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Immunotherapy for tuberculosis: future prospects
review-article
Publication date (Electronic):
20 April 2016
Journal:
ImmunoTargets and Therapy
Publisher:
Dove Medical Press
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Abstract
Tuberculosis (TB) is still a major global health problem. A third of the world’s population is infected with Mycobacterium tuberculosis. Only ~10% of infected individuals develop TB but there are 9 million TB cases with 1.5 million deaths annually. The standard prophylactic treatment regimens for latent TB infection take 3–9 months, and new cases of TB require at least 6 months of treatment with multiple drugs. The management of latent TB infection and TB has become more challenging because of the spread of multidrug-resistant and extremely drug-resistant TB. Intensified efforts to find new TB drugs and immunotherapies are needed. Immunotherapies could modulate the immune system in patients with latent TB infection or active disease, enabling better control of M. tuberculosis replication. This review describes several types of potential immunotherapies with a focus on those which have been tested in humans.
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An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence.
It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.
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Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells
Introduction Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), remains a leading cause of infectious disease mortality worldwide [1]. The majority of TB cases are exclusively pulmonary, suggesting a need for mucosal immunity in the control of Mtb. Th1-type immunity, including strong CD4+ Th1 cell and CD8+ T-cell responses, mediates control of Mtb infection [2]. Though many functions of CD4+ Th1 cells and CD8+ T cells are redundant, CD8+ T cells contrast with CD4+ cells in their ability to recognize MHC class II-negative cells and preferentially recognize cells heavily infected with Mtb [3]. In humans, Mtb-specific CD8+ T cells are present at high frequencies in both Mtb-infected and uninfected individuals [4],[5]. The presentation of peptide antigen bound to HLA-A, B, or C to CD8+ T cells is well characterized [4],[6] and has been termed HLA-Ia or classical antigen presentation. Several nonclassical MHC-Ib (HLA-Ib) systems have been described as well. In general, these systems utilize molecules of limited polymorphism to present antigens uniquely characteristic of an infectious pathogen. Examples include presentation of short formylated peptides by mouse H2-M3 [7], presentation of lipids and glycolipids by human group 1 CD1 (CD1a–c) molecules [8]–[11], and the presentation of bacterial glycolipids by CD1d [12],[13]. In some cases these nonclassically restricted T cells have been found at high frequency prior to pathogen exposure, suggesting an innate role. In our previous studies we have determined that human neonates have high frequencies of innate Mtb-reactive thymocytes that are not restricted by classical HLA-I molecules [14]. Functionally, such cells could either provide a direct role in the control of intracellular infection or could facilitate the acquisition of adaptive immunity. In humans, Mtb-reactive group 1 CD1 [15] and HLA–E restricted CD8+ T cells [16] have been described. We have observed that all individuals regardless of exposure to TB have CD8+ T cells capable of recognizing Mtb-infected cells [4],[5],[14]. Moreover, a proportion of these CD8+ T cells can be defined as nonclassically restricted [5]. Therefore, to define the relative contribution of classically versus nonclassically (NC) restricted CD8+ T cells, we used limiting dilution analysis (LDA) to characterize human, Mtb-specific CD8+ T cells in those with TB, those with latent TB infection (LTBI), and those with no evidence of prior exposure to Mtb. We show that CD8+ T-cell clones from individuals infected with Mtb are primarily HLA-Ia restricted. In contrast, NC restricted CD8+ T-cell clones that are neither HLA-Ia nor CD1-restricted, predominate in Mtb-uninfected donors but are nevertheless present in all donors. Furthermore, we demonstrate that these NC restricted CD8+ T-cell clones are restricted by MHC-related molecule 1 (MR1), an HLA-Ib molecule that displays striking evolutionary conservation in mammals [17]. These human Mtb-reactive T cells recognize Mtb-infected dendritic cells (DCs) and lung epithelial cells. Moreover, we show that Mtb-reactive mucosal associated invariant T (MAIT) clones cross react with cells infected with other mycobacteria as well as other bacteria such as Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. These clones express the semi-invariant Vα7.2 T cell receptor, are activated in a manner independent of the transporter associated with antigen processing and presentation (TAP), and have a mucosal homing phenotype. These phenotypic data lead us to designate these cells as MAIT cells [18],[19], a cell type with no previously known physiological function. Additionally, we demonstrate that infection with Mtb induces cell surface expression of MR1 on lung epithelium. Furthermore, Mtb-reactive MAIT cells are enriched in human lung and respond to Mtb-infected lung epithelial cells. Finally, we have performed direct ex vivo analysis of Mtb-reactive MR1-restricted MAIT cells, and find they are present at lower frequencies in the blood of those with active TB. These findings suggest that MAIT cells could play a direct role in the control of bacterial infection. Results Mtb-Reactive, NC Restricted CD8+ T Cells Predominate in TB-Uninfected Individuals In humans, direct ex vivo analysis of Mtb-specific CD8+ T cells reveals a strong association of HLA-Ia–restricted responses and infection with Mtb [4],[20]. Nonetheless, NC HLA-I–restricted CD8+ T cells comprise a substantial proportion of the overall response to Mtb in Mtb-infected individuals [4],[5],[20]. In individuals with no evidence of infection, we have consistently found high frequency CD8+ T cell responses against Mtb-infected DCs. To address the hypothesis that NC restricted CD8+ T cells comprise the dominant response in those without Mtb infection we performed LDA [5] using CD8+ T cells stimulated with Mtb-infected DCs. LDA was performed on individuals with no evidence of Mtb infection (uninfected controls, n = 5), individuals with evidence of latent infection with Mtb (LTBI, n = 5), and individuals with clinical TB (active TB, n = 6). From each of the 16 individuals, we screened an average of 128 clones per donor (Table 1) for their ability to specifically release interferon-γ (IFN-γ) in response to a panel of Mtb-infected but not uninfected targets. 10.1371/journal.pbio.1000407.t001 Table 1 Nonclassical CD8+ T-cell clones predominate in TB-uninfected individuals. Donor Status Donor ID Frequencies of Mtb-specific CD8+ cellsa n Clones Screened Percent Classical Percent Nonclassical Active D431 1/403 109 60 40 D432 1/1156 191 50 50 D435 1/664 17 24 76 D466 1/528 167 95 5 D480 1/418 192 59 41 D481 1/618 159 96 4 n = 6 — — — 64% 36% LTBI D426 1/6956 24 0 100 D443 1/1002 7 43 57 D450 1/1102 192 16 84 D454 1/1818 192 70 30 D504 1/1978 192 16 84 n = 5 — — — 29% 71% Uninfected D403 1/2148 92 16 84 D470 1/1774 192 4 96 D462 N.D. 86 18 83 D427 1/7568 192 27 73 D497 1/3126 53 10 90 n = 5 — — — 15% 85% a These frequencies were previously reported [4]. Abbreviations: N.D., not done. The antigen presenting cell (APC) target groups were: autologous DCs, HLA-mismatched DCs, or HLA-mismatched macrophages. HLA-Ia restricted clones were defined as those responding only to Mtb-infected HLA matched DCs. DCs were grown in X-Vivo media to ensure expression of cell surface CD1. NC-restricted clones were defined as those responding to all three Mtb-infected APC types. As macrophages do not express CD1, NC CD1-restricted T clones were defined as those responding only to infected DCs. Using this method, we have not observed CD1-restricted T-cell clones, resulting in categorization of all the non-HLA-Ia–restricted clones as NC-restricted T cells. None of the T-cell clones were stimulated by uninfected HLA mismatched targets ruling out responses due to alloreactivity. The results from the LDA analysis are presented in Table 1 and Figure 1. The proportion of NC-restricted T-cell clones from each group of donors is presented in Figure 1. As expected, HLA-Ia–restricted CD8+ T-cell clones were strongly associated with TB (p = 0.009) (Figure 1). Nonetheless, consistent with prior observation [5], a significant proportion of CD8+ T-cell clones from infected individuals were NC-restricted. Furthermore, CD8+ T-cell clones isolated from uninfected donors predominantly displayed a NC phenotype (Figure 1). 10.1371/journal.pbio.1000407.g001 Figure 1 LDA of Mtb-reactive CD8+ T-cell clones. Scatter plot demonstrating the proportion of NC restricted CD8+ T-cell clones obtained from individuals in the active, LTBI, and uninfected groups. Each symbol represents the average frequency from all clones screened from an individual donor (Table 1), which was categorized as NC restricted. The nonparametric Mann Whitney one-tailed t-test was used to assess statistical significant differences between groups. Significant differences were detected between active and uninfected groups (p = 0.0043) between the active and LTBI groups (p = 0.0411), but not between the LTBI and uninfected groups (p = 0.3362). To facilitate further analysis a representative subset of the NC-restricted T-cell clones from each donor was expanded and further characterized. Phenotypic analysis of expanded clones (n = 120) revealed uniform expression of CD8α and the αβ TCR (unpublished data). Additionally, we excluded potential activation by a soluble mediator by successfully using Mtb-infected paraformaldehyde-fixed DCs as stimulators. As a result, we have isolated 120 stable NC Mtb-reactive CD8+ αβ TCR+ T-cell clones. Mtb-Reactive NC CD8+ T-Cell Clones Are Restricted by the HLA-Ib Molecule MR1 To explain the high proportion of NC-restricted CD8+ T cells, we considered three hypotheses: presentation by an HLA-Ib molecule, natural killer (NK)-receptor mediated activation, and Toll-like receptor (TLR)-mediated activation. To exclude the possibility that TLR stimulation of DCs would be sufficient to activate the NC clones, we stimulated DCs with agonists to TLR2 (lipoteichoic acid) or to TLR4 (lipopolysaccharide) [14], as both TLRs have been associated with Mtb infection [21]. TLR stimulation of DCs did not result in T-cell activation (Figure 2A). To further evaluate the possibility that TLR2 and/or TLR4 stimulation was required for the recognition of Mtb-infected targets, antibody blockade was performed (Figure 2B). Neither TLR2 nor TLR4 blockade prevented Mtb-dependent T-cell activation. However, the TLR2 and TLR4 antibodies blocked 100% and 80% of interleukin-6 (IL-6) production by DCs treated with TLR2 and TLR4 agonists respectively (unpublished data). 10.1371/journal.pbio.1000407.g002 Figure 2 Mtb-specific NC CD8+ T cells are restricted by MR1. (A–E) Results of ELISPOT assays shown as IFN-γ spot forming units (SFU)/10,000 T cells in response to DCs (25,000/well) treated as described. (A) TLR agonist stimulation of DCs does not stimulate Mtb-reactive NC-restricted clones. DCs were treated (24 h) with TLR agonists specific for TLR2 (lipoteichoic acid, 10 µg/ml) and TLR4 (LPS; 100 ng/ml) at concentrations known to induce activation and cytokine production by DCs [14]. (B) TLR2 (5 µg/ml) or TLR4 (10 µg/ml) blocking antibodies were added to DCs that were uninfected or infected 1 h prior to the addition of Mtb-reactive NC T-cell clones. (C) Mtb-infected DCs were incubated with blocking antibodies (5 µg/ml) to NKG2D, ULBP1, MICA, CD94 for 1 h prior to the addition of the T-cell clones. (D) The pan HLA–I (W632) and CD1a, b, c, and d blocking antibodies were added to Mtb-infected DCs prior to the addition of T cells. (E) DCs infected with Mtb overnight were incubated with anti-MR1 blocking antibody (clone 26.5) or a mouse IgG2a isotype control (both at 5 µg/ml) for 1 h prior to the addition of T cells. (F–H) Cell surface phenotypic analyses of MR1-restricted clones and control clones. For cell surface detection, cells were incubated with antibodies specific for Vα7.2 (clone 3C10) (F), or CD8α, CD8β (G), or CD161 (H), and analyzed by flow cytometry. For (F) and (H), filled histograms represent the isotype control, bold lines represent antibody-specific staining. Columns 1, 2, and 3 represent MR1-restricted clones from different TB exposure groups: D470B1 (uninfected), D426B1 (latent), D466F5 (active), respectively. Column 4 represents HLA-E restricted clone D160 1–23 [16]. Column 5 represents HLA-B08-restricted clone D480C6 specific for the Mtb antigen CFP-103–11. Column 6 represents CD4+ HLA-II–restricted clone D454E12 specific for the Mtb antigen CFP-10. Error bars represent the mean and standard error from duplicate wells. N.D., not done. NK cells do not utilize a TCR but instead are regulated through opposing signals triggered through inhibitory or activating receptors. Mtb is known to induce the cell surface expression of stress molecules such as ULBP1 [22] and MICA [23], which are ligands for the activating NK receptor NKG2D. Antibody blockade of NKG2D/CD94 and the ligands ULBP1 and MICA did not alter recognition of Mtb-infected DCs by any of the T-cell clones (Figure 2C). We next tested the hypothesis that an HLA-Ib molecule was restricting the NC CD8+ T-cell clones. We previously isolated Mtb-specific human CD8+ T-cell clones restricted by the molecule HLA–E [16]. Human Mtb-specific CD8+ T cells restricted by the HLA-Ib molecules CD1a, CD1b, and CD1c [24] have been extensively characterized. To assess if these HLA-Ib molecules were restricting the NC T cells, we performed antibody blockade experiments. We previously showed that addition of the pan HLA-I blocking antibody W6/32 effectively blocks the HLA–E restricted clone (D160 1–23) [16]. While addition of W6/32 blocked recognition of Mtb-infected targets by the HLA–E-restricted clone, three NC-restricted Mtb-reactive CD8+ T-cell clones derived from different donors were unaffected (Figure 2D). In addition to blocking all HLA-Ia molecules and HLA–E, W6/32 also blocks the HLA-Ib molecule HLA–G. As expected, the addition of blocking antibodies previously shown to block responses to CD1a, CD1b, CD1c, or CD1d also had no effect on the clones (Figure 2D). We have extended these findings to all 120 NC CD8+ T-cell clones isolated from the 16 donors listed in Table 2. None of the 120 clones were blocked by the addition of W6/32 or CD1 blocking antibodies (unpublished data). These results suggest that neither CD1a, CD1b, CD1c, CD1d, HLA–E, HLA–G, nor HLA-Ia molecules restrict the panel of 120 Mtb-reactive CD8+ NC-restricted T-cell clones. These data suggest that a common Mtb-reactive CD8+ T subset is present in all individuals regardless of prior exposure to Mtb. 10.1371/journal.pbio.1000407.t002 Table 2 Phenotypic characterization of MR1-restricted Mtb-reactive T-cell clones. T-Cell Clone Donor Status Cell Surface Phenotype TCR Vα TCR Vβ CD161 D431G9 Active 7.2 17 − D432BA8 Active 7.2 U + D466 A3 Active 7.2 20 N.D. D466F5 Active 7.2 13.5 − D481A9 Active 7.2 2 + D426B1 LTBI 7.2 13.5 − D450C8 LTBI 7.2 13.2 + D454B6-2 LTBI 7.2 2 + D504H11 LTBI 7.2 17 N.D. D403C6 Uninfected 7.2 U + D427G10 Uninfected 7.2 U − D462D5 Uninfected 7.2 2 − D470B1 Uninfected 7.2 2 − D470C1-2 Uninfected 7.2 U − U, untypable indicates the Vβ chain could not be determined using the flow cytometric assay. Abbreviations: N.D., not done. We next postulated that the HLA-Ib molecule MR1 was the restricting allele for the NC clones. MR1 is a nonpolymorphic HLA-Ib molecule genetically linked with the CD1 locus in humans [25] and is the most evolutionarily conserved HLA–I molecule among mammals [26]. MR1 is required for the selection of a subset of T cells found primarily in the gut of mammals and thus named mucosal associated invariant T (MAIT) cells. The expansion of MAIT cells is dependent on the presence of gut flora suggesting that a bacterial derived or induced ligand is required for MR1-restricted T-cell expansion and activation [19]. Nevertheless, no bacterial or endogenous MR1-restricted antigen has been identified although considerable evidence supports an antigen presentation function by MR1 [27]–[29]. Furthermore, the biological role of MR1-restricted T cells is unknown, even though several parallels suggest a Natural Killer T-(NKT) cell–like regulatory role [30],[31]. As demonstrated in Figure 2E, addition of an anti-MR1 blocking antibody (26.5) [28] prior to the addition of NC clones abolished IFN-γ production by three different Mtb-reactive NC CD8+ T-cell clones and an additional 11 clones listed in Table 2. The addition of a different anti-MR1 blocking antibody (8F2.F9) resulted in similar blocking (unpublished data). In contrast, CD8+ T-cell clones restricted by HLA–E (D160 1–23) or HLA-B08 (D480F6), or a CD4+ HLA-II restricted clone (D454E12) were unaffected by the addition of the anti-MR1 blocking antibody (Figure 2E). MR1-Restricted, Mtb-Reactive CD8+ T-Cell Clones are MAIT Cells We next performed phenotypic analyses of Mtb-reactive MR1-restricted T-cell clones to determine if they shared properties of previously characterized MR1-restricted MAIT cells. We selected a subset of clones representative of TB exposed (active, n = 5; LTBI, n = 4) and uninfected donors (n = 5) (Table 2). One defining feature of both mouse and human MR1-restricted MAIT cells is the expression of a semi-invariant TCR Vα chain: Vα7.2/Jα33 for humans and the highly homologous Vα19/Jα33 for mice, respectively. Using an antibody that labels all T cells containing the Vα7.2 chain including those that pair with the Jα33 region [32], the Vα7.2 chain was detected by flow cytometry on all 14 MR1-restricted Mtb-specific T-cell clones as well as on an HLA-B08–restricted clone, but not on the HLA–E, or HLA-II–restricted clones (Figure 2F; Table 2). Given that 14 of 14 randomly selected clones were restricted by MR1, binomial analysis suggests a high prevalence of MR1 restriction among our panel of clones (>95%). Furthermore, we performed an analysis of Vα7.2 TCR expression on an additional 28 NC clones. Here all 28 clones expressed the Vα7.2 TCR suggesting that the remaining clones are MR1-restricted. The TCR of Vα7.2-expressing MAIT cells from the gut has been associated with the expression of the Vβ2 or Vβ13 TCR β chains [31]. However, we found at least 10% of the Mtb-specific MR1-restricted T-cell clones did not express either Vβ2 or Vβ13 (Table 2 and unpublished data). To determine if Vα7.2+ Mtb-reactive MR1-restricted T-cell clones expressed the canonical Vα7.2/Jα33 CDR3 region, the TCR alpha encoding cDNA was cloned from six representative Mtb-reactive clones chosen on the basis of their distinct patterns of Vβ TCR usage. All six T-cell clones were found to express the hAV72 segment as expected, and five of six expressed the hAJ33 segment (Table 3). Further, all six Mtb-reactive TCRs were found to have VJ junctional heterogeneity with two N additions, as previously reported for Vα7.2/Jα33 TCRs [33],[34]. Importantly all six TCRs from Mtb-reactive T-cell clones were found to encode CDR3α loops of the same length, which is highly conserved among all mammalian Vα7.2/Jα33+ cells studied thus far. And finally, each of the sequences of the CDR3α loops of the five Vα7.2/Jα33+ Mtb-reactive T cells matched a sequence from a previously reported Vα7.2/Jα33+ cell of undefined restriction and antigen specificity (Table 3) [33],[34]. 10.1371/journal.pbio.1000407.t003 Table 3 Genotypic TCR analysis of MR1-restricted Mtb-reactive T-cell clones. T-Cell Clone (Vβ) CDR3 J Chain D466 A3 (Vβ20) CAVLDSNYQLIWGAG hAJ33 D466F5 (Vβ13.5) CAVRDSNYQLIQWGAG hAJ33 D426B1 (Vβ13.5) CAVRDSNYQLIQWGAG hAJ33 D450C8 (Vβ13.2) CARSDSNYQLIWGAG hAJ33 D504H11 (Vβ17) CASMDSNYQLIWGAG hAJ33 D470B1 (Vβ2) CAVNGDDYKLSFGAG hAJ20 In humans, gut-derived MR1-restricted MAIT cells have been shown to express the CD8αα form of the CD8 co-receptor or lack CD4 or CD8 coreceptor expression [19],[34]. In peripheral blood, invariant TCR Vα7.2+ T cells were originally identified from and found to be overrepresented in the CD4−CD8− fraction of T cells [33]. More recently, MAIT CD8αβ T cells have also been described [32],[35]. As shown in Figure 2G (and unpublished data), all of the Mtb-reactive MR1-restricted clones tested (n = 14; Table 2) coexpressed CD8α and CD8β chains. Human and mouse MR1 lack residues associated with CD8 interaction [25], such that the functional significance of coreceptor expression on Mtb-reactive MR1-restricted T cells remains to be determined. In a recent analysis of MAIT cells from blood, the canonical Vα7.2+ cells were associated with expression of the NK receptor CD161 [32]. We found that Mtb-reactive MR1-restricted T-cell clones cells varied in their CD161 expression (Figure 2H; Table 2), although all cells expressed the mucosal homing integrin α4β7, CD45RO, and lacked CD45RA as previously described for MAIT cells (unpublished data) [32]. Prior work with mouse and human MR1-restricted MAIT cells has demonstrated that neither HLA-II nor TAP are required for thymic selection nor for antigen processing and presentation [18],[34]. To determine if TAP transport is required for presentation to Mtb-reactive MAIT cells we used an adenoviral vector expressing the TAP inhibitor ICP47 (Figure 3) [36],[37]. When ICP47-expressing DCs were subsequently infected with Mtb, neither representative MR1-restricted clones, nor an HLA-II restricted clone were affected by TAP inhibition. In contrast, TAP blockade resulted in over 85% inhibition of the response by the CFP-103–11 HLA-B08-restricted CD8+ T-cell clone. Hence, human Mtb-reactive MR1-restricted T cells, like previously described MAIT cells, do not require TAP for antigen processing and presentation. 10.1371/journal.pbio.1000407.g003 Figure 3 MR1-restricted recognition of Mtb-infected cells is TAP-independent. DCs autologous to D454 and expressing HLA-B08 were transduced with either a control adenoviral vector or adenoviral ICP47 using lipofectamine 2000. After 16 h, DCs were washed and either left uninfected, infected with Mtb, or pulsed with HLA-B08 specific peptide CFP103–11. Following overnight incubation, T cells were added (10,000) to DCs (25,000/well) and IFN-γ production was assessed by ELISPOT. Results are representative of three independent assays. No responses were detected from T cells incubated with uninfected DCs with or without adenoviral vectors. Error bars represent the mean and standard error from duplicate wells. The Mtb Cell Wall is Able to Stimulate Mtb-Reactive MAIT Cells in an MR1-Dependent Fashion To determine if an antigen from Mtb could stimulate the NC-restricted T-cell clones, we initially screened the panel of 120 stable NC clones for their ability to recognize autologous DCs loaded with the cell wall (CW) fraction from Mtb. In contrast to HLA-Ia restricted Mtb-specific T-cell clones, we found that all of the NC clones were stimulated by DCs loaded with Mtb CW (unpublished data). To delineate the antigen recognized by clones we compared the ability of CW to culture filtrate protein (CFP) from Mtb strain H37Rv (courtesy of K. Dobos) to induce a response by a panel of NC-restricted CD8+ T-cell clones (n = 21 and representative of the 16 donors). As expected, the CW fraction derived from Mtb induced robust responses by all the T-cell clones (Figure 4A), whereas the CFP was not stimulatory. To determine if the presentation of Mtb CW was dependent on MR1 we performed antibody blockade. Figure 4B shows that the CW response by three distinct MR1-restricted CD8+ T-cell clones was dependent on MR1. To further characterize the antigen associated with the CW fraction, we subjected the CW to a variety of treatments and tested the ability of the treated fractions to induce a response by the 21 NC-restricted CD8+ T-cell clones (Figure 4C). We have found that delipidated CW (dCW), compared to untreated CW, is strongly antigenic. To determine whether or not the MR1 antigen was proteinaceous, dCW was subjected to proteolytic digestion with a panel of proteases. With all but three NC T-cell clones, protease treatment of the dCW abrogated the antigenic activity. The mean and standard error for the respective treatment groups were: (dCW, 298.7+/−31.72); (subtilisin, 69.38+/−20.05); (trypsin, 79.57+/−17.25); (chymotrypsin, 74+/−17.93); (pronase, 33.38+/−10.86); (Glu-C, 113.5+/−27.48). In each case the Dunn's Multiple Comparison test showed significant differences between dCW and each of the protease-treated fractions (p 99%) by FACS and were added over a range of dilutions to the infected DCs (20,000/well) in the presence of irradiated autologous feeder PBMC (1−e5/well) and rhIL-2 (10 ng/ml). T cells were screened by ELISPOT 10–14 d later. All donors from which APCs and T cells were used in these assays were genetically haplotyped (Blood System Laboratory), thereby ensuring a complete mismatch of HLA-Ia alleles when necessary for screening. T-cell clones that retained Mtb specificity were subsequently expanded in the presence of irradiated allogeneic PBMC (25×106), irradiated allogeneic lymphoblastoid cell line (5×106), and anti-CD3 mAb (30 ng/ml) in RPMI 1640 media with 10% HS in a T-25 upright flask in a total volume of 30 ml. The cultures were supplemented with IL-2 (0.5 ng/ml) on days 1, 4, 7, and 10 of culture. The cell cultures were washed on day 4 to remove remaining soluble anti-CD3 mAb [51] and used no earlier than day 11. Assays IFN-γ ELISPOT assay All IFN-γ ELISPOT assays were performed as described [16]. Estimation of the frequency of Mtb-reactive CD8+ T cells using the IFN-γ ELISPOT was performed as described [4]. Intracellular cytokine staining assay T cells were isolated from single cell suspensions from blood, LNs, or lung using a negative selection isolation kit (Miltenyi-pan T cell kit). T cells were added to Mtb-infected or uninfected A549 cells at ratio of 3∶1 and incubated for 16 h in the presence of anti-CD28 (1 µg/ml) and CD49d (1 µg/ml). GolgiStop (BD Pharmingen) was added for the final 6 h of the assay. Cells were surface stained for expression of the Vα7.2 TCR (clone 3C10) and subsequently fixed and permeabilized with Cytofix/CytoPerm (BD Pharmingen) and stained in the presence of Perm/Wash (BD Pharmingen), with fluorochrome-conjugated antibodies to TNF-α and CD8α. Acquisition was performed with an LSRII flow cytometer with FACS Diva software (BD). All analyses were performed using FlowJo software (TreeStar). Reagents Antibodies to the following molecules were used: CD1a, CD1b, CD8a CD161 TCR αβ (BD Pharmingen); CD1c (MCA694), pan HLA-I antibody (W6/32) (Serotec); anti-TNF-α (Beckman Coulter); TCR Vβ usage was determined using the IOTest Beta Mark Kit (Beckman Coulter); TCRgd (5A6.E9-Endogen); CD1d (CD1d51, kindly provided by Steven Porcelli); CD8β (GenWay); CD49d (9F10), LEAF ms IgG1, LEAF msIgG2a, Integrin B7 (FIB504) (Biolegend); MR1 (26.5) [28]; Va7.2 (3C10) [32]; CD94 (MAB1058) NKG2D (MAB139), ULBP1 (MAB1380), MICA (MAB1300) TLR2 (MAB2616), TLR (AF1478) (R&D), Lamp1 (H5G11, SCBT); Tubulin (E1332Y, Abcam); TLR agonists: Lipoteichoic Acid (Sigma); LPS (Sigma), Pam3CysK4 (InVivo Gen); Fluoromount G (Southern Biotech).
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Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis.
Heui-Dong Park, Kristi M. Guinn, Maria I Harrell … (2003)
Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.
Author and article information
Journal
Journal ID (nlm-ta): Immunotargets Ther
Journal ID (iso-abbrev): Immunotargets Ther
Journal ID (publisher-id): ImmunoTargets and Therapy
Title:
ImmunoTargets and Therapy
Publisher:
Dove Medical Press
ISSN
(Electronic):
2253-1556
Publication date Collection: 2016
Publication date
(Electronic):
20
April
2016
Volume: 5
Pages: 37-45
Affiliations
Author notes
Correspondence: Getahun Abate, Department of Internal Medicine, Division of Infectious
Diseases, Allergy and Immunology, Saint Louis University, Doisy Research Center, 8th
floor, 1100 S. Grand Blvd, St. Louis, MO 63104, USA, Tel +1 314 977 5500, Email
abateg@
123456slu.edu
Daniel F Hoft, Department of Internal Medicine, Division of Infectious Diseases, Allergy
and Immunology, Saint Louis University, Doisy Research Center, 8th floor, 1100 S.
Grand Blvd, St. Louis, MO 63104, USA, Tel +1 314 977 5500, Fax +1 314 771 3816, Email
hoftdf@
123456slu.edu
Article
Publisher ID: itt-5-037DOI: 10.2147/ITT.S81892
PMC ID: 4970630
PubMed ID: 27529060
SO-VID: 5ce3ad49-4c1d-4028-bb75-b2ca34437db6
Copyright © © 2016 Abate and Hoft. This work is published and licensed by Dove Medical Press Limited
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