International Journal of Infectious Diseases 80 (2019) S58–S61 Contents lists available at ScienceDirect International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid Latent TB Infection (LTBI) – Mycobacterium tuberculosis pathogenesis and the dynamics of the granuloma battleground Martin Raoa , Giuseppe Ippolitob , Sayoki Mfinangac , Francine Ntoumid,e , Dorothy Yeboah-Manuf , Cristina Vilaplanag, Alimuddin Zumlah , Markus Maeureri,j,* a Champalimaud Centre for the Unknown, Lisbon, Portugal b National Institute for Infectious Diseases, Lazzaro Spallanzani, Rome, Italy c National Institute of Medical Research Muhimbili, Dar es Salaam, Tanzania d University Marien NGouabi and Fondation Congolaise pour la Recherche Médicale (FCRM), Brazzaville, Congo e Institute for Tropical Medicine, University of Tübingen, Germany f Department of Bacteriology, Noguchi Memorial Institute for Medical Research, Accra, Ghana g Experimental Tuberculosis Unit (UTE), Fundació Institut Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona (UAB), Badalona, Catalonia, Spain h Division of Infection and Immunity, University College London and NIHR Biomedical Research Centre, UCL Hospitals NHS Foundation Trust, London, UK i Champalimaud Centre for the Unknown, Avenida Brasília, 1400-038 Lisbon, Portugal j Department of Haematology and Oncology, Krankenhaus Nordwest, Frankfurt, Germany A R T I C L E I N F O A B S T R A C T Article history: Latent tuberculosis infection (LTBI) is established in over 90% of persons infected with Mycobacterium Received 14 January 2019 tuberculosis (Mtb), from whom new active TB cases will arise. Understanding the spatio-temporal Accepted 22 February 2019 dynamics of host immune responses in LTBI granulomas is essential to designing effective post-exposure Corresponding Editor: Eskild Petersen, therapies that inhibit progression to TB. Information arising from cancer studies and other modalities – Aarhus, Denmark where local chronic inflammation leads to immunopathology – can help provide insights into the biological pathways at play in LTBI granulomas. Translational studies using patient material as well as Keywords: LTBI+ donor-derived tissue samples are instrumental in understanding the various components of latent tuberculosis infection granuloma granuloma dynamics, immunological landscapes therein and how this could help to identify therapeutic mutations targets. Deep sequencing technologies may aid to decipher the genetic changes in lung granuloma and immune landscape blood samples from LTBI+ individuals associated with progression to active TB disease. This may lead to host-directed therapies advancement of development of targeted Host-Directed Therapies (HDTs) and their evaluation as adjunct TB therapies for improving treatment outcomes for LTBI and pulmonary TB. © 2019 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction pathophysiological events following primary or secondary infec- tion, remain a mystery (Zumla et al., 2011). The granuloma was Tuberculosis (TB) has plagued humankind for centuries and has thought to be a result of protective cell-mediated and has caused over one billion deaths. Today, an estimated 2 billion people been central to the study of pathogenesis of TB and other worldwide have latent TB infection (LTBI). Humans are the intracellular pathogens (James and Zumla, 1999). A spectrum of main reservoir of Mycobacterium tuberculosis (Mtb) and human granulomatous changes has been described from complete to human spread primarily occurs through inhalation of respira- resolution, to continuing granuloma turnover with protective tory aerosols and secretions containing Mtb exhaled from a patient and deleterious inflammation, caseous necrosis, fibrosis and with active pulmonary TB. Despite intense study over the past six scarring of tissue leading to long-term functional disability (Drain decades the sequence of human host-Mtb interactions and et al., 2018). Technological advances over the years have enabled more detailed studies at molecular and cellular level, several recent publications have questioned whether the granuloma or * Corresponding author at: Champalimaud Centre for the Unknown, Avenida its individual immune components make the granuloma a ‘friend’ Brasília, 1400-038 Lisbon, Portugal. or a ‘foe’ (Rubin, 2009; Galizia and Marti, 2018). Thus, gaining a E-mail addresses: martin.rao@research.fchampalimaud.org (M. Rao), deeper understanding of the host-pathogen interactions in giuseppe.ippolito@inmi.it (G. Ippolito), gsmfinanga@yahoo.com (S. Mfinanga), ffntoumi@hotmail.com (F. Ntoumi), dyeboah-manu@noguchi.ug.edu.dh persons harbouring LTBI may hold quintessential clues to (D. Yeboah-Manu), cvilaplana@gmail.com (C. Vilaplana), a.zumla@ucl.ac.uk achieving the WHO’s goal of eliminating TB as a public health (A. Zumla), markus.maeurer@fundacaochampalimaud.pt (M. Maeurer). threat by 2050. https://doi.org/10.1016/j.ijid.2019.02.035 1201-9712/© 2019 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Descargado para Anonymous User (n/a) en Autonomous University of Barcelona de ClinicalKey.es por Elsevier en marzo 26, 2021. Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2021. Elsevier Inc. Todos los derechos reservados. M. Rao et al. / International Journal of Infectious Diseases 80 (2019) S58–S61 S59 Imaging studies of the TB granulomas have shed light on how and dual recognition patterns with shared TCRs recognising individual lesions in the same patient evolve over time, just as how evolutionarily preserved relevant target structures (Pogorelyy cancer lesions do. This evolutionary process is mediated and et al., 2017) exists from early life while demanding further influenced by exposure to drugs, the hosts genetics as well as that investigation. The comparison of T-cell clones from twins, humans of the infecting Mtb species (Ankrah et al., 2016). Studies show that of different ages and cord blood showed that prenatally-formed T- position emission tomography (PET) with [18F]fluoro-2-deoxy-D- cell clones persist in the mature organism for decades, providing a glucose (FDG) is able to monitor lesions in patients with subclinical ‘pre-programmed’ immune-receptor repertoire (Pogorelyy et al., TB – which could have arisen from a previously LTBI state – and the 2017). This suggests that the TCR repertoire recognising potential behavioural pattern of individual lesions which are likely to antigenic targets, such as tumour antigens, or pathogens, has been perpetrate the development of symptomatic clinical disease (Drain shaped earlier in life. The consequences of prenatal infections or et al., 2018). Since most people infected with Mtb do not develop exposure to other target antigens and productive anti-tumour active clinical disease, LTBI presents a unique and challenging immune responses is now a field of active investigation. opportunity to understand the intricate relationship between host Comparative studies of TCR repertoires from the TB granuloma cells in the granuloma, the contribution of the stromal compart- with that from blood in conjunction with HLA allelic selection and ment to sustain infection and how the local immune responses is expression profiles across patient cohorts in different geographical placed therein. Thorough dissection of these biological component locations will yield valuable clinical data for mapping key host which collectively govern host-pathogen interactions and patho- response patterns and characteristics. genesis will reveal essential information necessary for developing next-generation drugs and therapies. The anatomy in granuloma lesions and lessons from tumour Genetic aberrations in the Mtb genome usually make the bacilli heterogeneity refractory to antibiotics and resistant to immune attack. However, we have yet to uncover whether some of these mutations aid their A fresh look into the functional diversity and anatomy in TB long survival in the LTBI granuloma without attracting much granulomas can be inspired by cancer studies, comprising different attention from immune cells. Furthermore, an individual’s human subgroups based on immune-reactivity patterns that may also be leukocyte antigen (HLA) background, naturally occurring varia- linked to sub-anatomical areas within the lesion (Gerlinger et al., tions in immune-related genes and selective pressure affecting 2012; Thorsson et al., 2018). The local anti-tumour immune gene expression patterns may influence local host-pathogen response landscape supports the notion that the spatial relation- interactions in the LTBI granuloma and later, the development of ship of inflammatory cells is vital in conferring in-situ protective clinical TB. Indeed, the genetic basis of susceptibility to TB is being immune responses (Thorsson et al., 2018). Adaptive cellular roped back into the limelight due to vaccine efficacy studies and immune responses herein may receive ‘bystander’ activation evaluation of new biomarkers for host-directed therapy (HDT) as signals via alternate ligands, molecular mimicry or even ‘in-built’ well as to predict clinical responses to standard anti-TB treatment cross-reactivity (Whiteside et al., 2018), as recently described for (Goletti et al., 2018). TCR gamma-delta (gd) exhibiting exquisite antigen specificity as Emerging data from clinical studies provide staunch ground for well as a broad recognition of stress signals via TCR germline- accounting for immune-cell types as well as surface or effector encoded motifs (Melandri et al., 2018). Endogenously-produced molecule expression profiles to better gauge the pathophysiologi- cytokines such as IL-15 and IL-7 expressed in TB granulomas cal status linking LTBI and clinical TB. Natural killer (NK) cells, (Maeurer et al., 1999) can stimulate memory CD8+ T cells and previously underemphasised in protection to TB, are now induce an effect comparable to specific TCR ligation, with acknowledged as important modulators of progression to active subsequent production of IFN-g, perforin and granzyme B (Liu TB from LTBI (Roy Chowdhury et al., 2018). An increase of CD4+ and et al., 2002). Thus, the interplay between various immune-cell CD8+ mucosal-associated invariant T cells (MAIT) in peripheral subsets in the disease microenvironment i.e. interaction of CD8+ T blood of individuals harbouring LTBI compared to uninfected cells and other immune cells, e.g. CD4+, NK cells, Tregs, macro- control subjects has also been reported (Paquin-Proulx et al., 2018). phages and myeloid-derived suppressor cells (Liu et al., 2002) is The existence of a clonotypic T-cell receptor (TCR) repertoire in the critical for protective cellular immune responses. Furthermore, LTBI is associated with protection against clinical disease (Tully spatial proximity of T cells to cancer cells has been strongly et al., 2005), which requires more detailed analysis in light of associated with increased survival in patients with cancer recent findings examining TCR diversity in patients with TB. More (Carstens et al., 2017). Similar studies examining the 3D interaction recent work has re-examined the structural diversity of the TCR of T cells in TB granulomas with other immune cell subsets, repertoire in blood from patients with TB where – in comparison to including Mtb-infected cells and epithelial cells – given the age- and exposure-matched controls – a restricted TCR-repertoire similarities between TB granulomas and cancerous lesions – exists and a broader TCR repertoire was associated with better warrant deeper insights. clinical performance among patients (Luo et al., 2012). In addition, Each TB granuloma appears to be ‘unique’ concerning the LTBI+ individuals exhibited a preferred usage of certain TCR overall cytokine production pattern as well as the different areas clonotypes, which was further analysed in a different study where within the same lesion, representing a challenge to applying the TCR complementarity-determining region 3 (CDR3), which is immuno-modulatory HDTs (Ndlovu and Marakalala, 2016). Some responsible for antigen specificity, was shared among individuals TB granulomas may contain dysfunctional T cells associated with in association with HLA restriction (Glanville et al., 2017). This chronic antigen exposure and immune exhaustion akin to helps elucidating the structural anatomy of the T-cell response in subpopulations of tumour-infiltrating T lymphocytes (TIL) in LTBI+ individuals, warrants a more detailed description of TB cancer (Anderson et al., 2017). The heterogeneity in TB granulo- granulomas at different stages. mas may also be associated with a simple sample bias of the Differences in the TCR repertoire may be governed by the sub- granuloma tissue area examined, such as described for tumour anatomy of the TB granuloma as well as infection with different heterogeneity (Gerlinger et al., 2012; Thorsson et al., 2018). T-cell Mtb species or re-infection – which occurs more frequently than populations which are already resident in the lung are also of previously thought (Cohen et al., 2012). The notion that seeding of great importance in orchestrating host responses in the LTBI commonly shared TCR clonotypes in lung tissue occurs early in granuloma based on information from other modalities, as embryonal development suggests that a pre-wired TCR repertoire described in Table 1. Descargado para Anonymous User (n/a) en Autonomous University of Barcelona de ClinicalKey.es por Elsevier en marzo 26, 2021. Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2021. Elsevier Inc. Todos los derechos reservados. S60 M. Rao et al. / International Journal of Infectious Diseases 80 (2019) S58–S61 Table 1 Features of tissue-resident memory (TRM) T cells which may be pertinent to LTBI. Main characteristics of TRM T cells Strongly CD103+ CD69+; some exist in CD103- CD69- compartments Comprise 40% of lung T-cell pool; not found in blood (among PBMCs) Produce more perforin and less granzyme B May clear dormant Mtb reservoirs if activated/mobilised TRM T-cell antigen specificity and Local antigen exposure drives TCR diversity of TRM T cells priming Genetic make-up of individuals plays a bid role i.e. mutational load, pathogen invasion, HLA background Some HLA elements may protect against infection but induce autoimmunity i.e. HLA-B57 is a ‘good antigen presenter’ for influenza-specific epitope recognition yet it is also associated with drug-induced autoimmunity Certain HLA restriction elements may be associated with superior Mtb clearance, thus enabling sterilising immunity in some people – in-depth genetic analysis of clinical samples required Effect of tissue microenvironment on TRM Molecular studies have identified 6 distinct immune profiles in cancer lesions: wound healing, IFN-g-dominant, T-cell biology inflammatory, lymphocyte depleted, immunologically ‘silent’ and TGF-β-dominant These phenotype-genotype combinations dictate the nature and biology of infiltrating as well as resident immune cells, response to immunotherapy as well as chemotherapy and survival Infecting pathogens also dictate the local immunological signature i.e. EBV is linked to lower B cells and HBV associates with high TCR gd numbers and IL-17 expression Exaggerated pro-inflammatory cytokine production i.e. TNF-α, IL-6, IFN-g, IL-18, type 1 interferons and vascular endothelial growth factor (VEGF) induces aberrant neovascularisation and affect immune-cell trafficking and movement within the tissue microenvironment – also shown in TB granulomas The number of PD-1+ tissue-infiltrating and TRM cells – particularly antigen-experienced CD8+ T cells – affects targeted immune responses to cancer and infected host cells alike IL-13, IL-4, IL-5 and TGF-β activate regulatory T-cell subsets, which dampen antigen-specific lymphocyte responses An intricate control of inflammatory responses by TRM subsets in LTBI lesions are likely to dictate whether active disease would ensure. TGF-β upregulation, for instance, is a known characteristic of patients with active TB Memory phenotypes of TRM T cells Effector memory cells (CD45- CCR7-) likely to be excellent at mediating disease regression in patients, based on adoptive cell therapy (ACT) studies in cancer Terminally-differentiated precursor-like T cells (CCR7+, CD45RA+) have reduced telomerase activity, shorter telomeres and lack tissue homing capacity i.e. tissue homing chemokine receptor CXCR3 expression CXCR3+ T cells in SCC tissue correlated positively with increased patient survival – also in the presence of pulmonary TB lesions Other TRM T-cell subsets Double-negative (DN) T cells may arise from CD4+ or CD8+ T cells May also exhibit a stem cell-like (CD103+, CD95+, CD117+) immunophenotype, consistent with favourable outcomes in cancer and TB TCR gd T cells, MAIT cells, invariant NK T cells There is already some evidence of these unconventional T cells playing a role in TB immunopathology as well as protection i.e. Vg9Vd2 T cells and MR1-restricted MAIT as well as iNKT cells in LTBI. Their role early after infection and during LTBI establishment in humans warrants further elucidation Changes in the gut and lung microbiomes and how this may Funding influence establishment of LTBI, local granuloma-associated immune responses and or progression to active TB disease is Giuseppe Ippolito, Sayoki Mfinanga, Francine Ntoumi, Dorothy worth investigating. The gut-lung axis is of particularly importance Yeboa-Manu and Alimuddin Zumla are members of the PANDORA-ID- in augmenting immune responses originating in the gut and NET consortium supported by the European and Developing exerting effects in the thoracic region (Bacher et al., 2019). Countries Clinical Trials Partnership (EDCTP2) programme (Grant Integrating the gut and lung microbiome profile for predicting Agreement RIA2016E-1609). GI received support from the Italian progressors from LTBI and patients with active TB who are likely to Ministry of Health, grant Ricerca Corrente, Research programme n.4: respond to therapy must be considered in future clinical studies. tuberculosis. The views and opinions of authors expressed herein do not necessarily state or reflect those of EDCTP. Markus Maeurer Conclusion acknowledges financial support from the Champalimaud Foundation. Dissecting the heterogeneity and dynamics of LTBI granulomas Ethical approval will yield a plethora of information regarding important aspects of biology: immune responses, mutational burden induced by Mtb Ethical approval was not required for this work. infection and an individual’s microbiome profile. Cutting-edge technology such as deep sequencing and multiplatform immuno- Appendix A. Supplementary data logical analysis will help develop a wholistic framework to better understand TB pathogenesis and the tenets of host protective Supplementary data associated with this article can be found, in mechanisms, which will collectively aid the development of the online version, at https://doi.org/10.1016/j.ijid.2019.02.035. effective HDTs. References Conflicts of interest Anderson KG, Stromnes IM, Greenberg PD. Obstacles posed by the tumor microenvironment to T cell activity: a case for synergistic therapies. Cancer The authors declare no conflicts of interest. Cell 2017;31(3):311–25. Descargado para Anonymous User (n/a) en Autonomous University of Barcelona de ClinicalKey.es por Elsevier en marzo 26, 2021. Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2021. Elsevier Inc. Todos los derechos reservados. M. 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