γδ T Cells Cross-Link Innate and Adaptive Immunity in Mycobacterium tuberculosis Infection

Protective immunity against mycobacterial infections such as Mycobacterium tuberculosis is mediated by interactions between specific T cells and activated antigen presenting cells. To date, many aspects of mycobacterial immunity have shown that innate cells could be the key elements that substantially may influence the subsequent adaptive host response. During the early phases of infection, innate lymphocyte subsets play a pivotal role in this context. Here we summarize the findings of recent investigations on γδ T lymphocytes and their role in tuberculosis immunity

Human macrophage model for selective evaluation of CD8⁺ and γδ⁺ cytotoxic T cell function in tuberculosis

The human macrophage cell line U937 was investigated as an in vitro model for human macrophage function in mycobacterial infections. This involved evaluating the ability of differentiated U937 cells to phagocytose Mycobacterium tuberculosis, control intracellular mycobacterial growth, and present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiation of U937 cells usmg IFN-γ, 1,25-(OH)₂ vitamin D₃, or PMA significantly enhanced their ability to phagocytose M. tuberculosis but failed to induce a subsequent respiratory burst response. Following infection, U937 cells were found to be permissive to the intracellular growth of both the virulent H37Rv strain of M. tuberculosis and the attenuated vaccine strain of M. bovis BCG. U937 cells have been shown to constitutively express high levels of cell surface HLA class I while expressing undetectable levels of HLA class II both at the mRN A level and at the cell surface. HLA class II expression was neither up-regulated following infection with M. tuberculosis nor inducible using IFN-γ, 1,25-(OH)₂ vitamin D₃, PMA, GM-CSF or a combination of these agents. In contrast, chronic infection of U937 cells with virulent H37Rv M. tuberculosis (but not with BCG) resulted in the cell surface expression of HLA class I being significantly up-regulated. Taken together, these characteristics made U937 cells a very attractive model for further investigations into their ability to present mycobacterial antigens to human HLA class I-restricted CTLs. Differentiation of U937 cells was found to completely abrogate their sensitivity to non-antigen specific cytolysis mediated by NK or LAK cells. Following infection with M. tuberculosis, U937 target cells were lysed by M. tuberculosis-primed CTLs from HLA class I-matched donors in an antigen-specific manner and with a similar efficiency to autologous macrophage targets. This cytolytic activity was restricted to live organisms since only U937 cells infected with virulent H37Rv M. tuberculosis and BCG but not those pulsed with soluble PPD were lysed by the HLA class I-matched effector cells. On the other hand, M. tuberculosisstimulated but ALA-mismatched CTLs failed to lyse infected U937 cells in an antigen-specific manner. T cell subset fractionation of the HLA class I-matched M. tuberculosis-primed CTL population and limiting dilution cloning demonstrated that the cytolytic activity was mediated by CD8⁺ cytolytic T cells and confirmed that CD4⁺ T cells showed no significant ability to lyse infected U937 target cells. Furthermore, this study found that M. tuberculosis-infected U937 target cells were lysed by CD8⁺ CTLs more rapidly and strongly than similarly infected autologous macrophage targets demonstrating the sensitivity of this in vitro model as an indicator for CD8⁺ cytolytic function in mycobacterial infections. M. tuberculosis-infected U937 cells were found to be highly sensitive to mycobacterial antigenspecific cytolysis mediated by γδ⁺ CTL. Mycobacterial antigen-specific γδ⁺ CTLs consistently showed stronger cytolytic activity against infected U937 target cells than γδ⁺ CTL but were not restricted to classical HLA class I or class II molecules. A panel of cytolytic human M. tuberculosis-reactive γδ⁺ CTL clones was established to investigate more thoroughly the role of γδ⁺ CTL lytic activity in human mycobacterial infections. This study examined the mechanism of cellular cytotoxicity used by these mycobacterial-specific γδ⁺ CTL clones against infected U93 7 targets and further investigated the effect of γδ⁺ T cell-mediated cytolysis on intracellular mycobacterial survival. Cytolysis mediated by the γδ⁺ T cell clones was found to be dependent on cell-to-cell contact. Furthermore, the ability of the γδ⁺ CTL clones to lyse infected targets was found to be strongly Ca²⁺-dependent, sensitive to cyclosporine A (a specific inhibitor of granule exocytosis ), and completely abrogated following Sr²⁺-induced de-granulation of the γδ⁺ T cell effectors, indicating that cytoxicity was mediated predominantly by the granule exocytosis/ perforin pathway. Despite being strongly cytolytic against infected U937 cells, however, the γδ⁺ CTL clones did not have any impact on the survival of intracellular M. tuberculosis. The major conclusions of this study are that U937 cells not only provide a useful in vitro human macrophage model allowing for selective evaluation of HLA class I-restricted CD8⁺ CTL function in mycobacterial infections but also provided a highly sensitive indicator for γδ⁺ CTL cytolytic activity

Interactions between dendritic cells and mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that has a major impact on human health. Control of tuberculosis has proved extremely difficult, particularly in developing and underdeveloped countries; this has been exacerbated by the variable efficacy of BCG, the current vaccine, and by the increasing prevalence of drug resistant strains. Effective immunity against Mtb involves cell-mediated mechanisms. Dendritic cells (DC) are likely to play a critical role in the induction of a cellular response to Mtb since they are the most efficient antigen-presenting cells (APC) for priming naive T cell lymphocytes. In this study we have investigated the interaction between Mtb and DC, and how that interaction might be used to generate protective immune responses. Working with a dendritic cell line and using electron microscopy we have shown that coincubation of DC with M.tuberculosis results in the rapid internalisation of the mycobacteria. Twelve hours after infection the mycobacteria are found within membrane-bound phagosomes. By 96 hours we could see some lysis of the DC although there was no evidence of apoptosis; the presence of Mtb was more difficult to detect by this stage. Over a 5-day period, the viability of M.tuberculosis that had been phagocytosed by DC was found to decline slightly, whereas an identical inoculum was able to replicate in cultured macrophages. We therefore investigated the mechanisms associated with this growth suppression and found that both oxygen and nitrogen radicals where involved. Changes in cytokine production by DC infected with Mtb were observed, with a significant up-regulation of cytokines involved in Th1 and Tc1 responses. Similar results were obtained when primary bone-marrow derived DC were infected with Mtb. Characterisation of surface molecules expressed by DC which had been infected with Mtb confirmed the maturation process of the cells with significant up-regulation of the costimulatory molecules B7-1 and B7-2 and increased expression of MHC class II molecules and ICAM-1. This response was found to be dependent on the rapid activation of the nuclear transcription factor NF-kB, and was independent of TNF-a release. We also demonstrated expression of c-Rel and Rel-B proteins in Mtb-activated DC. In addition to these in vitro studies, we have also demonstrated that Mtb-activated DC are extremely efficient in priming naive murine T cells and that this immune response does not require T cell help. Mtb-activation of DC also results in efficient cross priming of T cells specific for Mtb. These responses enable Mtb-activated DC to confer protection against challenge with viable Mtb, with levels of protection as good or better than those conferred by BCG. The further understanding of the mechanisms involved in the interaction of mycobacteria with DC, and the mechanisms underlying the transfer of protective immunity, should provide important insights for the development of novel approaches to immunotherapy or for the development of new vaccines

Distribuition of V δ1 and V δ2 T lymphocytes during the active disease and specific therapy of tuberculosis

A tuberculose é considerada um problema de saúde pública mundial. Estima-se que um terço da população mundial está infectada pelo Mycobacterium tuberculosis. Trabalhos avaliando a interação patógeno vs. hospedeiro tem incentivado o estudo dos mecanismos envolvidos na resposta imunológica inicial nos indivíduos infectados e doentes. A participação dos linfócitos T na resposta contra a micobactéria tem destacado a importância dessas células na imunopatogênese. Uma subpopulação de linfócitos T, as células T γδ têm sido investigada quanto à sua participação na resposta imunológica, principalmente frente a processos infecciosos e inflamatórios. Essas células carregam cadeia TCR γδ, atuam tanto na resposta inata como na adaptativa e são encontradas em epitélios de alguns órgãos e no sangue periférico. Duas subpopulações são predominantes no sangue periférico, aquelas expressando TCR Vγ2Vδ2 e outras expressando TCR VγnVδ1. A subpopulação Vδ2 tem demonstrado reatividade a produtos micobacterianos, enquanto que os mecanismos específicos das células T Vδ1 permanecem desconhecidos. Este trabalho propôs investigar através da citometria de fluxo a distribuição dos linfócitos T γδ, Vδ1 e Vδ2 na infecção pelo M. tuberculosis. Este trabalho foi realizado com amostras de pacientes com tuberculose pulmonar na fase aguda e durante terapia específica, indivíduos expostos ao M. tuberculosis e controles saudáveis. Nós encontramos uma diminuição na freqüência de células T Vδ2 nos pacientes com tuberculose, permanecendo estável durante terapia. Ao contrário as células T Vδ1 revelaram tanto uma freqüência aumentada na fase aguda, como também aumento contínuo ao longo da terapia específica. Com base no impacto da doença na freqüência desses linfócitos T γδ, acreditamos que essas células estejam envolvidas na resposta imunológica inicial contra a micobactéria, e que exista uma provável função complementar dessas células com as xi células T αβ. Nós recomendamos futuros estudos para explorar e avaliar a funcionalidade das células T Vδ1 e Vδ2, esses dados serão de grande valia para melhor compreender a contribuição dessas células na resposta imune durante a tuberculose.Tuberculosis is an enormous challenge to global health. One third of the world’s population is estimated to be infected by the Mycobacterium tuberculosis. Previous reports assessing the interaction between the pathogen and the host have promoted further studies on the mechanisms involved during the innate immune response of infected individuals. The participation of T lymphocytes in the response against the mycobacterium has revealed the importance of these cells in immunopathogenesis of the disease. The γδ T cells, a subset of T lymphocytes, have been investigated with regard to their participation in the immune response, mainly against infectious and inflammatory processes. These cells express TCR γδ chains, participating in innate as well as adaptive responses, and are found in the epithelium of some organs and in the peripheral blood. There are two predominant subsets of cells in the peripheral blood: those expressing TCR Vγ2Vδ2 chains and those expressing TCR VγnVδ1 chains. The Vδ2 subset has demonstrated reactivity to compounds produced by M. tuberculosis, while the specific mechanisms of Vδ1 T cells remain unknown. Using flow cytometry, this study aims were to investigate the frequency of both Vδ1 and Vδ2 subsets of γδ T cells in Mycobacterium tuberculosis infection. This study was performed with samples that include acute tuberculosis patients, those receiving specific therapy, exposed individuals, and healthy controls. We found that the frequency of Vδ2 T cells was diminished in tuberculosis patients, and remained steadily low during specific therapy. In contrast, the Vδ1 T cells subset displayed not only increased frequency at acute phase, but also continued to rise throughout the course of specific therapy. Based on the impact of the disease on the γδ T cells frequency, we believe that these cells may be involved in an innate immune response xiii against the mycobacterium. Additionally, it is likely that they play a complementary role to αβ T cells during infectious processes. We recommend future studies to explore and evaluate the funcionality of Vδ1 and Vδ2 T cells in order to better understand the contribution of these subsets in the immune response during tuberculosis infection.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)BV UNIFESP: Teses e dissertaçõe

Subcutaneous challenge of cattle with Mycobacterium avium subspecies paratuberculosis: an experimental system to study CD4+ T cell and gamma-delta T cell responses to infection

The cellular immune response in cattle to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis ) infection is poorly understood. To examine these early cellular events, we developed an experimental system in the cow to evaluate lymphocyte function following subcutaneous inoculation. Sixty days after infection, there was increased antigen-specific proliferation of lymphocytes from lymph nodes draining the site of infection in M. paratuberculosis-inoculated calves compared to those in saline-inoculated calves, but there were no differences in interferon (IFN)-gamma expression by lymph node-derived lymphocytes between these two groups, suggesting a lack of local T helper type 1 (Th1) polarization. All M. paratuberculosis-inoculated calves developed a strong delayed-type hypersensitivity response, consistent with development of Th1-polarized systemic effector/memory cells. Compared to antigen-stimulated draining lymph node cells, there was increased lymphocyte proliferation and IFN-gamma production by antigen-stimulated lymphocytes derived from the spleen in subcutaneously-infected calves. To test the hypothesis that the immune response at the site of infection is relatively impaired in comparison to the central immune response, we vaccinated calves with the commercial paratuberculosis vaccine, which induces a strong Th1 response, and then challenged the calves with live M. paratuberculosis . Whereas there was marked antigen-specific CD4+ T cell proliferation and IFN-gamma production by lymphocytes from the lymph node draining the vaccination site, there was no CD4+ T cell proliferation or IFN-gamma production from lymphocytes from the lymph node draining the challenge site in the same calf. The data suggest that live M. paratuberculosis impairs antigen-presenting cell (APC) function at the site of infection, such that there is defective signaling by the APC or reduced trafficking of effector CD4+ T cells into the lesion site. Draining lymph node cells were evaluated for their ability to activate M. paratuberculosis-infected macrophages. gammadelta T cells exposed to infected macrophages did not produce significant IFN-gamma, and nitric oxide production did not increase in cultures of infected macrophages containing gammadelta T cells; as such, gammadelta T cells do not appear to classically activate infected macrophages. In addition, antigen-stimulated CD4+ T cells producing significant amounts of IFN-gamma from infected calves also fail to induce macrophage activation

Chemical and immunological characterisation of glycophospholipid and phospho-oligosaccharide from mycobacteria

Following ligand binding by a wide variety of cytokines and growth factors, a glycosylphosphatidyl inositol phospholipase D (GPI-PLD) cleaves free membrane glycosylphosphatidyl inositol (GPI) to liberate water-soluble inositol phosphoglycan (IPG) second messengers. IPG are released outside cells and mediate many of the immediate metabolic effects of insulin. Many other cytokines and growth factors use IPG to signal, including IL-2, NGF, IGF-1 and ACTH. The composite structure of IPG molecules is known and they consist of hexose, hexosamine, inositol, phosphate and divalent cations. The development of clinical tuberculosis in a susceptible host appears to involve a highly complex interaction between the infecting organism and the host immune system, with much or indeed all the tissue damage characteristic of the disease being immune mediated. Many Mycobacterium tuberculosis colonised individuals do not develop active disease, and there are demonstrable immunological differences between the asymptomatic immune carrier state and the state of active disease in both humans and animal models. Mycobacterium tuberculosis contains PLD activity and contains phosphatidyl inositol linked glycans that could theoretically give rise to IPG like structures. Such glycolipids include lipoarabinomannan (LAM), lipomannan (LM) and phosphoinositol mannoside (PIM). Previously it has been shown in our laboratory and that of our collaborators that two strains of the pathogen M. tuberculosis and a non-pathogen, M. vaccae, contain IPG- like biological activity. This mycobacteria derived material has been provisionally named phospho-oligosaccharide (POS). The aim of this study was to examine the hypothesis that mycobacteria may contain a homologous system to the mammalian GPI/IPG signalling mechanism. Using an established protocol for the purification of mammalian GPI a glycophospholipid (GPL) was isolated from M. tuberculosis and M. vaccae that showed similar characteristics to mammalian GPI. This GPL was a substrate for (glycosyl) phosphatidylinositol phospholipase C (GPI-PLC), contained phosphate, sugar residues and did not contain amino groups. GPL was biologically active in a cell free pyruvate dehydrogenase (PDH) phosphatase activation assay and induced inflammatory mediator release from monocyte/macrophage cells. GPL is distinct from LAM, LM and PIM because it was sensitive to GPI-PLC and incorporated radioactive galactose. POS fractions were isolated from mycobacteria that were biologically active in PDH phosphatase, lipogenesis, cell proliferation and nitric oxide production assays, contained carbohydrate, phosphate (except for one fraction) and amino groups. On the basis of amino group analysis, which revealed that POS contained amino groups and GPL did not, POS is not a cleavage product of mycobacterial GPL, as is the case with GPI and IPG in mammalian cells. However, there were interesting similarities in biological activity between GPL and POS. Serological studies were undertaken to characterise mycobacterial POS both structurally and immunologically. Although POS exhibit biological activity in systems where mammalian IPG are also active, there are clearly structural differences between POS and IPG because some antibodies that bind IPG do not bind POS. POS are immunogenic: administration of complete Freund's adjuvant induced an anti-POS antibody response in rabbits (but not in mice) and sera from tuberculosis patients contained significantly higher levels of anti-POS antibody than healthy controls. POS is proliferogenic for human peripheral blood mononuclear cells in vitro, in particular, POS activates human B cells in vitro causing a greater increase in B cell CD25 expression than T cell CD25 expression. Thus the material described has some interesting immunological properties, although its role in the pathogenesis of tuberculosis remains to be determined. The growth factors IL-2 and insulin have both been shown to signal using IPG cleaved from GPI, it is therefore intriguing that mycobacteria appear to contain a homologue of an immunologically relevant mammalian second messenger

GENOMIC ORGANIZATION AND EXPRESSION OF THE WC1 HYBRID CORECEPTOR AND PATTERN RECOGNITION RECEPTOR ON PORCINE GAMMA DELTA T CELLS

gd T cells are a crucial component of the immune response to a number of increasingly relevant and largely zoonotic pathogens to which efficacious vaccination is lacking. In ruminants and swine, gd T cells represent a major population of peripheral blood and epithelial tissue-resident lymphocytes. gdT cells respond to both protein and non-protein antigens independently of MHC presentation and possess immunological memory. Upon activation, gamma delta T cells illicit a variety of effector functions and play an indispensable role of orchestrating the downstream immune response. These characteristics make gamma delta T cells a promising candidate for recruitment by vaccination, however, methods for effectively priming these cells remain to be elucidated. The type I transmembrane receptor Workshop Cluster One (WC1) is expressed as a multigenic array on gd T cells in swine and ruminants. In cattle there are 13 unique WC1 genes (WC1-1 to WC1-13) each comprised of 6-11 SRCR domains that selectively bind unprocessed antigen in a manner that resembles a pattern recognition receptor (PRRs). WC1 functions as a hybrid PRR and co-receptor for the gamma delta TCR as it potentiates activation signals from the TCR and dictates antigen specificity of expressing gd T cells. cDNA evidence suggests that porcine WC1 is expressed as a multigenic array consisting of 9 genes (WC1-1 to WC1-9) each encoding 6 SRCR domains with unique pathogen binding potential. The objective of this study is to characterize the multigenic array of porcine WC1, investigate its propensity for pathogen binding, and evaluate its expression on gd T cells. Using the MAKER annotation pipeline, we annotated Sscrofa11.1 for sequence derived from full-length cDNA transcripts representing the 9 porcine WC1 genes. We were able to map 8 of the 9 genes, leaving one (WC1-8) unplaced in the current assembly. We defined three subpopulations of porcine gd T cells based on expression of WC1 and CD2. Finally, we confirmed that porcine WC1 SRCR domains are capable of directly binding whole fixed bacteria including Leptospira spp and Mycobacterium bovis

Dendritic cell mediated modulation of immune responses by Mycobacterium vaccae

The contemporary hygiene hypothesis suggests that certain microorganisms that were present throughout human evolution modulate the host immune system to reduce allergy associated T helper 2 (Th2) responses and inflammatory diseases by augmenting regulatory T cells. The prototypic environmental mycobacterium, M. vaccae has been used in mouse models of asthma to support this hypothesis, but data from human models and possible mechanisms are very limited. In view of the role of dendritic cells (DCs) in shaping adaptive T cell responses, the effect of innate immune interactions between human DCs and M. vaccae on allogeneic and antigen specific DC-dependent polarisation of T cells was tested. M. vaccae can stimulate cellular activation via Toll-like receptor 2 (TLR2) and therefore was compared to a specific TLR2 ligand (Pam3CSK4) and alternative stimulation with a TLR4 ligand (LPS). M. vaccae alone induced DC-dependent inhibition of Th2 responses, in contrast to Pam3CSK4, which had the opposite effect and LPS, which had no polarising effect. Comparison of DC maturation, genome-wide transcriptional response, and cytokine production in response to each stimulus did not correlate with the specific functional effects. In particular, directly comparable DC transcriptional responses to M. vaccae and Pam3CSK4 suggested that TLR2-mediated transcriptional regulation was not sufficient for inhibition of Th2 responses. Exclusive transcriptional responses to M. vaccae implicated a role for CREB1-dependent gene expression and analysis of signalling events confirmed selective early activation of the CREB pathway by M. vaccae. Collectively, this work has established that M. vaccae interaction with DCs does inhibit human Th2 responses and that further study of the CREB pathway in this model may provide novel insight into the molecular mechanisms of DC-dependent T cell polarisation. The final chapter of results presents development and validation of a novel approach for using short interspersed elements (SINEs) as a tool for normalisation of RT-qPCR data

Kinetics of the Immune Response to Mycobacterium Paratuberculosis of Resistant and Susceptible Mice.

The resistance and immunologic responses of a Bcg susceptible (C57BL/6) and Bcg resistant (C3H/He) strain of mouse were compared after infection with Mycobacterium paratuberculosis. Bacterial resistance was evaluated by bacterial counts and histopathology of chronically infected mice. Levels of non-specific macrophage activation and the response of various T cell subsets were also evaluated following infection. Susceptible mice orally infected with Mycobacterium paratuberculosis developed granulomatous lesions containing acid-fast bacteria in the mesenteric lymph nodes. Significant differences in CFU of M. paratuberculosis were observed between C57BL/6 and C3H/He mice following an i.p. infection. Susceptible mice failed to limit bacterial proliferation, while bacterial counts progressively declined in resistant mice. Susceptible mice had numerous granulomas in the liver and developed mesenteric lymph node lesions. Resistant mice had fewer hepatic granulomas and did not develop mesenteric lymph node lesions. Both strains of mice developed similar levels of non-specific macrophage activation 10 days after i.p. infection with M. paratuberculosis as determined by a listeria challenge assay. Differences were detected in the proportions of T cell subsets and activation markers between the two strains. Both control and infected C3H/He mice had higher percentages of CD4+ cells, whereas C57BL/6 mice had higher proportions of CD8+ and $\gamma\delta$ cells. Both strains responded to the initial infection with increased numbers of CD8+ and/or $\gamma\delta$ cells, but resistant mice responded with higher proportions of CD4+ cells, whereas C57BL/6 mice responded with higher percentages of $\gamma\delta$ T and/or CD8+ cells. Differences in the expression of CD25+ and CD44+ cells were also detected between the two strains of mice. Expression of CD25 increased in resistant mice, but decreased in susceptible mice after infection. Overall CD44 expression was higher in susceptible mice but increased after infection in both mice. These data suggest that resistance to M. paratuberculosis is associated with higher proportions of CD4+ T cells, while CD8+ and/or $\gamma\delta$ T cells are associated with susceptibility. The decrease in CD25 receptor expression in susceptible mice suggests that a dysregulation in IL-2 or its receptor may also be involved in the pathogenesis of paratuberculosis

Pre-clinical assessment of novel candidate HIV-1 vaccines using the Chacma baboon

Includes bibliographical references (leaves 176-221)