Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity.

Innate immune memory is an emerging area of research. However, innate immune memory at major mucosal sites remains poorly understood. Here, we show that respiratory viral infection induces long-lasting memory alveolar macrophages (AMs). Memory AMs are programed to express high MHC II, a defense-ready gene signature, and increased glycolytic metabolism, and produce, upon re-stimulation, neutrophil chemokines. Using a multitude of approaches, we reveal that the priming, but not maintenance, of memory AMs requires the help from effector CD8 T cells. T cells jump-start this process via IFN-γ production. We further find that formation and maintenance of memory AMs are independent of monocytes or bone marrow progenitors. Finally, we demonstrate that memory AMs are poised for robust trained immunity against bacterial infection in the lung via rapid induction of chemokines and neutrophilia. Our study thus establishes a new paradigm of immunological memory formation whereby adaptive T-lymphocytes render innate memory of mucosal-associated macrophages.

Airway Macrophages Mediate Mucosal Vaccine-Induced Trained Innate Immunity against Mycobacterium tuberculosis in Early Stages of Infection.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis (TB), is responsible for millions of infections and deaths annually. Decades of TB vaccine development have focused on adaptive T cell immunity, whereas the importance of innate immune contributions toward vaccine efficacy has only recently been recognized. Airway macrophages (AwM) are the predominant host cell during early pulmonary M. tuberculosis infection and, therefore, represent attractive targets for vaccine-mediated immunity. We have demonstrated that respiratory mucosal immunization with a viral-vectored vaccine imprints AwM, conferring enhanced protection against heterologous bacterial challenge. However, it is unknown if innate immune memory also protects against M. tuberculosis In this study, by using a murine model, we detail whether respiratory mucosal TB vaccination profoundly alters the airway innate immune landscape associated with AwM prior to M. tuberculosis exposure and whether such AwM play a critical role in host defense against M. tuberculosis infection. Our study reveals an important role of AwM in innate immune protection in early stages of M. tuberculosis infection in the lung.

CD11b+ Dendritic Cell-Mediated Anti-Mycobacterium tuberculosis Th1 Activation Is Counterregulated by CD103+ Dendritic Cells via IL-10.

Mycobacterium tuberculosis, the pathogen causing pulmonary tuberculosis (TB) in humans, has evolved to delay Th1 immunity in the lung. Although conventional dendritic cells (cDCs) are known to be critical to the initiation of T cell immunity, the differential roles and molecular mechanisms of migratory CD11b+ and CD103+ cDC subsets in anti-M. tuberculosis Th1 activation remain unclear. Using a murine model of pulmonary M. tuberculosis infection, we found that slow arrival of M. tuberculosis-bearing migratory CD11b+ and CD103+ cDCs at the draining lymph nodes preceded the much-delayed Th1 immunity and protection in the lung. Contrary to their previously described general roles in Th polarization, CD11b+ cDCs, but not CD103+ cDCs, were critically required for Th1 activation in draining lymph nodes following M. tuberculosis infection. CD103+ cDCs counterregulated CD11b+ cDC-mediated Th1 activation directly by producing the immune-suppressive cytokine IL-10. Thus, our study provides new mechanistic insights into differential Th immune regulation by migratory cDC subsets and helps to develop novel vaccines and therapies.

Single-Dose Mucosal Immunotherapy With Chimpanzee Adenovirus-Based Vaccine Accelerates Tuberculosis Disease Control and Limits Its Rebound After Antibiotic Cessation.

BACKGROUND: The development of strategies to accelerate disease resolution and shorten antibiotic therapy is imperative in curbing the global tuberculosis epidemic. Therapeutic application of novel vaccines adjunct to antibiotics represents such a strategy. METHODS: By using a murine model of pulmonary tuberculosis (TB), we have investigated whether a single respiratory mucosal therapeutic delivery of a novel chimpanzee adenovirus-vectored vaccine expressing Ag85A (AdCh68Ag85A) accelerates TB disease control in conjunction with antibiotics and restricts pulmonary disease rebound after premature (nonsterilizing) antibiotic cessation. RESULTS: We find that immunotherapy via the respiratory mucosal, but not parenteral, route significantly accelerates pulmonary mycobacterial clearance, limits lung pathology, and restricts disease rebound after premature antibiotic cessation. We further show that vaccine-activated antigen-specific T cells, particularly CD8 T cells, in the lung play an important role in immunotherapeutic effects. CONCLUSIONS: Our results indicate that a single-dose respiratory mucosal immunotherapy with AdCh68Ag85A adjunct to antibiotic therapy has the potential to significantly accelerate disease control and shorten the duration of conventional treatment. Our study provides the proof of principle to support therapeutic applications of viral-vectored vaccines via the respiratory route.

CXCR3 Signaling Is Required for Restricted Homing of Parenteral Tuberculosis Vaccine-Induced T Cells to Both the Lung Parenchyma and Airway.

Although most novel tuberculosis (TB) vaccines are designed for delivery via the muscle or skin for enhanced protection in the lung, it has remained poorly understood whether systemic vaccine-induced memory T cells can readily home to the lung mucosa prior to and shortly after pathogen exposure. We have investigated this issue by using a model of parenteral TB immunization and intravascular immunostaining. We find that systemically induced memory T cells are restricted to the blood vessels in the lung, unable to populate either the lung parenchymal tissue or the airway under homeostatic conditions. We further find that after pulmonary TB infection, it still takes many days before such T cells can enter the lung parenchymal tissue and airway. We have identified the acquisition of CXCR3 expression by circulating T cells to be critical for their entry to these lung mucosal compartments. Our findings offer new insights into mucosal T cell biology and have important implications in vaccine strategies against pulmonary TB and other intracellular infections in the lung.

Enhancement of Antituberculosis Immunity in a Humanized Model System by a Novel Virus-Vectored Respiratory Mucosal Vaccine.

Background: The translation of preclinically promising novel tuberculosis vaccines to ultimate human applications has been challenged by the lack of animal models with an immune system equivalent to the human immune system in its genetic diversity and level of susceptibility to tuberculosis. Methods: We have developed a humanized mice (Hu-mice) tuberculosis model system to investigate the clinical relevance of a novel virus-vectored (VV) tuberculosis vaccine administered via respiratory mucosal or parenteral route. Results: We find that VV vaccine activates T cells in Hu-mice as it does in human vaccinees. The respiratory mucosal route for delivery of VV vaccine in Hu-mice, but not the parenteral route, significantly reduces the humanlike lung tuberculosis outcomes in a human T-cell-dependent manner. Conclusions: Our results suggest that the Hu-mouse can be used to predict the protective efficacy of novel tuberculosis vaccines/strategies before they proceed to large, expensive human trials. This new vaccine testing system will facilitate the global pace of clinical tuberculosis vaccine development.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin-induced pulmonary fibrosis.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Role of B Cells in Mucosal Vaccine-Induced Protective CD8+ T Cell Immunity against Pulmonary Tuberculosis.

Emerging evidence suggests a role of B cells in host defense against primary pulmonary tuberculosis (TB). However, the role of B cells in TB vaccine-induced protective T cell immunity still remains unknown. Using a viral-vectored model TB vaccine and a number of experimental approaches, we have investigated the role of B cells in respiratory mucosal vaccine-induced T cell responses and protection against pulmonary TB. We found that respiratory mucosal vaccination activated Ag-specific B cell responses. Whereas respiratory mucosal vaccination elicited Ag-specific T cell responses in the airway and lung interstitium of genetic B cell-deficient (Jh(-/-) knockout [KO]) mice, the levels of airway T cell responses were lower than in wild-type hosts, which were associated with suboptimal protection against pulmonary Mycobacterium tuberculosis challenge. However, mucosal vaccination induced T cell responses in the airway and lung interstitium and protection in B cell-depleted wild-type mice to a similar extent as in B cell-competent hosts. Furthermore, by using an adoptive cell transfer approach, reconstitution of B cells in vaccinated Jh(-/-) KO mice did not enhance anti-TB protection. Moreover, respiratory mucosal vaccine-activated T cells alone were able to enhance anti-TB protection in SCID mice, and the transfer of vaccine-primed B cells alongside T cells did not further enhance such protection. Alternatively, adoptively transferring vaccine-primed T cells from Jh(-/-) KO mice into SCID mice only provided suboptimal protection. These data together suggest that B cells play a minimal role, and highlight a central role by T cells, in respiratory mucosal vaccine-induced protective immunity against M. tuberculosis.

Restoration of innate immune activation accelerates Th1-cell priming and protection following pulmonary mycobacterial infection.

The immune mechanisms underlying delayed induction of Th1-type immunity in the lungs following pulmonary mycobacterial infection remain poorly understood. We have herein investigated the underlying immune mechanisms for such delayed responses and whether a selected innate immune-modulating strategy can accelerate Th1-type responses. We have found that, in the early stage of pulmonary infection with attenuated Mycobacterium tuberculosis (M.tb H37Ra), the levels of infection in the lung continue to increase logarithmically until days 14 and 21 postinfection in C57BL/6 mice. The activation of innate immune responses, particularly DCs, in the lung is delayed. This results in a delay in the subsequent downstream immune responses including the migration of antigen-bearing DCs to the draining lymph node (dLN), the Th1-cell priming in dLN, and the recruitment of Th1 cells to the lung. However, single lung mucosal exposure to the TLR agonist FimH postinfection is able to accelerate protective Th1-type immunity via facilitating DC migration to the lung and draining lymph nodes, enhancing DC antigen presentation and Th1-cell priming. These findings hold implications for the development of immunotherapeutic and vaccination strategies and suggest that enhancement of early innate immune activation is a viable option for improving Th1-type immunity against pulmonary mycobacterial diseases.

Marked improvement of severe lung immunopathology by influenza-associated pneumococcal superinfection requires the control of both bacterial replication and host immune responses.

Bacterial superinfection and associated lung immunopathology are major contributors to hospitalizations and mortality after influenza. However, the underlying mechanisms and effective intervention strategies remain poorly defined. By using a model of influenza and pneumococcal superinfection, we found that dual-infected animals experienced rapid weight loss and succumbed to infection. Bacterial outgrowth, dysregulated cytokines, including keratinocyte-derived chemokine and macrophage inflammatory protein 2, and severe lung neutrophilia and immunopathology were linked to the poor clinical outcome. In vivo neutralization of highly induced macrophage inflammatory protein 2 did not affect clinical outcome, bacterial loads, or lung immunopathology. On the other hand, in vivo neutrophil depletion did not alter the clinical outcome and bacterial burden, although it moderately improved lung immunopathology. Treatment with a bacteriostatic antibiotic, azithromycin, alone significantly improved clinical outcome and bacterial clearance, but failed to reduce lung immunopathology. In comparison, treatment with a global inflammation inhibitor, dexamethasone, alone failed to alter clinical outcome, bacterial infection, and immunopathology, despite its moderate reducing effects on neutrophilic and cytokine responses. In contrast, combined treatment with both azithromycin and dexamethasone best improved clinical outcome, bacterial clearance, lung cellular and cytokine responses, and immunopathology. Our study suggests that marked improvement of clinical outcome and lung immunopathology caused by bacterial superinfection requires the control of both bacterial infection and aberrant host immune responses. Our findings hold implications in clinical management for influenza-associated bacterial superinfections.

Heterogeneity in lung macrophage control of Mycobacterium tuberculosis is modulated by T cells.

Following Mycobacterium tuberculosis infection, alveolar macrophages are initially infected but ineffectively restrict bacterial replication. The distribution of M. tuberculosis among different cell types in the lung changes with the onset of T cell immunity when the dominant infected cellular niche shifts from alveolar to monocyte-derived macrophages (MDM). We hypothesize that changes in bacterial distribution among different cell types is driven by differences in T cell recognition of infected cells and their subsequent activation of antimicrobial effector mechanisms. We show that CD4 and CD8 T cells efficiently eliminate M. tuberculosis infection in alveolar macrophages, but they have less impact on suppressing infection in MDM, which may be a bacterial niche. Importantly, CD4 T cell responses enhance MDM recruitment to the lung. Thus, the outcome of infection depends on the interaction between the T cell subset and the infected cell; both contribute to the resolution and persistence of the infection.

Key advances in vaccine development for tuberculosis-success and challenges.

Breakthrough findings in the clinical and preclinical development of tuberculosis (TB) vaccines have galvanized the field and suggest, for the first time since the development of bacille Calmette-Guérin (BCG), that a novel and protective TB vaccine is on the horizon. Here we highlight the TB vaccines that are in the development pipeline and review the basis for optimism in both the clinical and preclinical space. We describe immune signatures that could act as immunological correlates of protection (CoP) to facilitate the development and comparison of vaccines. Finally, we discuss new animal models that are expected to more faithfully model the pathology and complex immune responses observed in human populations.

Heterogeneity in lung macrophage control of Mycobacterium tuberculosis is determined by T cells.

Following Mycobacterium tuberculosis infection, alveolar macrophages are initially infected but ineffectively restrict bacterial replication. The distribution of M. tuberculosis among different cell types in the lung changes with the onset of T cell immunity when the dominant infected cellular niche shifts from alveolar to monocyte-derived macrophages (MDM). We hypothesize that changes in bacterial distribution among different cell types is driven by differences in T cell recognition of infected cells and their subsequent activation of antimicrobial effector mechanisms. We show that CD4 and CD8 T cells efficiently eliminate M. tuberculosis infection in alveolar macrophages, but they have less impact on suppressing infection in MDM, which may be a bacterial niche. Importantly, CD4 T cell responses enhance MDM recruitment to the lung. Thus, the outcome of infection depends on the interaction between the T cell subset and the infected cell; both contribute to the resolution and persistence of the infection.

Host genetic background is a barrier to broadly effective vaccine-mediated protection against tuberculosis.

Heterogeneity in human immune responses is difficult to model in standard laboratory mice. To understand how host variation affects Bacillus Calmette Guerin-induced (BCG-induced) immunity against Mycobacterium tuberculosis, we studied 24 unique collaborative cross (CC) mouse strains, which differ primarily in the genes and alleles they inherit from founder strains. The CC strains were vaccinated with or without BCG and challenged with aerosolized M. tuberculosis. Since BCG protects only half of the CC strains tested, we concluded that host genetics has a major influence on BCG-induced immunity against M. tuberculosis infection, making it an important barrier to vaccine-mediated protection. Importantly, BCG efficacy is dissociable from inherent susceptibility to tuberculosis (TB). T cell immunity was extensively characterized to identify components associated with protection that were stimulated by BCG and recalled after M. tuberculosis infection. Although considerable diversity is observed, BCG has little impact on the composition of T cells in the lung after infection. Instead, variability is largely shaped by host genetics. BCG-elicited protection against TB correlated with changes in immune function. Thus, CC mice can be used to define correlates of protection and to identify vaccine strategies that protect a larger fraction of genetically diverse individuals instead of optimizing protection for a single genotype.

Intranasal multivalent adenoviral-vectored vaccine protects against replicating and dormant M.tb in conventional and humanized mice.

Viral-vectored vaccines are highly amenable for respiratory mucosal delivery as a means of inducing much-needed mucosal immunity at the point of pathogen entry. Unfortunately, current monovalent viral-vectored tuberculosis (TB) vaccine candidates have failed to demonstrate satisfactory clinical protective efficacy. As such, there is a need to develop next-generation viral-vectored TB vaccine strategies which incorporate both vaccine antigen design and delivery route. In this study, we have developed a trivalent chimpanzee adenoviral-vectored vaccine to provide protective immunity against pulmonary TB through targeting antigens linked to the three different growth phases (acute/chronic/dormancy) of Mycobacterium tuberculosis (M.tb) by expressing an acute replication-associated antigen, Ag85A, a chronically expressed virulence-associated antigen, TB10.4, and a dormancy/resuscitation-associated antigen, RpfB. Single-dose respiratory mucosal immunization with our trivalent vaccine induced robust, sustained tissue-resident multifunctional CD4+ and CD8+ T-cell responses within the lung tissues and airways, which were further quantitatively and qualitatively improved following boosting of subcutaneously BCG-primed hosts. Prophylactic and therapeutic immunization with this multivalent trivalent vaccine in conventional BALB/c mice provided significant protection against not only actively replicating M.tb bacilli but also dormant, non-replicating persisters. Importantly, when used as a booster, it also provided marked protection in the highly susceptible C3HeB/FeJ mice, and a single respiratory mucosal inoculation was capable of significant protection in a humanized mouse model. Our findings indicate the great potential of this next-generation TB vaccine strategy and support its further clinical development for both prophylactic and therapeutic applications.

Understanding delayed T-cell priming, lung recruitment, and airway luminal T-cell responses in host defense against pulmonary tuberculosis.

Mycobacterium tuberculosis (M.tb), the causative bacterium of pulmonary tuberculosis (TB), is a serious global health concern. Central to M.tb effective immune avoidance is its ability to modulate the early innate inflammatory response and prevent the establishment of adaptive T-cell immunity for nearly three weeks. When compared with other intracellular bacterial lung pathogens, such as Legionella pneumophila, or even closely related mycobacterial species such as M. smegmatis, this delay is astonishing. Customarily, the alveolar macrophage (AM) acts as a sentinel, detecting and alerting surrounding cells to the presence of an invader. However, in the case of M.tb, this may be impaired, thus delaying the recruitment of antigen-presenting cells (APCs) to the lung. Upon uptake by APC populations, M.tb is able to subvert and delay the processing of antigen, MHC class II loading, and the priming of effector T cell populations. This delay ultimately results in the deferred recruitment of effector T cells to not only the lung interstitium but also the airway lumen. Therefore, it is of upmost importance to dissect the mechanisms that contribute to the delayed onset of immune responses following M.tb infection. Such knowledge will help design the most effective vaccination strategies against pulmonary TB.

Limited recognition of Mycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice.

Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination are frequently assessed by measuring T-cell recognition of crude Mtb antigens, recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-specific T cells recognize Mtb-infected macrophages. Thus, an important question is what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. We address this question by developing a modified elispot assay using viable Mtb-infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of T cells that recognize Mtb-infected macrophages were determined using interferon-γ elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the relatively high frequencies of T cells specific for antigens such as ESAT-6 and TB10.4, low frequencies of total pulmonary T cells elicited by aerosolized Mtb infection recognize Mtb-infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that recognize Mtb-infected macrophages. We propose that the frequency of T cells that recognize infected macrophages could correlate with protective immunity and may be an alternative approach to measuring T-cell responses to Mtb antigens.

Functionally Overlapping Variants Control Tuberculosis Susceptibility in Collaborative Cross Mice.

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis infection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-γ) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) × CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes (Tip1 to Tip4). These included QTL that were associated with bacterial burden, IFN-γ production following infection, and an IFN-γ-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L (αL; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross.IMPORTANCE The variable outcome of Mycobacterium tuberculosis infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.

Spray dried human and chimpanzee adenoviral-vectored vaccines are thermally stable and immunogenic in vivo.

Cold chain-free vaccine technologies are needed to ensure effective vaccine delivery and coverage, particularly in resource-poor countries. However, the immunogenicity and thermostability of spray dried live viral vector-based vaccines such as recombinant adenoviral-vectored vaccines remain to be investigated. To address this issue, we have spray dried human adenoviral (AdHu5)- and chimpanzee adenoviral (AdCh68)-vectored tuberculosis vaccines in a mannitol and dextran matrix. Spray dried powders containing these two vaccines display the morphologic and chemical properties desired for long-term thermostability and vaccination. Upon reconstitution, they effectively transfected the cells in vitro with relatively small losses in viral infectivity related to the spray drying process. Following in vivo vaccination, AdHu5- and AdCh68-vectored vaccines were as immunogenic as the conventional fresh, cryopreserved liquid vaccine samples. Of importance, even after cold chain-free storage, at ambient temperatures and relatively low humidity for 30 and 90days, the vaccines retained their in vivo immunogenicity, while the liquid vaccine samples stored under the same conditions lost their immune-activating capability almost entirely. Our results support further development of our spray drying technologies for generating thermally stable adenoviral-vectored and other viral-vectored vaccines.

Mucosal immunity and novel tuberculosis vaccine strategies: route of immunisation-determined T-cell homing to restricted lung mucosal compartments.

Despite the use of bacille Calmette-Guérin (BCG) for almost a century, pulmonary tuberculosis (TB) continues to be a serious global health concern. Therefore, there has been a pressing need for the development of new booster vaccines to enhance existing BCG-induced immunity. Protection following mucosal intranasal immunisation with AdHu5Ag85A is associated with the localisation of antigen-specific T-cells to the lung airway. However, parenteral intramuscular immunisation is unable to provide protection despite the apparent presence of antigen-specific T-cells in the lung interstitium. Recent advances in intravascular staining have allowed us to reassess the previously established T-cell distribution profile and its relationship with the observed differential protection. Respiratory mucosal immunisation empowers T-cells to home to both the lung interstitium and the airway lumen, whereas intramuscular immunisation-activated T-cells are largely trapped within the pulmonary vasculature, unable to populate the lung interstitium and airway. Given the mounting evidence supporting the safety and enhanced efficacy of respiratory mucosal immunisation over the traditional parenteral immunisation route, a greater effort should be made to clinically develop respiratory mucosal-deliverable TB vaccines.