Gwendolyn Swarbrick

Bacterial sepsis is a leading cause of neonatal mortality. Pro-inflammatory MR1-restricted T (MR1T) cells may help protect from sepsis by recognizing bacterial pathogens producing the canonical MR1 antigen 5-OP-RU. Most adult MR1T cells are mucosal-associated invariant T (MAIT) cells expressing a semi-invariant TCRα, while neonatal MR1T cells express diverse TCRα chains. Here, we perform combined single-cell RNA-sequencing and TCR repertoire analyses on MR1/5-OP-RU tetramer-positive cells from neonatal cord blood (CB) and adult blood. Compared to adult MR1T cells, CB MR1T cells exhibit greater TCR diversity, reduced cytotoxic and proinflammatory gene expression, diminished bacterial recognition and reduced binding to MR1/5-OP-RU. Structural analysis of a CB MAIT TCR reveals decreased β chain contribution to the TCR–MR1 interface relative to an adult MAIT TCR. These findings demonstrate developmental stage-specific differences in MR1T cell repertoire, function and MAIT TCR structure with implications for neonatal sepsis.

Neonatal sepsis is a leading cause of childhood mortality. Understanding immune cell development can inform strategies to combat this. MR1-restricted T (MR1T) cells can be defined by their recognition of small molecules derived from microbes, self, and drug and drug-like molecules, presented by the MHC class 1-related molecule (MR1). In healthy adults, the majority of MR1T cells express an invariant α-chain; TRAV1-2/TRAJ33/12/20 and are referred to as mucosal-associated invariant T (MAIT) cells. Neonatal MR1T cells isolated from cord blood (CB) demonstrate more diversity in MR1T TCR usage, with the majority of MR1-5-OP-RU-tetramer(+) cells being TRAV1-2(-). To better understand this diversity, we performed single-cell-RNA-seq/TCR-seq (scRNA-seq/scTCR-seq) on MR1-5-OP-RU-tetramer(+) cells from CB (n=5) and adult participants (n=5). CB-derived MR1T cells demonstrate a less cytotoxic/pro-inflammatory phenotype, and a more diverse TCR repertoire. A panel of CB and adult MAIT and TRAV1-2(-) MR1T cell clones were generated, and CB-derived clones were unable to recognize several common riboflavin-producing childhood pathogens (S. aureus, S. pneumoniae, M. tuberculosis). Biochemical and structural investigation of one CB MAIT TCR (CB964 A2; TRAV1-2/TRBV6-2) showed a reduction in binding affinity toward the canonical MR1-antigen, 5-OP-RU, compared to adult MAIT TCRs that correlated with differences in β-chain contribution in the TCR-MR1 interface. Overall, this data shows that CB MAIT and TRAV1-2(-) MR1T cells, express a diverse TCR repertoire, a more restricted childhood pathogen recognition profile and diminished cytotoxic and pro-inflammatory capacity. Understanding this diversity, along with the functional ability of TRAV1-2(-) MR1T cells, could provide insight into increased neonatal susceptibility to infections.

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

In response to microbial infection, the nonclassical Ag-presenting molecule MHC class I–related protein 1 (MR1) presents secondary microbial metabolites to mucosal-associated invariant T (MAIT) cells. In this study, we further characterize the repertoire of ligands captured by MR1 produced in Hi5 (Trichoplusia ni) cells from Mycobacterium smegmatis via mass spectrometry. We describe the (to our knowledge) novel MR1 ligand photolumazine (PL)V, a hydroxyindolyl-ribityllumazine with four isomers differing in the positioning of a hydroxyl group. We show that all four isomers are produced by M. smegmatis in culture and that at least three can induce MR1 surface translocation. Furthermore, human MAIT cell clones expressing distinct TCR β-chains differentially responded to the PLV isomers, demonstrating that the subtle positioning of a single hydroxyl group modulates TCR recognition. This study emphasizes structural microheterogeneity within the MR1 Ag repertoire and the remarkable selectivity of MAIT cell TCRs.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of pediatric morbidity and mortality. Young children are at high risk of TB following Mtb exposure, and this vulnerability is secondary to insufficient host immunity during early life. Our primary objective was to compare CD4+ and CD8+ T-cell production of proinflammatory cytokines IFN-gamma, IL-2, and TNF-alpha in response to six mycobacterial antigens and superantigen staphylococcal enterotoxin B (SEB) between Ugandan adults with confirmed TB (n = 41) and young Ugandan children with confirmed (n = 12) and unconfirmed TB (n = 41), as well as non-TB lower respiratory tract infection (n = 39). Flow cytometry was utilized to identify and quantify CD4+ and CD8+ T-cell cytokine production in response to each mycobacterial antigen and SEB. We found that the frequency of CD4+ and CD8+ T-cell production of cytokines in response to SEB was reduced in all pediatric cohorts when compared to adults. However, T-cell responses to Mtb-specific antigens ESAT6 and CFP10 were equivalent between children and adults with confirmed TB. In contrast, cytokine production in response to ESAT6 and CFP10 was limited in children with unconfirmed TB and absent in children with non-TB lower respiratory tract infection. Of the five additional mycobacterial antigens tested, PE3 and PPE15 were broadly recognized regardless of TB disease classification and age. Children with confirmed TB exhibited robust proinflammatory CD4+ and CD8+ T-cell responses to Mtb-specific antigens prior to the initiation of TB treatment. Our findings suggest that adaptive proinflammatory immune responses to Mtb, characterized by T-cell production of IFN-gamma, IL-2, and TNF-alpha, are not impaired during early life.

Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8⁺ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8⁺ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine- related molecules distinct from other lung-resident CD8⁺ T cells, such as interferon-γ- and IL-12- receptors. These data highlight MAIT cells’ predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

MR1-restricted T (MR1T) cells are defined by their recognition of metabolite antigens presented by the monomorphic MHC class 1-related molecule, MR1, the most highly conserved MHC class I related molecule in mammalian species. Mucosal-associated invariant T (MAIT) cells are the predominant subset of MR1T cells expressing an invariant TCR α-chain, TRAV1-2. These cells comprise a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, including Mycobacterium tuberculosis. Here, we sought to characterize development of circulating human MR1T cells as defined by MR1-5-OP-RU tetramer labeling and of the TRAV1-2⁺ MAIT cells defined by expression of TRAV1-2 and high expression of CD26 and CD161 (TRAV1-2⁺CD161⁺⁺CD26⁺⁺ cells). We analyzed postnatal expansion, maturation, and functionality of peripheral blood MR1-5-OP-RU tetramer⁺ MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection with M. tuberculosis. Early after birth, frequencies of MR1-5-OP-RU tetramer⁺ MR1T cells increased rapidly by several fold. This coincided with the transition from a predominantly CD4⁺ and TRAV1-2⁻ population in neonates, to a predominantly TRAV1-2⁺CD161⁺⁺CD26⁺⁺ CD8⁺ population. We also observed that tetramer⁺ MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased ~10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer⁺TRAV1-2⁺ and highly expressed CD161 and CD26, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naïve T cell markers on tetramer⁺ TRAV1-2⁺ MR1T cells more rapidly than tetramer⁺TRAV1-2⁻ MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer⁺TRAV1-2⁺ population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood.

MR1-restricted T (MR1T) cells are defined by their recognition of metabolite antigens presented by the monomorphic MHC class 1-related molecule, MR1, the most highly conserved MHC class I related molecule in mammalian species. Mucosal-associated invariant T (MAIT) cells are the predominant subset of MR1T cells expressing an invariant TCR α-chain, TRAV1-2. These cells comprise a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, includingMycobacterium tuberculosis. Here, we sought to characterize development of circulating human MR1T cells as defined by MR1-5-OP-RU tetramer labelling and of the TRAV1-2⁺MAIT cells defined by expression of TRAV1-2 and high expression of CD26 and CD161 (TRAV1-2⁺CD161⁺⁺CD26⁺⁺cells). We analysed postnatal expansion, maturation and functionality of peripheral blood MR1-5-OP-RU tetramer⁺MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection withM. tuberculosis.Early after birth, frequencies of MR1-5-OP-RU tetramer⁺MR1T cells increased rapidly by several fold. This coincided with the transition from a predominantly CD4⁺and TRAV1-2⁻population in neonates, to a predominantly TRAV1-2⁺CD161⁺⁺CD26⁺⁺CD8⁺population. We also observed that tetramer⁺MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased ∼10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer⁺TRAV1-2⁺and highly expressed CD161 and CD26, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naïve T cell markers on tetramer⁺TRAV1-2⁺MR1T cells more rapidly than tetramer⁺TRAV1-2⁻MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer⁺TRAV1-2⁺population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood.