Biology subjects

Lewinsohn, D. M.

Publications and source records attributed to Lewinsohn, D. M..

12 recordsLinked to original sources

bioRxiv2026

Human memory CD4+ T cells recognize non-infected macrophage bystanders exposed to Mycobacterium tuberculosis-infected cells

Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T cell recognition of infected macrophages. However, T cells also colocalize with non-infected macrophages in granulomas. We investigated whether these bystander macrophages present Mtb antigens and shape human CD4+ T cell responses. Using ex vivo co-culture systems, non-infected monocyte-derived macrophages (MDMs) were exposed to Mtb-infected MDMs or infection-conditioned supernatants before co-incubation with autologous memory CD4+ T cells from individuals with latent Mtb infection (LTBI). Bystander macrophages activated memory CD4+ T cells through MHC-II-dependent antigen presentation. Single-cell T cell receptor (TCR) sequencing and TCR-transduced reporter cell lines identified Mtb-specific clonotypes recognizing both infected and bystander macrophages, as well as clonotypes preferentially recognizing infected cells. Strikingly, a subset of TCRs recognized infected but not bystander macrophages. Antigen transfer occurred through soluble Mtb proteins rather than extracellular vesicles. Compared with responses to infected macrophages, bystander macrophages induced attenuated effector responses. These findings reveal antigen-specific recognition of bystander macrophages and suggest that antigens preferentially presented by infected cells may inform TB vaccine design.

immunology↗

bioRxiv2026

Lung Resident T Cells Across the Spectrum of TB Infection and Disease

Tuberculosis (TB) remains the leading infectious cause of death worldwide, yet immunological protection against Mycobacterium tuberculosis (Mtb) remain poorly understood. We performed integrated single-cell transcriptomics and functional T-cell cloning on paired bronchoalveolar lavage (BAL) and peripheral blood samples from those exposed to TB, using IGRA and PET-CT to study potentially protective responses across the spectrum of TB infection/disease. Recent Mtb exposure alone was sufficient to remodel the pulmonary T-cell compartment. Longitudinal follow-up demonstrated that all participants who progressed to active TB had baseline PET-CT abnormalities, showing that PET-CT scan can be used to find those at high risk of progression. Furthermore, those who were PET-positive, but did not progress to active TB provide a natural model for protection, and exhibited enrichment of pulmonary cytotoxic CD8-associated T cells, identifying a candidate protective immune program. TAR-seq identified a discrete population of clonally expanded Mtb-responsive T cells and enabled direct linkage of antigen-responsive TCRs to transcriptional state through T cell cloning. Functional T-cell cloning further established a framework connecting antigen specificity, TCR sequence, and pulmonary cell state. Together, these findings provide a comprehensive atlas of human pulmonary T-cell immunity across the spectrum of TB, identify pulmonary immune features associated with durable control of infection, and establish an integrated platform for defining protective T-cell responses to inform next-generation TB vaccine development.

immunology↗

bioRxiv2026

Opposing roles for SNAP23 and SNAP25 in mediating MR1 trafficking and antigen presentation

MHC class I-related protein 1 (MR1) is a highly conserved antigen presenting molecule that presents small molecule metabolites derived from diverse microbial pathogens to mucosal-associated invariant T (MAIT) cells. We have shown previously that MR1 traffics through endosomal compartments via soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including Syntaxin 4 and vesicle-associated membrane protein (VAMP) 4. Here, we investigate the role of synaptosome-associated proteins (SNAPs), which pair with Syntaxins and VAMPs to form functional SNARE complexes, in MR1-mediated antigen presentation. Among SNAP homologs, we identify that SNAP23 contributes to the presentation of Mycobacterium tuberculosis (Mtb)-derived antigens and loss of SNAP23 reduces the number of MR1-containing vesicles during infection. In contrast, SNAP25 suppresses MR1 presentation for both intracellular pathogens Mtb and Mycobacterium avium, as well as extracellular pathogen Candida albicans. This study demonstrates opposing roles for SNAP23 and SNAP25 in MR1 antigen presentation to MAIT cells, and extends our understanding of how SNAP family proteins regulate MR1 trafficking.

immunology↗

bioRxiv2026

Antigenic stimulation in conjunction with cytokine is required for mediating IL-17A production in human MAIT cells

Mucosal-associated invariant T (MAIT) cells are donor unrestricted T cells capable of both antigen-specific adaptive responses and cytokine driven innate-like functions. Although human MAIT cells uniformly express ROR{gamma}t and IL23R, they generally produce IFN-{gamma}, and only a small fraction produces IL-17. Recent studies show that combined TCR and cytokine stimulation can elicit functional heterogeneity in blood-derived MAIT cells. Here, we investigate the role of IL-23/IL-23R signaling in mediating the function and transcriptional profiles of lung MAIT cell clones. We demonstrate that BAL-derived lung MAIT cell clones exhibit distinct cytokine profiles and variable IL23R expression. Short-term IL-23 stimulation triggers clone-specific transcriptional programs and IL23R-dependent upregulation of type 17-associated genes. Prolonged conditioning of lung MAIT cell clones with TCR (5-OP-RU) and cytokine (IL-23) stimulation induces stable IL-17A production along with unique transcriptional changes. TCR + IL-23 conditioning alone upregulates clone-specific and shared cytoskeletal/structural gene programs, whereas subsequent PMA/Ionomycin stimulation further induces IL-12 family signaling and metabolic genes. Together, these findings demonstrate that IL23R expression and TCR signaling are required for IL-17A production, highlighting that these conditions may be met in tissue environments where MR1-specific antigens and proinflammatory cytokines coexist.

immunology↗

bioRxiv2025

Sorting endosomes play key roles in presentation of Mycobacterium tuberculosis-derived ligands to MAIT cells

The immune system has developed specialized mechanisms to recognize intracellular pathogens such as Mycobacterium tuberculosis (Mtb). Major Histocompatibility Complex Class I-Related molecule (MR1) is a conserved nonclassical antigen presenting molecule that presents ligands derived from microbial riboflavin synthesis to Mucosal Associated Invariant T (MAIT) cells. While endosomal trafficking facilitates MR1 antigen presentation during Mtb infection, the exact mechanisms by which MR1 loading of Mtb-derived ligands occurs are not known. We found that trafficking through sorting endosomes mediates MR1 antigen presentation during Mtb infection. Sorting endosomes utilize trafficking proteins such as Syntaxin 6, Syntaxin 12, Syntaxin 16 and VAMP4. Prior work demonstrates the importance of VAMP4 for MR1 presentation during Mtb infection; we have found that Stx12 and Stx16 are also important. Interference with Stx12 or Stx16 via siRNA-mediated knockdown reduces MR1 antigen presentation of Mtb. Using RFP-tagged constructs, we found Stx16 co-localized more with MR1 vesicles compared to Stx12 in MR1-GFP expressing airway epithelial cells. Stx12 and Stx16 blockade increase MR1 surface stabilization and total expression, indicating that impaired endosomal trafficking hinders MR1 internalization. Together, these findings support a role for sorting endosomes in the selective sampling of the intracellular environment and MR1-mediated recognition of Mtb-infected cells.

immunology↗

bioRxiv2025

Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can be sensed by T cells, which are essential for the control of infection. In comparison to viral infections, Mtb antigens are relatively limited and hence, challenging to sample. Specialized antigen presentation pathways enable the presentation of such scarce antigens to CD8+ T cells, which are, thus, uniquely poised to survey intracellular environments. A subset of CD8+ T cells prevalent in the airways, known as mucosal-associated invariant T (MAIT) cells, can be activated through the presentation of Mtb antigens via the MHC class I-related protein 1 (MR1) molecule. Prior work demonstrates that endosomal calcium signaling is critical for MR1-mediated presentation of Mtb-derived antigens. Here, we show that the calcium-sensing trafficking proteins Synaptotagmin (Syt) 1 and Syt7 specifically promote MAIT cell activation in response to Mtb-infected cells. In bronchial epithelial cells, Syt1 and Syt7 localize to late endo-lysosomes and MR1 vesicles. Loss of Syt1 and Syt7 results in enlarged MR1 vesicles and an increased number of MR1 vesicles in close proximity to Mtb-containing vacuoles during infection. This study identifies a specialized pathway in which Syt1 and Syt7 facilitate the translocation of MR1 from Mtb-containing vacuoles, potentially to the cell surface for antigen presentation.

immunology↗

bioRxiv2025

Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

The antigen presenting molecule MHC class I-related protein 1 (MR1) binds small molecule metabolites derived from microbial riboflavin biosynthetic pathways and presents them at the cell surface for surveillance by MR1-restricted mucosal-associated invariant T cells (MAIT cells). MR1 ligands can originate in the extracellular space or in endosomal compartments that contain microbial pathogens. Distinct, complementary antigen processing and presentation pathways enable MR1 to survey diverse intracellular locations and present both exogenous and intracellular antigens. Here, we generated a panel of BEAS-2B MR1 KO cells reconstituted with MR1 proteins mutated at amino acids 9 - 16. The mutated MR1 molecules differentially translocated to the cell surface in response to 6-formylpterin and differed in their ability to present mycobacterial antigens to MAIT cell clones. While they barely presented Mycobacterium smegmatis supernatant and other exogenous MAIT cell antigens, their ability to present antigens derived from mycobacterial infection and a 5-A-RU prodrug requiring endosomal processing remained largely intact. Protein co-immunoprecipitation and mass spectrometry-based proteomic analysis showed that mutated MR1 differentially associated with calnexin and {beta}2-microglobulin (B2M). Knock-down of B2M in cells over-expressing MR1 phenocopied the loss of exogenous antigen presentation but did not impact presentation of intracellular antigens. Thus, the MR1-mediated presentation of exogenous antigen appears to be limited by binding to B2M whereas the lower sensitivity to B2M deficiency implies that MAIT cell activation via the endosomal antigen presentation pathway may be limited by the availability of MR1 itself.

immunology↗

bioRxiv2022

Delivery of loaded MR1 Monomer Results in Efficient Ligand Exchange to Host MR1 and Subsequent MR1T cell activation

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.

immunology↗

bioRxiv2022

Deaza-modification of MR1 ligands modulates recognition by MR1-restricted T cells

MR1-restricted T (MR1T) cells recognize microbial small molecule metabolites presented on the MHC Class I-like molecule MR1 and have been implicated in early effector responses to microbial infection. As a result, there is considerable interest in identifying chemical properties of metabolite ligands that permit recognition by MR1T cells, for consideration in therapeutic or vaccine applications. Here, we made chemical modifications to known MR1 ligands to evaluate the effect on MR1T cell activation. Specifically, we modified 6,7-dimethyl-8-D-ribityllumazine (DMRL) to generate 6,7-dimethyl-8-D-ribityldeazalumazine (DZ), and then further derivatized DZ to determine the requirements for retaining MR1 surface stabilization and agonistic properties. Interestingly, the IFN-{gamma} response toward DZ varied widely across a panel of T cell receptor (TCR)-diverse MR1T cell clones; while one clone was agnostic toward the modification, most displayed either an enhancement or depletion of IFN-{gamma} production when compared with its response to DMRL. To gain insight into a putative mechanism behind this phenomenon, we used in silico molecular docking techniques for DMRL and its derivatives and performed molecular dynamics simulations of the complexes. In assessing the dynamics of each ligand in the MR1 pocket, we found that DMRL and DZ exhibit differential dynamics of both the ribityl moiety and the aromatic backbone, which may contribute to ligand recognition. Together, our results support an emerging hypothesis for flexibility in MR1:ligand-MR1T TCR interactions and enable further exploration of the relationship between MR1:ligand structures and MR1T cell recognition for downstream applications targeting MR1T cells.

immunology↗

bioRxiv2020

MR1-restricted MAIT cells from the human lung mucosal surface have distinct phenotypic, functional, and transcriptomic features that are preserved in HIV infection

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant {beta} T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of MR1 restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in healthy individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-{gamma} and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with atypical MAIT cells and tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in healthy people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.

immunology↗

bioRxiv2019

Postnatal expansion, maturation and functionality of MR1 T cells in humans

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 -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 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 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 naive 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.

immunology↗

bioRxiv2019

Alternative Splicing of MR1 regulates antigen presentation to MAIT cells

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are not well characterized.\n\nIn this report, we sought to characterize these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. Despite the widespread expression of MR1A and MR1B, only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A serves to decrease MAIT cell activation following bacterial infection. However, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Relative MR1A/MR1B expression in MR1-expressing thymocytes is associated with their prevalence.\n\nOur results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand.\n\nFundingThis work was supported by NIH T32HL083808 (EK, GAN, EM), VA Merit Award I01CX001562 (MJH), NIH R01AI29976 (MJH), NIH R01AI048090 (DML), NIH R21AI124225-01A1 (FT) and VA Merit Award I01BX000533 (DML). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

immunology↗