A Pulmonary Lactobacillus murinus Strain Induces Th17 and RORγt+ Regulatory T Cells and Reduces Lung Inflammation in Tuberculosis.

The lungs harbor multiple resident microbial communities, otherwise known as the microbiota. There is an emerging interest in deciphering whether the pulmonary microbiota modulate local immunity, and whether this knowledge could shed light on mechanisms operating in the response to respiratory pathogens. In this study, we investigate the capacity of a pulmonary Lactobacillus strain to modulate the lung T cell compartment and assess its prophylactic potential upon infection with Mycobacterium tuberculosis, the etiological agent of tuberculosis. In naive mice, we report that a Lactobacillus murinus (Lagilactobacillus murinus) strain (CNCM I-5314) increases the presence of lung Th17 cells and of a regulatory T cell (Treg) subset known as RORγt+ Tregs. In particular, intranasal but not intragastric administration of CNCM I-5314 increases the expansion of these lung leukocytes, suggesting a local rather than systemic effect. Resident Th17 and RORγt+ Tregs display an immunosuppressive phenotype that is accentuated by CNCM I-5314. Despite the well-known ability of M. tuberculosis to modulate lung immunity, the immunomodulatory effect by CNCM I-5314 is dominant, as Th17 and RORγt+ Tregs are still highly increased in the lung at 42-d postinfection. Importantly, CNCM I-5314 administration in M. tuberculosis-infected mice results in reduction of pulmonary inflammation, without increasing M. tuberculosis burden. Collectively, our findings provide evidence for an immunomodulatory capacity of CNCM I-5314 at steady state and in a model of chronic inflammation in which it can display a protective role, suggesting that L. murinus strains found in the lung may shape local T cells in mice and, perhaps, in humans.

C-type lectin receptor DCIR modulates immunity to tuberculosis by sustaining type I interferon signaling in dendritic cells.

Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs.

B Cells Producing Type I IFN Modulate Macrophage Polarization in Tuberculosis.

RATIONALE: In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understood. OBJECTIVES: To document the role of B cells in TB in an unbiased manner. METHODS: We generated the transcriptome of B cells isolated from Mycobacterium tuberculosis (Mtb)-infected mice and validated the identified key pathways using in vitro and in vivo assays. The obtained data were substantiated using B cells from pleural effusion of patients with TB. MEASUREMENTS AND MAIN RESULTS: B cells isolated from Mtb-infected mice displayed a STAT1 (signal transducer and activator of transcription 1)-centered signature, suggesting a role for IFNs in B-cell response to infection. B cells stimulated in vitro with Mtb produced type I IFN, via a mechanism involving the innate sensor STING (stimulator of interferon genes), and antagonized by MyD88 (myeloid differentiation primary response 88) signaling. In vivo, B cells expressed type I IFN in the lungs of Mtb-infected mice and, of clinical relevance, in pleural fluid from patients with TB. Type I IFN expression by B cells induced an altered polarization of macrophages toward a regulatory/antiinflammatory profile in vitro. In vivo, increased provision of type I IFN by B cells in a murine model of B cell-restricted Myd88 deficiency correlated with an enhanced accumulation of regulatory/antiinflammatory macrophages in Mtb-infected lungs. CONCLUSIONS: Type I IFN produced by Mtb-stimulated B cells favors macrophage polarization toward a regulatory/antiinflammatory phenotype during Mtb infection.

The direction of plasma membrane exchange between lymphocytes and accessory cells by trogocytosis is influenced by the nature of the accessory cell.

Exchange of plasma membrane fragments, including cell-surface proteins and lipids, in conjugates formed between lymphocytes and their cellular partners is a field of intense investigation. Apart from its natural occurrence during Ag recognition, the process of membrane transfer can be triggered in experimental or therapeutic settings when lymphocytes targeted by Abs are conjugated to FcgammaR-expressing accessory cells. The direction of membrane capture (i.e., which of the two cells is going to donate or accept plasma membrane fragments) can have important functional consequences, such as insensitivity of tumor cells to treatment by therapeutic mAbs. This effect, called antigenic modulation or shaving, occurs as a result of a process in which the FcgammaR-expressing cells remove the mAb and its target protein from the tumor cells. We therefore analyzed this process in conjugates formed between various FcgammaR-expressing cells and a series of normal or tumor T and B cells opsonized with different Abs capable of triggering membrane exchange (including the therapeutic Ab rituximab). Our results show that the direction of membrane capture is dictated by the identity of the FcgammaR-expressing cell, much more so than the type of lymphocyte or the Ab used. We found that monocytes and macrophages are prone to be involved in bidirectional trogocytosis with opsonized target cells, a process they can perform in parallel to phagocytosis. Our observations open new perspectives to understand the mechanisms involved in trogocytosis and may contribute to optimization of Ab-based immunotherapeutic approaches.

A lentiviral vector expressing a dendritic cell-targeting multimer induces mucosal anti-mycobacterial CD4+ T-cell immunity.

Most viral vectors, including the potently immunogenic lentiviral vectors (LVs), only poorly direct antigens to the MHC-II endosomal pathway and elicit CD4+ T cells. We developed a new generation of LVs encoding antigen-bearing monomers of collectins substituted at their C-terminal domain with the CD40 ligand ectodomain to target and activate antigen-presenting cells. Host cells transduced with such optimized LVs secreted soluble collectin-antigen polymers with the potential to be endocytosed in vivo and reach the MHC-II pathway. In the murine tuberculosis model, such LVs induced efficient MHC-II antigenic presentation and triggered both CD8+ and CD4+ T cells at the systemic and mucosal levels. They also conferred a significant booster effect, consistent with the importance of CD4+ T cells for protection against Mycobacterium tuberculosis. Given the pivotal role of CD4+ T cells in orchestrating innate and adaptive immunity, this strategy could have a broad range of applications in the vaccinology field.

ILC precursors differentiate into metabolically distinct ILC1-like cells during Mycobacterium tuberculosis infection.

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces, and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis (Mtb) infection alters the phenotype and function of lung IL-18Rα+ ILC toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program. Moreover, a BCG-driven type I milieu enhances the early generation of ILC1-like cells during secondary challenge with Mtb. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen-tailored immune response.

Ligand binding but undetected functional response of FcR after their capture by T cells via trogocytosis.

Intercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcgammaR as well as the associated FcRgamma subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcgammaR-expressing APC. Capture of FcgammaR occurred during coculture of T cells with FcgammaR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcgammaR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcgammaR captured by T cells, and biochemical studies suggested the improper integration of FcgammaR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcgammaR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation.

Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in T cells but not in B cells.

Upon recognition of their respective cellular partners, T and B cells acquire their antigens by a process of membrane capture called trogocytosis. Here, we report that various inhibitors of actin polymerization or of kinases involved in intracellular signaling partially or fully inhibited trogocytosis by CD8(+) and CD4(+) T cells, whereas they had no effect on trogocytosis by B cells. Similarly, trogocytosis by T cells was inhibited at 4 degrees C, whereas in B cells it was independent of temperature, indicating that trogocytosis by B cells does not rely on active processes. By contrast, most inhibitors we tested impaired both T-cell and B-cell activation. The differential effect of inhibitors on T-cell and B-cell trogocytosis was not due to the higher affinity of the B-cell receptor for its cognate antigen compared with the affinity of the T-cell receptor for its own antigen, but it correlated tightly with the abilities of T cells and B cells to form conjugates with their target cells in the presence of inhibitors. Trogocytosis thus has different requirements in different cell types. Moreover, the capture of membrane antigen by B cells is identified as a novel signaling-independent event of B-cell biology.

Could CD4 capture by CD8+ T cells play a role in HIV spreading?

CD8(+) T cells have been shown to capture plasma membrane fragments from target cells expressing their cognate antigen, a process termed "trogocytosis". Here, we report that human CD4, the Human Immunodeficiency Virus (HIV) receptor, can be found among the proteins transferred by trogocytosis. CD4 is expressed in a correct orientation after its capture by CD8(+) T cells as shown by its detection using conformational antibodies and its ability to allow HIV binding on recipient CD8(+) T cells. Although we could not find direct evidence for infection of CD8(+) T cells having captured CD4 by HIV, CD4 was virologically functional on these cells as it conferred on them the ability to undergo syncytia formation induced by HIV-infected MOLT-4 cells. Our results show that acquisition of CD4 by CD8(+) T cells via trogocytosis could play a previously unappreciated role for CD8(+) T cells in HIV spreading possibly without leading to their infection.

Host-Derived Lipids from Tuberculous Pleurisy Impair Macrophage Microbicidal-Associated Metabolic Activity.

Mycobacterium tuberculosis (Mtb) regulates the macrophage metabolic state to thrive in the host, yet the responsible mechanisms remain elusive. Macrophage activation toward the microbicidal (M1) program depends on the HIF-1α-mediated metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Here, we ask whether a tuberculosis (TB) microenvironment changes the M1 macrophage metabolic state. We expose M1 macrophages to the acellular fraction of tuberculous pleural effusions (TB-PEs) and find lower glycolytic activity, accompanied by elevated levels of OXPHOS and bacillary load, compared to controls. The eicosanoid fraction of TB-PE drives these metabolic alterations. HIF-1α stabilization reverts the effect of TB-PE by restoring M1 metabolism. Furthermore, Mtb-infected mice with stabilized HIF-1α display lower bacillary loads and a pronounced M1-like metabolic profile in alveolar macrophages (AMs). Collectively, we demonstrate that lipids from a TB-associated microenvironment alter the M1 macrophage metabolic reprogramming by hampering HIF-1α functions, thereby impairing control of Mtb infection.

Suitability of various membrane lipophilic probes for the detection of trogocytosis by flow cytometry.

Trogocytosis is a recently discovered phenomenon whereby lymphocytes capture fragments of the plasma membrane from antigen presenting cells (APCs). Using APCs labeled with widely used fluorescent lipophilic probes, we previously described a trogocytosis analysis protocol (TRAP) useful to understand the mechanisms and biological consequences of this process and to identify lymphocytes reacting specifically with antigen-bearing APCs. We have compared the suitability of 22 different fluorescent lipophilic probes for use in TRAP assays with cytotoxic T lymphocytes (CTL). The criteria we used were: simple and efficient incorporation in APC membranes, minimal passive diffusion among cells but efficient transfer onto T cells during trogocytosis. Sphingosin-based probes were found to incorporate inefficiently into cells. For others with unsaturated lipid chains, we found a tendency for extensive passive diffusion. In the end, about a third of the probes tested were found to be suitable in TRAP assays, which all carry either C16 or C18 saturated carbon chains, including some that can be excited with a red laser. Moreover, we found it possible to combine TRAP assays based on lipophilic probes with intracellular cytokine detection. We have identified a set of new lipophilic fluorescent probes suitable for TRAP assays in combination with intracellular staining.

The Host Microbiota Contributes to Early Protection Against Lung Colonization by Mycobacterium tuberculosis.

Tuberculosis (TB), caused by the airborne bacterial pathogen Mycobacterium tuberculosis, remains a major source of morbidity and mortality worldwide. So far, the study of host-pathogen interactions in TB has mostly focused on the physiology and virulence of the pathogen, as well as, on the various innate and adaptive immune compartments of the host. Microbial organisms endogenous to our body, the so-called microbiota, interact not only with invading pathogens, but also with our immune system. Yet, the impact of the microbiota on host defense against M. tuberculosis remains poorly understood. In order to address this question, we adapted a robust and reproducible mouse model of microbial dysbiosis based on a combination of wide-spectrum antibiotics. We found that microbiota dysbiosis resulted in an increased early colonization of the lungs by M. tuberculosis during the first week of infection, correlating with an altered diversity of the gut microbiota during this time period. At the cellular level, no significant difference in the recruitment of conventional myeloid cells, including macrophages, dendritic cells and neutrophils, to the lungs could be detected during the first week of infection between microbiota-competent and -deficient mice. At the molecular level, microbiota depletion did not impact the global production of pro-inflammatory cytokines, such as interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)-1ß in the lungs. Strikingly, a reduced number of mucosal-associated invariant T (MAIT) cells, a population of innate-like lymphocytes whose development is known to depend on the host microbiota, was observed in the lungs of the antibiotics-treated animals after 1week of infection. These cells produced less IL-17A in antibiotics-treated mice. Notably, dysbiosis correction through the inoculation of a complex microbiota in antibiotics-treated animals reversed these phenotypes and improved the ability of MAIT cells to proliferate. Altogether, our results demonstrate that the host microbiota contributes to early protection of lung colonization by M. tuberculosis, possibly through sustaining the function(s) of MAIT cells. Our study calls for a better understanding of the impact of the microbiota on host-pathogen interactions in TB. Ultimately, this study may help to develop novel therapeutic approaches based on the use of beneficial microbes, or components thereof, to boost anti-mycobacterial immunity.

Chemical labels metabolically installed into the glycoconjugates of the target cell surface can be used to track lymphocyte/target cell interplay via trogocytosis: comparisons with lipophilic dyes and biotin.

Trogocytosis, the process whereby lymphocytes capture membrane components from the cells they interact with, is classically evidenced by the transfer of fluorescent lipophilic compounds or biotinylated proteins from target cells to T or B cells. A particular class of molecules, not studied explicitly so far in the context of trogocytosis is glycoconjugates. Here, we used a method to metabolically install chemical labels in target cell glycoconjugates. Working with those target cells, we describe the conditions allowing CTL to be detected based on glycoconjugate trogocytosis triggered by antigen or stimulatory antibodies. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this approach to the detection of CD4(+) T and B cells. Overall, glycoconjugates were transferred between target cells and lymphocytes during trogocytosis with efficiencies comparable or higher than measured for biotinylated proteins or lipophilic dyes incorporated into general membrane lipids. From a technological point of view, our approach can be employed to detect reactive lymphocytes via glycoconjugate trogocytosis. More generally, we believe that the ever-growing ability to employ chemistry in living systems to label particular compounds will be powerful in unraveling the contributions of glycosylation to various aspects of T and B cells biology.

Wheeling and Dealing With Antigen Presentation in Tuberculosis.

In tuberculosis, antigens are transferred from infected to uninfected dendritic cells. Does this favor T lymphocyte response and anti-mycobacterial host defense? In a recent report published in Cell Host & Microbe, Ernst and colleagues show that Mycobacterium tuberculosis seems to have hijacked this mechanism for its own benefit.

Intercellular transfer of antigen-presenting cell determinants onto T cells: molecular mechanisms and biological significance.

Upon physiological stimulation, receptors with tyrosine kinase activity (RTK) are rapidly internalized together with their soluble ligands. T cell activation is the consequence of recognition by the T cell receptor (TCR) of specific peptide-major histocompatibility protein complexes (peptide-MHC) present at the membrane of antigen-presenting cells (APC). The TCR belongs to the RTK family and is known to be endocytosed upon ligand recognition. It differs from most other RTK in that its ligand, the peptide-MHC complex, is membrane bound and the TCR-ligand interaction is quite weak. Recent experiments have shown that the TCR ligand becomes internalized by T cells upon stimulation. Here we review current knowledge on the molecular mechanisms by which the membrane-bound MHC molecules can be transferred onto T cells, and propose hypotheses on the role this phenomenon could play in physio-pathological situations involving T cells.

Collectin CL-LK Is a Novel Soluble Pattern Recognition Receptor for Mycobacterium tuberculosis.

Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.

Antigen smuggling in tuberculosis.

The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells.

Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

Improving administration regimens of CyaA-based vaccines using TRAP assays to detect antigen-specific CD8(+) T cells directly ex vivo.

Vaccination with recombinant adenylate cyclase of Bordetella pertussis (CyaA) carrying antigen is a promising approach to target antigen-presenting cells. We have used Trogocytosis Analysis Protocol (TRAP) assays to monitor immune responses raised by different vaccination regimens with recombinant CyaA carrying the ovalbumin antigen. We find that the intradermal, intramuscular or subcutaneous routes are all superior to intravenous injections, and actually lead to a sufficiently high frequency of reactive CTL to be detected and characterized directly ex vivo by TRAP assay or other standard assays. Finally, for all routes, we find a clear boosting effect upon re-injection of the vaccine.