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The granuloma is an elaborated aggregate of immune cells found in non-infectious as well as infectious diseases. It is a hallmark of tuberculosis (TB). Predominantly thought as a hostdriven strategy to constrain the bacilli and prevent dissemination, recent discoveries indicate the granuloma can also be modulated into an efficient tool to promote microbial pathogenesis. The aim of future studies will certainly focus on better characterization of the mechanisms driving the modulation of the granuloma functions. Here, we provide unique perspectives from both the innate and adaptive immune system in the formation and the role of the TB granuloma. As macrophages (MΦs) comprise the bulk of granulomas, we highlight the emerging concept of MΦ polarization and its potential impact in the microbicide response, and other activities, that may ultimately shape the fate of granulomas. Alternatively, we shed light on the ability of B cells to influence inflammatory status within the granuloma. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes recently stimulated by antigen-presenting cells (APC) display major histocompatibility class (MHC) I and II molecules inherited from APC. We have previously reported that, in vitro, transfer of MHC molecules and several other proteins occurs through trogocytosis, i.e. the active acquisition of target cell membrane fragments by T lymphocytes. Here, using the model of viral antigen LCMVgp33-41 recognition in transgenic P14 mice, we show that CD8+ T cells perform trogocytosis in vivo, as detected by the capture of biotin- or fluorescence-labeled components of the APC surface. Trogocytosis occurs during interactions of CD8+ T cells with at least two kinds of cells: target cells and dendritic cells (DC). In lymph nodes, CD8+ T cells having performed trogocytosis with DC express the CD69 activation marker indicating that trogocytosis detects recently activated cells. Taken together, our findings suggest that trogocytosis may be a new in vivo marker of the recent interaction between a CD8+ T cell and its cellular partners or targets. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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)-1β 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.

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.

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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The pattern of receptors sensing pathogens onto host cells is a key factor that can determine the outcome of the infection. This is particularly true when such receptors belong to the family of pattern recognition receptors involved in immunity. Mycobacterium tuberculosis, the etiologic agent of tuberculosis interacts with a wide range of pattern-recognition receptors present on phagocytes and belonging to the Toll-like, Nod-like, scavenger and C-type lectin receptor families. A complex scenario where those receptors can establish cross-talks in recognizing pathogens or microbial determinants including mycobacterial components in different spatial and temporal context starts to emerge as a key event in the outcome of the immune response, and thus, the control of the infection. In this review, we will focus our attention on the family of calcium-dependent carbohydrate receptors, the C-type lectin receptors, that is of growing importance in the context of microbial infections. Members of this family appear to be key innate immune receptors of mycobacteria, capable of cross-talk with other pattern recognition receptors to induce or modulate the inflammatory context upon mycobacterial infection.

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.

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.

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.

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.

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., ((c) 2008 International Society for Advancement of Cytometry.)

Objective: Compared with cell-free viral infection, virological synapses increase HIV capture by target cells, viral infectivity and cytopathicity, and are believed to be less sensitive to antibody neutralization. We have evaluated the impact of antibodies against HIV envelope glycoproteins (gp120 and gp41) on cell-to-cell HIV transmission., Methods: We analyzed the role of trogocytosis in cell-to-cell HIV transmission and the inhibitory mechanisms of antigp120 antibody IgGb12 and antigp41 antibodies 4E10 and 2F5 using cocultures of NL4-3 or BaL-infected MOLT/CCR5 cells with primary CD4 T cells., Results: Analysis of early steps of HIV transmission in these cocultures showed that IgGb12, but not 4E10 and 2F5, inhibited the formation of virological synapses. Consequently, IgGb12 but not antigp41 antibodies blocked the transfer of HIV particles from infected to target cells and the trogocytic transfer of CD4 molecules from target to infected cells. Interestingly, trogocytic transfer of membranes was not detected in the HIV transmission direction. Furthermore, analysis of late events of HIV transmission showed that all neutralizing antibodies blocked productive infection of target cells, suggesting that HIV infection between T cells is transmitted by a neutralization-sensitive mechanism involving HIV budding from infected cells and capture by target cells., Conclusion: Despite mechanistic differences, antigp120 and antigp41 antibodies block infectious cell-to-cell HIV transmission. Our data suggest that eliciting high titers of neutralizing antibodies in vivo should be maintained as a main end of HIV vaccine design.

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.

A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.

Key events of T and B cell biology are regulated through direct interaction with APC or target cells. Trogocytosis is a process whereby CD4(+) T, CD8(+) T, and B cells capture their specific membrane-bound Ag through the acquisition of plasma membrane fragments from their cellular targets. With the aim of investigating whether the ability to trigger trogocytosis was a selective property of Ag receptors, we set up an assay that allowed us to test the ability of many different cell surface molecules to trigger trogocytosis. On the basis of the analysis of a series of surface molecules on CD4(+) T, CD8(+) T, and B cells, we conclude that a set of cell type-specific surface determinants, including but not limited to Ag receptors, do trigger trogocytosis. On T cells, these determinants include components of the TCR/CD3 as well as that of coreceptors and of several costimulatory molecules. On B cells, we identified only the BCR and MHC molecules as potentials triggers of trogocytosis. Remarkably, latrunculin, which prevents actin polymerization, impaired trogocytosis by T cells, but not by B cells. This was true even when the same Abs were used to trigger trogocytosis in T or B cells. Altogether, our results indicate that although trogocytosis is performed by all hemopoietic cells tested thus far, both the receptors and the mechanisms involved can differ depending on the lineage of the cell acquiring membrane materials from other cells. This could therefore account for the different biological consequences of Ag capture via trogocytosis proposed for different types of cells.

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.

Detection, quantification, separation and characterization of T and B cells reactive to specific antigens are important tasks in both basic and clinical immunology. Here, we describe an approach allowing the performance of all four tasks on a functional basis by flow cytometry. The assay is based on the property of lymphocytes to capture membrane components from the cells they interact with, in a process we call trogocytosis. Working with CD8+ CTL and target cells labeled with membrane markers, we describe the conditions allowing reactive lymphocytes to be detected rapidly and inexpensively within mixed populations. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this method to the detection of antigen-specific CD4+ T and B cells. While our method is, for the time being, not as sensitive as staining of CTL with MHC class I multimers, it allows the simultaneous quantitative identification of reactive CD8+, CD4+ and B cells. Altogether, our method offers a simple and general alternative to other methods previously described to detect and quantify lymphocyte reactivity, and it can also be used in combination with those.

We have developed a method exploiting the phenomenon of trogocytosis to detect lymphocytes reacting specifically with target cells by flow cytometry. Trogocytosis is a process by which lymphocytes capture fragments of the plasma membrane from the antigen-presenting cells (APCs) expressing their cognate antigen. For this method, a label (such as a fluorescent lipid or biotin) is first incorporated in the membrane of APCs. These labeled cells are then co-cultured for a few hours with a population of cells containing the lymphocytes to be detected. After this period of stimulation, lymphocytes that have performed trogocytosis are identified by their acquisition of the label initially present on the APC membrane using flow cytometry. A major advantage of this method is its compatibility with the simultaneous detection of phenotypic and/or functional markers on the lymphocytes. Furthermore, cells can be recovered alive and active after detection of trogocytosis, and are therefore available for further characterization or even conceivably for therapeutic purposes.

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.

We have investigated the density of peptides required to elicit different biological responses in cytotoxic T lymphocytes (CTL), including trogocytosis (i.e., the phenomenon whereby the lymphocytes actively capture fragments of plasma membrane from those cells with which they establish an immune synapse). We have used two separate mouse models of CTL recognising defined peptides presented by MHC class I molecules. In both systems, triggering of cytotoxicity and capture of membrane components reached saturation with low densities of ligand. On the other hand, down-modulation of cell-surface levels of TCR, induction of IFN-gamma production and detection of peptide captured required much higher ligand densities. Interestingly, fratricide (i.e., killing between CTL sharing the same specificity), a mechanism proposed to account for CTL exhaustion, was detected only at antigen concentrations still well above that second threshold leading to full blown activation. Taken together, our results show that the different thresholds that govern the elicitation of different CTL functions correlate with different proportions of antigen among the target cell components being captured via trogocytosis.

Thymus-derived regulatory T lymphocytes of CD4(+)CD25(+) phenotype regulate a large variety of beneficial and deleterious immune responses and can inhibit lethal graft-versus-host disease in rodents. In vitro, CD4(+)CD25(+) T cells require specific major histocompatibility complex (MHC)/peptide ligands for their activation, but once activated they act in an antigen-nonspecific manner. In vivo, regulatory T cells are also activated in an antigen-specific fashion, but nothing is known about antigen specificity of their suppressor-effector function. Here we show that CD4(+)CD25(+) regulatory T lymphocytes isolated from naive mice and activated in vitro with allogeneic antigen-presenting cells (APCs) induced specific long-term tolerance to bone marrow grafts disparate for major and minor histocompatibility antigens; whereas "target" bone marrow was protected, third-party bone marrow was rejected. Importantly, in mice injected with a mix of target and third-party bone marrows, protection and rejection processes took place simultaneously. These results indicate that CD4(+)CD25(+) regulatory T cells can act in an antigen-specific manner in vivo. Our results suggest that CD4(+)CD25(+) regulatory T cells could in the future be used in clinical settings to induce specific immunosuppression.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.