Publications

The development of mucosal-associated invariant T (MAIT) cells is dependent upon the class Ib molecule MHC-related protein 1 (MR1), commensal bacteria, and a thymus. Furthermore, recent studies have implicated MR1 presentation to MAIT cells in bacteria recognition, although the mechanism remains undefined. Surprisingly, however, surface expression of MR1 has been difficult to detect serologically, despite ubiquitous detection of MR1 transcripts and intracellular protein. In this article, we define a unique mAb capable of stabilizing endogenous mouse MR1 at the cell surface, resulting in enhanced mouse MAIT cell activation. Our results demonstrated that under basal conditions, endogenous MR1 transiently visits the cell surface, thus reconciling the aforementioned serologic and functional studies. Furthermore, using this approach, double-positive thymocytes, macrophages, and dendritic cells were identified as potential APCs for MAIT cell development and activation. Based on this pattern of MR1 expression, it is intriguing to speculate that constitutive expression of MR1 may be detrimental for maintenance of immune homeostasis in the gut and/or detection of pathogenic bacteria in mucosal tissues.

Mucosal-associated invariant T lymphocytes (MAIT lymphocytes) are characterized by two evolutionarily conserved features: an invariant T cell antigen receptor (TCR) alpha-chain and restriction by the major histocompatibility complex (MHC)-related protein MR1. Here we show that MAIT cells were activated by cells infected with various strains of bacteria and yeast, but not cells infected with virus, in both humans and mice. This activation required cognate interaction between the invariant TCR and MR1, which can present a bacteria-derived ligand. In humans, we observed considerably fewer MAIT cells in blood from patients with bacterial infections such as tuberculosis. In the mouse, MAIT cells protected against infection by Mycobacterium abscessus or Escherichia coli. Thus, MAIT cells are evolutionarily conserved innate-like lymphocytes that sense and help fight off microbial infection.

Several nonclassical major histocompatibilty antigens (class Ib molecules) have emerged as key players in the early immune response to pathogens or stress. Class Ib molecules activate subsets of T cells that mount effector responses before the adaptive immune system, and thus are called innate T cells. MR1 is a novel class Ib molecule with properties highly suggestive of its regulation of mucosal immunity. The Mr1 gene is evolutionarily conserved, is non-Mhc linked, and controls the development of mucosal-associated invariant T (MAIT) cells. MAIT cells preferentially reside in the gut, and their development is dependent on commensal microbiota. Although these properties suggest that MAIT cells function as innate T cells in the mucosa, this has been difficult to test, due to the (i) paucity of MAIT cells that display MR1-specific activation in vitro and (ii) lack of knowledge of whether or not MR1 presents antigen. Here we show that both mouse and human MAIT cells display a high level of cross-reactivity on mammalian MR1 orthologs, but with differences consistent with limited ligand discrimination. Furthermore, acid eluates from recombinant or cellular MR1 proteins enhance MAIT cell activation in an MR1-specific and cross-species manner. Our findings demonstrate that the presentation pathway of MR1 to MAIT cells is highly evolutionarily conserved.

Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation. Unlike iNKT cells, MAIT cells require the class Ib molecule MHC-related protein I (MR1), B cells, and gut flora for development and/or expansion, and they preferentially reside in the gut lamina propria. Evidence strongly suggests that MAIT cell activation is ligand-dependent, but the nature of MR1 ligand is unknown. In this study, we define a mechanism of endogenous antigen presentation by MR1 to MAIT cells. MAIT cell activation was dependent neither on a proteasome-processed ligand nor on the chaperoning by the MHC class I peptide loading complex. However, MAIT cell activation was enhanced by overexpression of MHC class II chaperones Ii and DM and was strikingly diminished by silencing endogenous Ii. Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation. The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes. These findings demonstrate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to sample both endocytosed and endogenous antigens.

The major outer membrane protein (MOMP) of Campylobacter jejuni is an abundant surface protein with a pore-forming function and may be a potential candidate for vaccine development. Despite the fact that MOMP is immunogenic and the recombinant MOMP (rMOMP) can be readily produced in Escherichia coli, the nature of the antibody response to MOMP during in vivo infection is not well understood. In this study, various methods involving detergent replacement and liposome reconstitution were used to refold rMOMP, and antibody responses to MOMP elicited in Campylobacter-colonized chickens were evaluated using sera from chickens either naturally or experimentally infected by C. jejuni. The results demonstrated that proteoliposomes restored the reactivity of rMOMP to rabbit antibodies elicited by native MOMP, indicating the recovery of native MOMP conformation by this refolding method. Importantly, sera from naturally or experimentally infected chickens reacted weakly with denatured rMOMP, but strongly with rMOMP reconstituted in proteoliposome, suggesting that the chicken antibody response to MOMP is predominantly directed against conformational epitopes. These observations provide direct evidence for conformation-dependent humoral responses to MOMP induced by Campylobacter infection, demonstrate that C. jejuni MOMP is immunogenic in its natural host and suggest that proteoliposomes may be potentially used for the evaluation of rMOMP-based vaccines.

The identification of pattern-recognition receptors that selectively respond to evolutionarily conserved chemical (often pathogen-derived) moieties has provided key insight into how innate immune cells facilitate rapid and relatively specific antimicrobial immune activity. In contrast, relatively slower adaptive immune responses rely on T cell clonal expansion that develops in response to variable peptides bound to the groove of classical major histocompatibility complex (MHC) proteins. For certain nonclassical 'MHC-like' class Ib proteins, such as H2-M3 and CD1d, their respective binding grooves seem to have been adapted to present to T cells unique molecular patterns analogous to those involved in innate signaling. Here we propose that another MHC class Ib protein, MR1, which is required for the gut flora-dependent development of mucosa-associated invariant T cells, presents either a microbe-produced or a microbe-induced pattern.

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.

Thermophilic Campylobacter, particularly Campylobacter jejuni, is one of the major foodborne human pathogens of animal origin. Reliable and sensitive typing tools are required for understanding the epidemiology and ecology of this zoonotic bacteria agent. Currently, several molecular typing methods are available for differentiating Campylobacter strains, but each of them has limitations. Our previous study revealed that considerable sequence polymorphism exists in the cmp gene encoding the major outer membrane protein of Campylobacter and suggested that sequence variation of cmp may be utilized for discrimination of Campylobacter strains. In this study, we evaluated the feasibility of the cmp-based typing tool, using pulsed-field gel electrophoresis (PFGE) as the "gold" standard for comparison. The cmp alleles were sequenced from multiple Campylobacter strains, grouped, and compared with the PFGE profiles of these strains using Bionumerics. Results showed that 43 cmp sequence types and 43 PFGE types existed among the 60 Campylobacter isolates. Typeability of these strains is 100% using either the cmp-based method or PFGE. The discrimination indices are 0.973 for the cmp-based method and 0.969 for PFGE, respectively. The cmp sequence types are 77.6% congruent with the PFGE types. These results indicate that the cmp-based typing is a simple, yet highly discriminatory approach for molecular differentiation of C. jejuni strains.

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.

Campylobacter jejuni, a major foodborne human pathogen, has become increasingly resistant to fluoroquinolone (FQ) antimicrobials. By using clonally related isolates and genetically defined mutants, we determined the fitness of FQ-resistant Campylobacter in chicken (a natural host and a major reservoir for C. jejuni) in the absence of antibiotic selection pressure. When monoinoculated into the host, FQ-resistant and FQ-susceptible Campylobacter displayed similar levels of colonization and persistence in the absence of FQ antimicrobials. The prolonged colonization in chickens did not result in loss of the FQ resistance and the resistance-conferring point mutation (C257 –> T) in the gyrA gene. Strikingly, when coinoculated into chickens, the FQ-resistant Campylobacter isolates outcompeted the majority of the FQ-susceptible strains, indicating that the resistant Campylobacter was biologically fit in the chicken host. The fitness advantage was not due to compensatory mutations in the genes targeted by FQ and was linked directly to the single point mutation in gyrA, which confers on Campylobacter a high-level resistance to FQ antimicrobials. In certain genetic backgrounds, the same point mutation entailed a biological cost on Campylobacter, as evidenced by its inability to compete with the FQ-susceptible Campylobacter. These findings provide a previously undescribed demonstration of the profound effect of a resistance-conferring point mutation in gyrA on the fitness of a major foodborne pathogen in its natural host and suggest that the rapid emergence of FQ-resistant Campylobacter on a worldwide scale may be attributable partly to the enhanced fitness of the FQ-resistant isolates.