T cells was assessed using an autologous APC-T cell co-culture as previously described [40, 45]. Purification of CD4+ and CD8+ T cells was conducted by using negative-isolation kits (Dynabeads 450, Dynal Biotech) . The purity of the CD4+ and CD8+ T cells was more than 95% when assessed using FACSCalibur. Naïve CD4+ and CD8+ T cells were produced by further treatment of these T cells with mAb to CD45RO, which were followed by beads coated with goat anti-mouse IgGs Ab (Dynal Biotech). The purity of both subsets of naïve T cells was more than 97%. However, there was no contamination of memory type T cells in the naïve T cell preparations. More than 98% of CD45RA+ T cells was positive in the expression of CCR7 molecule. Memory type T cells were similarly produced by the treatment of cells with mAb to CD45RA Ag. The purified responder cells (1 × 105 per well) were plated in 96-well round-bottom tissue culture plates, and APCs infected with rBCG or pulsed with protein were added to give the indicated APC: T cell ratio. Supernatants of APC-T cell co-cultures were collected on day 4 and the cytokine levels were determined. In some cases, rBCG-infected DC and macrophages were treated with mAb to HLA-ABC (W6/32, Mouse IgG2a, kappa), HLA-DR (L243, Mouse IgG2a, kappa), CD86 (IT2.2, Mouse IgG2b, kappa, BD Biosciences) or normal mouse IgG. Also, in some cases, immature DC and macrophages were treated with 50 μM of chloroquine (Sigma-Aldrich) for 2 h and subsequently infected with BCG-dHCM. The optimal dose of the Abs and reagents was determined in advance.
Measurement of cytokine production
Levels of the following cytokines were measured; IFN-γ produced by CD4+ and CD8+ T cells, and IL-12p70, IL-12p40, TNFα, IL-1β and GM-CSF produced by DC or macrophages stimulated for 24 or 48 h with rBCGs. The concentrations of these cytokines were quantified using the enzyme assay kits, Opt EIA Human ELISA Set (BD Bioscience).
For inoculation into mice, rBCG and MTB H37Rv strain were cultured in Middlebrook 7H9 medium supplemented with Middlebrook ADC enrichment to log phase and stored at 108 CFU/ml at -80°C. Before the aliquots were used for inoculation, the concentration of viable bacilli was determined by plating on Middlebrook 7H10 agar plate supplemented with Middlebrook OADC enrichment. Three 5-week-old C57BL/6 J mice (Clea Japan Inc., Tokyo, Japan) per group were inoculated subcutaneously with 0.1 ml of PBS or PBS containing 1 × 103 rBCGs. The animals were kept in specific pathogen free conditions and were supplied with sterilized food and water. Four or 12 weeks after inoculation, the spleens were removed and the splenocytes were suspended at a concentration of 2 × 106 cells per ml in culture medium. The splenocytes were stimulated with an indicated concentration of rMMP-II, rHSP70 (HyTest), rCysO or H37Rv-derived cytosolic protein in triplicates in 96-well round bottom microplates [20, 30]. The individual culture supernatants were collected 3–4 days after stimulation and IFN-γ was measured using Opt EIA Mouse ELISA Set (BD Bioscience). For observing the effect of BCG vaccination on MTB infection, five C57BL/6 mice per group were vaccinated with 1 × 103 CFU/mouse either BCG-261H or BCG-dHCM for 6 weeks, and were challenged with 100 CFU/lungs of H37Rv by aerosol infection using an automated inhalation exposure apparatus (Glas-Col Corp., IN, Model 099C A4212). Six weeks later, bacterial burden in lungs and spleen was assessed by mechanical disruption in PBS with 0.05% v/v Tween 80 and enumerated by colony assay. Animal studies were reviewed and approved by the Animal Research Committee of Experimental Animals of the National Institute of Infectious Diseases, and were conducted according to their guidelines.
Student’s t-test was applied to determine the statistical differences.
Immunological characterization of rCysO protein
Activation of Ag-presenting cells by BCG-dHCM
Activation of naïve and memory type T cells by BCG-dHCM
Production of T cells responsive to the secondary stimulation by BCG-dHCM in vivo
Effect of BCG-dHCM vaccination on the multiplication of aerosol challenged M. tuberculosis
IFN-γ produced by activated type 1 CD4+ T cells and CD8+ T cells has an essential function in both antimicrobial activity against MTB and limitation of lung inflammation associated with massive accumulation of neutrophils which are recruited by Th17 cells [7–9, 46]. Therefore, one of the important aims of vaccination is to produce memory type CD4+ T cells as well as memory type CD8+ T cells capable of producing abundant dose of IFN-γ by responding rapidly to MTB-infected DC and macrophages. From the studies using T cell receptor-transgenic animal model, it is known that it needs several weeks for host T cells to start inhibiting the multiplication of aerosol infected MTB in primary mice [5, 6]. It is unknown whether alveolar APCs could be primed by vaccination, so that time required for APC to move to the regional LN after phagocytosing MTB in lung could be reduced. However, it could certainly reduce the time necessary for T cells to be activated by the interaction with MTB-infected APCs in the LN by differentiating naïve T cells into memory subset by vaccination. BCG has been used widely as a vaccine against tuberculosis, but its effect is quite limited. BCG can only prevent the development of miliary tuberculosis and tuberculosis meningitis in child, but is not effective for prevention of adult lung tuberculosis . However, BCG has many antigenic molecules common to that present in MTB, and moreover, the safety of BCG is well established [16, 17]. Thus, we are of the opinion that the improvement of BCG by producing rBCGs would be the fastest route to produce more reliable single injection vaccine against tuberculosis. In this respect, rBCG should have some antigenic molecules which are present in MTB, and also should highly activate not only naïve T cells of both CD4 and CD8 subsets, but also APCs including DC and macrophages. Since both DC and macrophages express MMP-II-related peptide on their surface upon an infection with both H37Ra and H37Rv , and also MMP-II can ligate TLR2 and consequently activates NF-κB pathway of APCs [28–31], MMP-II is considered to be good target that could be used as an active vaccinating agent. In this study, we produced new rBCG termed BCG-dHCM, that is a urease-deficient rBCG that secrete the fusion protein composed of HSP70, CysO and MMP-II. In the production of BCG-dHCM, we used MMP-II as a central component, HSP70 as an adjuvant, and CysO as an element necessary to release Ag in APCs. BCG-dHCM secreted the fusion protein in both lysosome in which abundant enzyme is available and in phagosomes of BCG-susceptible cells. Thus BCG-dHCM strongly activated both subsets of naïve T cells and APCs. The efficient activation of APCs by BCG-dHCM is revealed through up-regulation of APC-associated molecules and by enhanced production of various proinflammatory cytokines including IL-12, IL-1β and TNFα from DC and macrophages. The efficient activation of these APCs can be assumed to be directly linked with the effective activation of adaptive immunities. Actually macrophages-infected with BCG-dHCM activated memory type CD4+ T cells. Previous rBCG termed BCG-70M that is BCG introduced with HSP70-MMP-II fusion gene failed to activate memory type T cells through macrophages , thus, the high immunostimulatory function of BCG-dHCM seems to be owing to the high antigenic load on the surface of BCG-dHCM-infected macrophages, that is due to efficient translocation of BCG-dHCM into lysosome. The effective memory T cells are ones capable of responding to the molecules expressed on the surface of MTB-infected APCs, and are produced by activating naïve T cells in an Ag-specific manner [27, 31]. In this respect, the activation of both naïve CD4+ T cells and naïve CD8+ T cells by BCG-dHCM was dependent on MHC and CD86 molecules expressed on APCs. Thus, BCG-dHCM seems to be stimulating naïve T cells in an Ag-specific fashion. Presumably, APCs infected MTB express various epitopes on their surface, indicating that the presence of clonal diversity of T cells, might provide better control of MTB. Subcutaneous inoculation of BCG-dHCM into C57BL/6 mice produced long lasting T cells responsive to in vitro secondary stimulation. Upon stimulation with not only MMP-II, CysO and HSP70, but also H37Rv-derived cytosolic (Figure 6) and membrane (not shown) protein, T cells obtained from mice inoculated with BCG-dHCM were efficiently reactivated and produced high concentration of IFN-γ. Although the exact reason why these T cells were capable of responding to MTB components is not clear, it may be reasonable to speculate that the translocation of BCG-dHCM into lysosome induced the degradation of BCG that leads to production of epitope derived from BCG itself. CysO is engaged in alternative cysteine biosynthesis pathway, which plays an essential role for the MTB survival in the oxidative condition [33, 34]. Also, CysO is categorized in ubiquitin superfamily and is possible to direct protein towards proteosome degradation pathway . In fact, in this study, we found that rCysO, produced by using M. smegmatis in a LPS-free condition, activated both innate and adaptive cellular responses, since it induced some cytokine production from APCs and phenotypic changes in DC, and also stimulated IFN-γ production from both subsets of T cells. When we introduced CysO gene into urease-deficient rBCG accompanying with HSP70-MMP-II fusion gene, CysO was produced as a part of fusion protein, in fact, the immunogenic function of CysO may be up-regulated because of the chaperone activity of HSP70, that may lead to production of CysO-specific memory T cells in vivo, although we could not elucidate the detailed mechanisms of the immunological function of CysO. Actually, mice inoculated with BCG-dHCM produced T cells that respond to CysO vigorously. These results indicate that CysO protein was certainly secreted from rBCG in APC and was used for T cell activation. CysO expression in MTB is inhibited in hypoxic condition and is induced by reaeration , thus, CysO may be highly expressed in MTB in active phase, and, also, is involved in the survival of MTB in macrophages. Therefore, production of CysO reactive T cells may be advantageous for the induction of host defense reaction against MTB which is re-activated in macrophages. In this study, we used small dose of BCG, one fifth of usual dose , and by which the parent BCG cannot inhibit MTB growth in lungs in our hands (not shown), for vaccination of mice in order to elucidate the difference between BCG-261H and BCG-dHCM. As expected BCG-261H did not inhibit the MTB growth, but even a small dose of BCG-dHCM at least partially inhibits multiplication of MTB in both lungs and spleen. In this respect, BCG-dHCM seems to be superior to BCG-261H. The reason why BCG-261H did not inhibit the multiplication of MTB may be because we tested bacterial burden in lung and spleen at 6 wks, but not 4 wks which is a frequently used time point , after MTB challenge, because report suggests that 4 ~ 5 wks are necessary to reach the stable level of pulmonary bacterial burden even in naïve mice . Also, due to BCG strain differences, there may be differences in protective effect in mice experiments. However, more detailed studies, for example, the difference between BCG-DHTM and BCG-dHCM, and the relation between the presence of CysO-reactive T cells and MTB replication in macrophages, are absolutely required, to prove the usefulness of CysO involvement for the production of vaccine against tuberculosis.
These results indicate that the secretion of polycomponent antigenic molecules can efficiently produce polyclonal Ag-specific T cells responsive to secondary stimulation in vivo, and may provide one possible tool for the development of better vaccine against tuberculosis.
Mycobacterium bovis BCG
Heat shock protein
Major membrane protein
Major histcompatibility complex
Peripheral blood mononuclear cell
Multiplicity of infection
Colony forming unit.
We thank Ms. M. Kujiraoka for her technical support, and the Japanese Red Cross Society for kindly providing PBMCs from healthy donors. This work was supported in part by a Grant-in-Aid for "Research on Emerging and Re-emerging Infectious Diseases" (Grant no. H24-Shinko-Ippan-009 and H25-Shinko-Ippan-005 to M.M) from the Ministry of Health, Labour, and Welfare of Japan.
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