Human Lung Epithelial Cells Contain Mycobacterium tuberculosis in a Late Endosomal Vacuole and Are Efficiently Recognized by CD8+ T Cells

Mycobacterium tuberculosis (Mtb) is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells). Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.     

Evidence for MR1 antigen presentation to mucosal-associated invariant T cells

The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its alpha1/alpha2 domains. Recent studies demonstrated that MR1 expression is required for development and expansion of a small population of T cells expressing an invariant T cell receptor (TCR) alpha chain called mucosal-associated invariant T (MAIT) cells. Despite these intriguing properties it has been difficult to determine whether MR1 expression and MAIT cell recognition is ligand-dependent. To address these outstanding questions, monoclonal antibodies were produced in MR1 knock-out mice immunized with recombinant MR1 protein, and a series of MR1 mutations were generated at sites previously shown to disrupt the ability of class Ia molecules to bind peptide or TCR. Here we show that 1) MR1 molecules are detected by monoclonal antibodies in either an open or folded conformation that correlates precisely with peptide-induced conformational changes in class Ia molecules, 2) only the folded MR1 conformer activated 2/2 MAIT hybridoma cells tested, 3) the pattern of MAIT cell activation by the MR1 mutants implies the MR1/TCR orientation is strikingly similar to published major histocompatibility complex/alphabetaTCR engagements, 4) all the MR1 mutations tested and found to severely reduce surface expression of folded molecules were located in the putative ligand binding groove, and 5) certain groove mutants of MR1 that are highly expressed on the cell surface disrupt MAIT cell activation. These combined data strongly support the conclusion that MR1 has an antigen presentation function.     

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery

The compartmental organization of eukaryotic cells is?maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the?cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface.     

MAIT Cell Recognition of MR1 on Bacterially Infected and Uninfected Cells

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.     

Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells

Vitamin B2 (riboflavin) is essential for metabolic functions and is synthesized by many bacteria, yeast, and plants, but not by mammals and other animals, which must acquire it from the diet. In mammals, modified pyrimidine intermediates from the microbial biosynthesis of riboflavin are recognized as signature biomarkers of microbial infection. This recognition occurs by specialized lymphocytes known as mucosal associated invariant T (MAIT) cells. The major histocompatibility class I-like antigen-presenting molecule, MR1, captures these pyrimidine intermediates, but only after their condensation with small molecules derived from glycolysis and other metabolic pathways to form short-lived antigens. The resulting MR1-Ag complexes are recognized by MAIT cell antigen receptors (αβ T cell receptors (TCRs)), and the subsequent MAIT cell immune responses are thought to protect the host from pathogens at mucosal surfaces. Here, we review our understanding of how these novel antigens are generated and discuss their interactions with MR1 and MAIT TCRs.     

Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells

Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8⁺ T cells play a unique role. High frequency Mtb-reactive CD8⁺ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8⁺ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Vα7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an “innate” T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.     

Syntaxin7 is required for lytic granule release from cytotoxic T lymphocytes

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL-mediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.     

Mucosal associated invariant T cells: Don't forget your vitamins

T lymphocytes express clonal receptors, called T cell receptors (TCRs), which specifically recognize antigens presented in combination with major histocompatibility molecules (MHC). To date, T cell antigens can be broadly categorized into two classes: peptides and lipids. A recent paper published in Nature by Kjer-Nielsen and colleagues reveals that a unique population of T lymphocytes expresses TCRs that recognize a completely new and unexpected class of antigens, vitamin metabolites.The immune system has evolved a variety of defense mechanisms against foreign pathogens, including both innate and adaptive processes that work in concert to eliminate potential threats. The innate arm of the immune system includes cells that express a variety of non-polymorphic, generic receptors, which recognize structurally conserved molecules derived from microbes. In contrast, the adaptive arm includes cells that express clonally distributed variable receptors generated through somatic rearrangements of gene segments, which recognize specific antigens derived from pathogens. Engagement of these receptors at the surface of lymphocytes by their specific antigens results in clonal division and the production of cellular mediators. The variable receptors are immunoglobulin, expressed by B lymphocytes, and the αβ T cell receptor (TCR), expressed by the vast majority of T lymphocytes.In contrast with immunoglobulins, which can recognize virtually any antigenic structure, αβ TCRs recognize antigens that are displayed by antigen-presenting molecules, such as the ones encoded by the major histocompatibility complex (MHC). MHC class I and class II are polymorphic molecules that present a multitude of antigens in the form of peptides derived from pathogens. However, it is now clear that a significant fraction of T lymphocytes bear αβ TCRs that do not recognize conventional MHC molecules plus peptides but instead are directed at what has been labeled as “non-classical” MHC-like molecules. These “non-classical” MHC-like molecules are often encoded in the genome outside of the MHC locus itself and display little to no polymorphism. As such, a unique role in antigen presentation is usually expected from these “non-classical” MHC-like molecules. For example, H2-M3 molecules have the unique capacity to present bacteria-derived N-formylated peptides¹, while members of the CD1 family, which includes the well-studied CD1d molecule, present lipid antigens².While CD1d plus lipid complexes can be recognized by a variety of lymphocytes bearing different αβ TCRs, they are also the target of a unique innate-like T lymphocyte population called natural killer T (NKT) cells. The NKT TCR is somewhat of an anomaly in the world of classical αβ TCRs in that is formed through the usage of a restricted set of gene segments. The α chain of the NKT TCR is always comprised of a single canonical rearrangement between the TRAV11 and TRAJ18 gene segments in mice (or the orthologs genes TRAV10 and TRAJ18 in human), which pairs with a limited set of Vβ segments. The NKT TCR has been shown to recognize a variety of self and foreign lipids presented by CD1d and its engagement at the surface of NKT cells leads to a rapid and diverse cytokine secretion storm. As such, NKT cells have been implicated in the regulation of a multitude of immunological processes, including infections, cancer, and autoimmunity³.Another subset of T cells bearing a restricted αβ TCR repertoire was recently identified^4,5. Due to their preferential localization in the gut lamina propria, these cells were deemed mucosal-associated invariant T (MAIT) cells⁶. Their ''semi-invariant'' TCRα chain is composed of a limited set of rearrangements between the TRAV1 and the TRAJ33 gene segments, which pair with a limited set of Vβ chains. The generation of a monoclonal antibody directed at the human TRAV1 chain allowed for the enumeration and tracking of MAIT cells. Surprisingly, it was found that MAIT cells can constitute up to 10% of human peripheral blood T cells and up to 40% of human liver T cells⁷.The TCRs expressed by MAIT cells were shown to recognize the MHC-related protein 1, MR1, a very intriguing non-classical MHC class I molecule in its infancy of characterization⁶. MR1 is encoded outside of the MHC locus in human, mouse and rat, and shows 90% sequence identity in its putative ligand-binding domains (α1/α2) between the human and the mouse, which far exceeds the 70% similarity shared by this region of human and mouse classical MHC class I molecules. The strict conservation of both MR1 and MAIT cells among mammalian species, as well as the important proportion of MAIT cells within the human T lymphocyte population, are all suggestive of stringent evolutionary pressure for important function(s) fulfilled by MAIT cells. In support of this hypothesis, it was shown that MAIT cells are activated by cells infected with various strains of bacteria and yeast in both human and mouse^8,9. This activation required cognate interaction between the invariant TCR and MR1, which was proposed to present a bacteria-derived ligand. In this way, these lymphocytes can rapidly sense and help fight off microbial infections. However, the exact nature of this putative bacteria-derived ligand has remained elusive.In a recent issue of Nature, Kjer-Nielsen et al.¹⁰ shed some new light on the nature of the MAIT antigens. Owing to the fact that, in general, MHC class I molecules are extremely unstable unless they have engaged a ligand, Kjer-Nielsen et al. found that refolding of MR1 in the presence of vitamin-containing solutions substantially increased their yield of refolded MR1 proteins. Taking advantage of this finding, they further refined their candidate ligands and identified the presence of the folic acid (vitamin B9) metabolite, 6-formyl pterin (6-FP), bound to MR1. Further, they provided the first crystal structure of the MR1 protein in complex with 6-FP, thereby revealing how the MR1 antigen-binding groove appears ideally suited to present small organic compounds. Interestingly, 6-FP was found in the MR1 antigen-binding cleft where it sits horizontally with no residues extending over the α helices for potential recognition by a TCR. Indeed, the authors showed that although 6-FP can clearly be presented by MR1 molecules, it is non-stimulatory for MAIT cells.The authors were also able to refold MR1 in the presence of culture supernatant from Salmonella typhimurium, a bacterial strain known to stimulate MAIT cells. Mass spectrometry analysis of MR1-complexed ligands revealed metabolites from the riboflavin (vitamin B2) biosynthesis pathway. The riboflavin metabolites are structurally similar to 6-FP, but possess an extra ribityl moiety, which is postulated to extend up into the groove of MR1 and be accessible for TCR recognition. In support of this hypothesis, the riboflavin metabolites are able to stimulate human MAIT cells as well as Jurkat cells engineered to express three different human MAIT TCRs.These findings have important implications, not only for the emerging field of MAIT cell biology but also for immunologists in general. In addition to peptides and lipids, the immune system contains T cells that have the ability to recognize and survey a third class of antigens: vitamin B metabolites (Figure 1).Open in a separate windowFigure 1Three broad classes of ligands recognized by TCRs: peptides, lipids, and vitamin metabolites. Examples of each class of ligands are shown. Peptides from MCMV and flagellin are presented by MHC class I or class II to classical αβ T cells, respectively. Vitamin metabolites are presented to MAIT cells by MR1. The lipids, α-galactosylceramide (αGalCer) and dideoxymycobactins presented to NKT cells and CD1a-restricted T cells, respectively.Mammals, including humans, have lost the capacity for de novo synthesis of B vitamins and rely on diet as well as intestinal bacteria and yeast species that can synthesize them for their acquisition via intestinal absorption. B vitamins are essential components of many cellular processes, with many functioning as precursors for enzyme cofactors or playing the role of coenzymes that carry chemical groups or electrons between molecules. Importantly, riboflavin (vitamin B2) itself, which is found in all mammalian cells, did not stimulate human MAIT cells, but its metabolic precursor, 6,7-dimethyl-8-ribityllumazine, did. These results suggest that the role of MAIT cells might be to survey microbial infections or overgrowth at mucosal sites by sensing the overall quantity of riboflavin metabolites in an MR1-restricted manner. In support of this idea, the authors noted that bacteria and yeast species that were found to stimulate MAIT cells all possess a complete riboflavin synthesis pathway, while other non-stimulatory species did not have this ability.The findings by Kjer-Nielsen et al. raise many new interesting and intriguing questions. The study highlights that MR1 molecules can present both vitamin B2 and B9 metabolites, yet only vitamin B2 metabolites can stimulate MAIT cells. These results raise the possibility that several different metabolites might compete for MR1 binding and thereby modulate the activation of MAIT cells. To date, the mechanisms of antigen presentation by MR1 molecules remain largely unexplored.Like conventional αβ T cells that undergo positive selection by self-peptide-MHC complexes in the thymus, MAIT cells also develop in the thymus, where they must recognize MR1 molecules, presumably loaded with antigens, for proper development^6,11. Are these antigens really “self” or are they, as the absence of MAIT cells in germ-free mice could perhaps suggest, metabolic products derived from the microbiota? Although riboflavin transporters have been identified¹², it remains unclear whether and how its metabolites might be transported throughout the organism. Furthermore, certain clones of MAIT cells can detect non-infected MR1-expressing antigen-presenting cells (APCs), suggesting that some MAIT TCRs might have a dual specificity for both microbe-derived metabolites as well as APC-derived, or media-provided, antigen(s). These results imply that perhaps other antigenic structures distinct from vitamin metabolites might exist for MAIT cells. Identification of the antigen(s) that are involved in intrathymic MAIT cell selection will certainly remain a central goal in the future.Finally, the preferential localization of MAIT cells in the lining of mucosal surfaces and their protective role in several infections^8,9,13,14 open new avenues for the development of vaccine strategy that specifically targets MAIT cells but also call for the exploration of a potential role of MAIT cells in mucosal disorders such as Crohn''s disease and ulcerative colitis.     

Cell surface expression of MR1B,a splice variant of the MHC class I-related molecule MR1, revealed with antibodies

The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αβ T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αβTCR footprints.     

MR1-restricted V alpha 19i mucosal-associated invariant T cells are innate T cells in the gut lamina propria that provide a rapid and diverse cytokine response

Mucosal-associated invariant T (MAIT) cells reside primarily in the gut lamina propria and require commensal flora for selection/expansion. They are restricted by the highly conserved MHC class I-related molecule MR1 and, like most NK T cells, express an invariant TCRalpha chain. Although they probably contribute to gut immunity, MAIT cells have not been functionally characterized because they are so rare. To create a model in which they are more abundant, we generated transgenic mice expressing only the TCRalpha chain (Valpha19i) that defines MAIT cells. By directly comparing Valpha19i transgenic mice on MR1+/+ and MR1-/- backgrounds, we were able to distinguish and characterize a population of Valpha19i T cells dependent on MR1 for development. MR1-restricted Valpha19i transgenic T cells recapitulate what is known about MAIT cell development. Furthermore, a relatively high proportion of transgenic MAIT cells express NK1.1, and most have a cell surface phenotype similar to that of Valpha14i NK T cells. Finally, MR1-restricted Valpha19i T cells secrete IFN-gamma, IL-4, IL-5, and IL-10 following TCR ligation, and we provide evidence for what may be two functionally distinct MAIT cell populations. These data strongly support the idea that MAIT cells contribute to the innate immune response in the gut mucosa.     

Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.     

An efficient co-expression and purification system for the complex of Stx4 and C-terminal domain of Synip

Synip and Stx4 complex plays a key role in GLUT4 vesicle trafficking and fusion with plasma membrane. The interaction of Synip with Stx4 prevents interaction of VAMP2 located in GLUT4 vesicle with Stx4 in basal state. Insulin induces the dissociation of the Synip and Stx4 complex, and then triggers VAMP2 to interact with Stx4 to form the SNARE complex, thus promoting the vesicle fusion. In this report, we adopt a novel system for co-expression of the Synip and Stx4 by using two common vectors pGEX6p-1 and pET28a(+) to investigate their expression, purification, and interaction. Through this co-expression system, we successfully co-expressed the Synip and Stx4 complex with high yield, and co-purified at an approximate 1:1 molar ratio with high purity (95%). We also demonstrate that the 1-28 residues of Stx4 are dispensable for interaction with Synip using this co-expression system.     

Structure-function analysis of VPS9-ankyrin-repeat protein (Varp) in the trafficking of tyrosinase-related protein 1 in melanocytes

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking.     

Morphological and histochemical characterization of the secretory epithelium in the canine lacrimal gland

In the present study, the expression of secretory components and vesicular transport proteins in the canine lacrimal gland was examined and morphometric analysis was performed. The secretory epithelium consists of two types of secretory cells with different morphological features. The secretory cells constituting acinar units (type A cells) exhibited higher levels of glycoconjugates, including β-GlcNAc, than the other cell type constituting tubular units (type T cells). Immunoblot analysis revealed that antimicrobial proteins, such as lysozyme, lactoferrin and lactoperoxidase, Rab proteins (Rab3d, Rab27a and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins (VAMP2, VAMP4, VAMP8, syntaxin-1, syntaxin-4 and syntaxin-6), were expressed at various levels. We immunohistochemically demonstrated that the expression patterns of lysozyme, lactoferrin, Rab27a, Rab27b, VAMP4, VAMP8 and syntaxin-6 differed depending on the secretory cell type. Additionally, in type T cells, VAMP4 was confined to a subpopulation of secretory granules, while VAMP8 was detected in almost all of them. The present study displayed the morphological and histochemical characteristics of the secretory epithelium in the canine lacrimal gland. These findings will help elucidate the species-specific properties of this gland.Key words: dog, lacrimal gland, glycoconjugate, Rab protein, SNARE protein, electron microscopy     

A gamma interferon independent mechanism of CD4 T cell mediated control of M. tuberculosis infection in vivo

CD4 T cell deficiency or defective IFNγ signaling render humans and mice highly susceptible to Mycobacterium tuberculosis (Mtb) infection. The prevailing model is that Th1 CD4 T cells produce IFNγ to activate bactericidal effector mechanisms of infected macrophages. Here we test this model by directly interrogating the effector functions of Th1 CD4 T cells required to control Mtb in vivo. While Th1 CD4 T cells specific for the Mtb antigen ESAT-6 restrict in vivo Mtb growth, this inhibition is independent of IFNγ or TNF and does not require the perforin or FAS effector pathways. Adoptive transfer of Th17 CD4 T cells specific for ESAT-6 partially inhibited Mtb growth while Th2 CD4 T cells were largely ineffective. These results imply a previously unrecognized IFNγ/TNF independent pathway that efficiently controls Mtb and suggest that optimization of this alternative effector function may provide new therapeutic avenues to combat Mtb through vaccination.     

Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B,Slp4a,Vamp7, and Syntaxin 3

Mutations in the motor protein Myosin Vb (Myo5B) or the soluble NSF attachment protein receptor Syntaxin 3 (Stx3) disturb epithelial polarity and cause microvillus inclusion disease (MVID), a lethal hereditary enteropathy affecting neonates. To understand the molecular mechanism of Myo5B and Stx3 interplay, we used genome editing to introduce a defined Myo5B patient mutation in a human epithelial cell line. Our results demonstrate a selective role of Myo5B and Stx3 for apical cargo exocytosis in polarized epithelial cells. Apical exocytosis of NHE3, CFTR (cystic fibrosis transmembrane conductance regulator), and GLUT5 required an interaction cascade of Rab11, Myo5B, Slp4a, Munc18-2, and Vamp7 with Stx3, which cooperate in the final steps of this selective apical traffic pathway. The brush border enzymes DPPIV and sucrase-isomaltase still correctly localize at the apical plasma membrane independent of this pathway. Hence, our work demonstrates how Myo5B, Stx3, Slp4a, Vamp7, Munc18-2, and Rab8/11 cooperate during selective apical cargo trafficking and exocytosis in epithelial cells and thereby provides further insight into MVID pathophysiology.     

VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.