Biology subjects

Behar, S. M.

Publications and source records attributed to Behar, S. M..

18 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

The CD8 immgenT framework as a universal reference of mouse CD8 Tαβ cell differentiation states

Mouse CD8+ T cell differentiation has been studied extensively in models of infections and tumors, yet no unified framework spans the full spectrum of immunological contexts. Within the immgenT project, we profiled RNA, surface markers, and TCR clonotypes in conventional CD8+ T cells across >600 samples, spanning multiple perturbations, tissues, and timepoints. Twenty-one clusters across naive, effector, circulating memory, tissue-resident memory, progenitor-exhausted, and terminally-exhausted CD8+ T cell compartments emerged, with striking molecular convergence across acute and chronic infections, tumors, autoimmunity, aging, and homeostasis, illustrating that shared transcriptional states support protective or dysfunctional outcomes depending on developmental history and microenvironment. We validate immgenT as a comprehensive reference by integrating external datasets from conditions not represented in immgenT and by defining a flow cytometry panel spanning the CD8+ T cell landscape. Thus, immgenT-CD8 provides a molecular framework for harmonizing CD8+ T cell literature and clarifies relationships across diverse immune challenges.

immunology↗

bioRxiv2026

immgenT: A Comprehensive Reference of Convergent T-cell States in the Mouse

The immgenT collaborative project generated a comprehensive molecular atlas of T cells spanning virtually all mouse organs and disease states, profiling ~800,000 cells from 750 samples with RNA, 128-plex surface protein, and {beta}TCR sequence. Applying a deep generative model to joint RNA and protein data defined the landscape of T-cell states organized into eight lineages and 107 robust clusters, integrating similar cells from different contexts, and resolving prior nomenclatures. Analysis of effector molecules, transcription factors and modules showed that both immunological functions and regulatory programs are shared across cell states. This framework provides a stable, reusable reference, demonstrated by computationally integrating 16 external datasets from diverse biological contexts. A set of public web tools supports browsing of these data and mapping of any dataset onto the immgenT framework. These results propose a molecular classification of T cells organized around a set of shared states reused across immunological contexts.

immunology↗

bioRxiv2025

SP140 limits type I interferon-driven pathology, preserving T cell motility and promoting resistance in tuberculosis

CD8+ T cells are elicited during tuberculosis yet how susceptible lung environments shape their maintenance and behavior is unclear. We examined pulmonary T cell responses to Mycobacterium tuberculosis in resistant control and Sp140-/- mice, a model of type I interferon (IFN-I)-driven susceptibility. Resistant mice generated diverse CD8+ effector- and memory-like subsets producing TNF and IFN{gamma}, whereas Sp140-/- mice showed a broad loss of pulmonary CD8+ T cells. Single-cell RNA sequencing revealed altered states in CD8+ T cells from susceptible mice with exhaustion and IFN-I transcriptional programs. IFNAR blockade rescued CD8+ T cell numbers, diversity, cytokine production, reduced bacterial burden and lung pathology, with comparable benefits also valid for CD4+ T cells. Intravital microscopy of infected lungs further showed that T cell dynamics and motility within lesions were restricted under exuberant IFN-I signaling but fully restored by IFNAR blockade. Thus, IFN-I-driven tissue pathology restricts T cell accumulation and motility during tuberculosis. TeaserIn tuberculosis, excessive type I interferon signaling reshapes the lung environment, limiting T cell accumulation and motility within infected lesions.

immunology↗

bioRxiv2025

BCG vaccination elicits protection against Mtb infection mediated by two phases of T cell immunity

Abstract/SummaryVaccine development for tuberculosis is a global priority. Our studies using Collaborative Cross (CC) mice show that genetic diversity influences the efficacy of BCG, the most widely used TB vaccine. BCG vaccination of CC042 mice reduces their lung bacillary burden and increases their survival following low-dose aerosol Mycobacterium tuberculosis infection (MTBI), despite impaired T cell trafficking from a defective Itgal gene. Early protection requires the presence of T cells at the time of BCG vaccination but is not mediated by B cell or T cell recall responses following MTBI. In contrast, T cell depletion following BCG vaccination reduces survival after MTBI. Thus, CC042 mice reveal two phases of immunity induced by BCG that require T cells: an early phase mediated by innate responses and a later phase mediated by effector CD4 and/or CD8 T cells. Although measurement of vaccine-induced protection 30 days after MTBI is a standard measure of vaccine efficacy in the murine TB model, we find this time point is independent of memory T cells. Our results suggest that vaccine-elicited innate responses have a larger role in protection than previously considered. The concordance between lung CFU, pathology, and survival make CC042 mice a useful model for vaccine evaluation.

immunology↗

bioRxiv2025

NRF2 inhibition of alveolar macrophage MHC II expression during Mycobacterium tuberculosis infection

During Mycobacterium tuberculosis (Mtb) infection, infected alveolar macrophages (AMs) initially up-regulate a NRF2 regulated cell-protective program, which is detrimental to host control and impedes AM activation, including MHC II expression. MHC II is critical for CD4+ T cell activation and host immunity during Mtb infection. We hypothesized that NRF2 regulates the MHC II pathway and AM antigen presentation to T cells. We found that NRF2 inhibits MHC II, but not MHC I, specifically in AMs, following Mtb infection in vitro and in vivo. NRF2 dampens Ciita and H2-Ab1 gene expression in uninfected AMs, and MHC II inhibition by NRF2 is retained following innate stimuli and IFN{gamma} exposure. NRF2 expression in Mtb-infected AMs impedes their ability to activate ESAT6-specific CD4+ T cells. Thus, although NRF2 expression enhances cell-protective functions, it has the unexpected consequence of limiting innate-adaptive crosstalk, which can impair CD4+ T cell activation and host immunity during Mtb infection. HIGHLIGHTSO_LINRF2 inhibits MHC II on AMs, but not DCs, MDMs, or PMNs, during Mtb infection C_LIO_LINRF2 inhibits Ciita and H2-Ab1 and total MHC II protein in AMs, but not in BMDMs C_LIO_LINRF2 blockade of AM MHC II is retained following PAMP and IFN{gamma} stimulation C_LIO_LINRF2 impedes the activation of antigen-specific CD4+ T cells by Ams C_LI

immunology↗

bioRxiv2025

CD226 identifies effector CD8+ T cells during tuberculosis and costimulates recognition of Mycobacterium tuberculosis-infected macrophages

CD8+ T cells defend against Mycobacterium tuberculosis (Mtb) infection but variably recognize Mtb-infected macrophages. To define how the diversity of lung parenchymal CD8+ T cells changes during chronic infection, cells from C57BL/6J mice infected for 6- and 41-weeks were analyzed by scRNA-seq. We identified an effector lineage, including a cluster that expresses high levels of cytotoxic effectors and cytokines, and dysfunctional lineage that transcriptionally resembles exhausted T cells. The most significant differentially expressed gene between two distinct CD8+ T cell lineages is CD226. Mtb-infected IFN{gamma}-eYFP reporter mice revealed IFN{gamma} production is enriched in CD226+CD8+ T cells, confirming these as functional T cells in vivo. Purified CD226+ but not CD226- CD8+ T cells recognize Mtb-infected macrophages, and CD226 blockade inhibits IFN{gamma} and granzyme B production. Thus, CD226 costimulation is required for efficient CD8+ T cell recognition of Mtb-infected macrophages, and its expression identifies CD8+ T cells that recognize Mtb-infected macrophages. One Sentence SummaryShinkawa et al. discover that CD226 is a functional marker that distinguishes effector from dysfunctional CD8+ T cells in the Mycobacterium tuberculosis (Mtb)-infected lung and has a crucial role in costimulating CD8+ T cell recognition of Mtb-infected macrophages.

immunology↗

bioRxiv2025

CD4 T cell dysfunction is associated with bacterial recrudescence during chronic tuberculosis.

While most people contain Mycobacterium tuberculosis infection, some individuals develop active disease, usually within two years of infection. Why immunity fails after initially controlling infection is unknown. C57BL/6 mice control Mycobacterium tuberculosis for up to a year but ultimately succumb to disease. We hypothesize that the development of CD4 T cell dysfunction permits bacterial recrudescence. We developed a reductionist model to assess antigen-specific T cells during chronic infection and found evidence of CD4 T cell senescence and exhaustion. In C57BL/6 mice, CD4 T cells upregulate coinhibitory receptors and lose effector cytokine production. Single cell RNAseq shows that only a small number of CD4 T cells in the lungs of chronically infected mice are polyfunctional. While the origin and causal relationship between T- cell dysfunction and recrudescence remains uncertain, we propose T cell dysfunction leads to a feed-forward loop that causes increased bacillary numbers, greater T cell dysfunction, and progressive disease.

immunology↗

bioRxiv2023

Heterogeneity in lung macrophage control of Mycobacterium tuberculosis is determined by T cells

Following Mycobacterium tuberculosis infection, alveolar macrophages are initially infected but ineffectively restrict bacterial replication. The distribution of M. tuberculosis among different cell types in the lung changes with the onset of T cell immunity when the dominant infected cellular niche shifts from alveolar to monocyte-derived macrophages (MDM). We hypothesize that changes in bacterial distribution among different cell types is driven by differences in T cell recognition of infected cells and their subsequent activation of antimicrobial effector mechanisms. We show that CD4 and CD8 T cells efficiently eliminate M. tuberculosis infection in alveolar macrophages, but they have less impact on suppressing infection in MDM, which may be a bacterial niche. Importantly, CD4 T cell responses enhance MDM recruitment to the lung. Thus, the outcome of infection depends on the interaction between the T cell subset and the infected cell; both contribute to the resolution and persistence of the infection.

immunology↗

bioRxiv2022

High bacillary burden and the ESX-1 type VII secretion system promote MHC class I presentation to CD8 T-cells during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) subverts host defenses to persist in macrophages despite immune pressure. CD4 T-cells can recognize macrophages infected with a single bacillus in vitro. Under identical conditions, CD8 T-cells inefficiently recognize infected macrophages and fail to restrict Mtb growth, although they can inhibit Mtb growth during high burden intracellular infection. We show that high intracellular Mtb numbers cause macrophage death, leading other macrophages to scavenge cellular debris and cross-present the TB10.4 antigen to CD8 T-cells. Presentation by infected macrophages requires Mtb to have a functional ESX-1 type VII secretion system. These data indicate that phagosomal membrane damage and cell death promote class I MHC presentation of the immunodominant antigen TB10.4 by macrophages. Although this mode of antigen-presentation stimulates cytokine production that we presume would be host beneficial; killing of uninfected cells could worsen immunopathology. We suggest that shifting the focus of CD8 T-cell recognition to uninfected macrophages would limit the interaction of CD8 T-cells with infected macrophages and impair CD8 T-cell mediated resolution of tuberculosis.

immunology↗

bioRxiv2022

Identification and characterization of the T cell receptor (TCR) repertoire of the Cynomolgus macaque (Macaca Fascicularis)

BackgroundNon-human primates (NHP) are desirable as animal models of human disease because they share behavioral, physiological, and genomic traits with people. Hence, NHP recapitulate manifestations of disease not observed in other animal species. The Macaca fascicularis (i.e., Cynomolgus macaque) is an NHP species extensively used for biomedical research, but the TCR repertoire hasnt been characterized yet. ResultWe used the genomic sequences to design primers to identify the expressed TCR repertoire by single cell RNAseq. The data analysis from 22 unique samples were used to assign a functional status to each TCR genes. We identified and analyzed the TRA/D, TRB and TRG loci of the Cynomolgus macaque. ConclusionThe genomic organization of the Cynomolgus macaque has great similarity with Macaca mulatta (i.e., Rhesus macaque) and they shared >90% sequence similarity with the human TCR repertoire. These data will facilitate the analysis of T cell immunity in Cynomolgus macaques.

immunology↗

bioRxiv2021

IFNγ and iNOS-mediated alterations in the bone marrow and thymus and its impact on Mycobacterium avium-induced thymic atrophy

Disseminated infection with the high virulence strain of Mycobacterium avium 25291 lead to progressive thymic atrophy. We previously uncovered that M. avium-induced thymic atrophy is due to increased levels of glucocorticoids synergizing with nitric oxide (NO) produced by interferon gamma (IFN{gamma}) activated macrophages. Where and how these mediators are playing, was yet to be understood. We hypothesized that IFN{gamma} and NO might be affecting bone marrow (BM) T cell precursors and/or T cell differentiation in the thymus. We show that M. avium infection causes a reduction on the percentage of lymphoid-primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLP). Additionally, BM precursors from infected mice are unable to reconstitute thymi of RAGKO mice in an IFN{gamma}-dependent way. Thymi from infected mice presents a NO-dependent inflammation. When transplanted under the kidney capsule of non-infected mice, thymic stroma from infected mice is unable to sustain T cell differentiation. Finally, we observed increased thymocyte death via apoptosis after infection, independent of both IFN{gamma} and iNOS, and a decrease on activated caspase-3 positive thymocytes, that was not observed in the absence of iNOS expression. Together our data suggests that M. avium-induced thymic atrophy results from a combination of impairments, mediated by IFN{gamma} and NO, affecting different steps of T cell differentiation from T cell precursor cells in the BM to the thymic stroma and thymocytes.

immunology↗

bioRxiv2021

CD4 T cell help prevents CD8 T cell exhaustion and promotes control of Mycobacterium tuberculosis infection

CD4 T cells are essential for immunity to tuberculosis because they produce cytokines including interferon-{gamma}. Whether CD4 T cells act as "helper" cells to promote optimal CD8 T cell responses during Mycobacterium tuberculosis is unknown. Using two independent models, we show that CD4 T cell help enhances CD8 effector functions and prevents CD8 T cell exhaustion. We demonstrate synergy between CD4 and CD8 T cells in promoting the survival of infected mice. Purified helped, but not helpless, CD8 T cells efficiently restrict intracellular bacterial growth in vitro. Thus, CD4 T cell help plays an essential role in generating protective CD8 T cell responses against M. tuberculosis infection in vitro and in vivo. We infer vaccines that elicit both CD4 and CD8 T cells are more likely to be successful than vaccines that elicit only CD4 or CD8 T cells.

immunology↗

bioRxiv2020

Mitochondrial respiration contributes to the interferon gamma response in antigen presenting cells

The immunological synapse allows antigen presenting cells (APC) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While toll-like receptor ligation relies on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFN{gamma}). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in macrophages to identify the regulators of IFN{gamma}-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multi-screen approach enabled us to identify novel pathways that control these functionally distinct markers. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFN{gamma} signaling pathway. We report that the IFN{gamma} response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFN{gamma} and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity.

immunology↗

bioRxiv2020

Single-cell profiling of tuberculosis lung granulomas reveals functional lymphocyte signatures of bacterial control

Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. There is also a temporal aspect to bacterial control, in that granulomas that arise later in infection (in the context of an established immune response) share the functional characteristics of restrictive granulomas and are more capable of killing Mtb. Taken together, our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB. One-Sentence SummaryBacterial control in TB lung granulomas correlates with distinct cellular immune microenvironments and time of formation after infection.

immunology↗

bioRxiv2019

Functionally overlapping variants control TB susceptibility in Collaborative Cross mice

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis (Mtb) infection. We previously found that Collaborative Cross mouse strains differ in their susceptibility to Mtb, and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine, IFN{gamma}, in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 susceptibility. Using a population derived from a CC001/Unc (CC001) x CC042 intercross, we mapped four QTL underlying Tuberculosis ImmunoPhenotypes (Tip1-4). These included 2 major effect QTL on Chromosome 7 (Tip1 and Tip2) that were associated with bacterial burden. Tip2, along with Tip3 (Chromosome 15) and Tip4 (Chromosome 16) also correlated with IFN{gamma} production following infection, whereas Tip1 appeared to control an IFN{gamma}-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and these T cells failed to express the Integrin alpha L (L; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a, and is found within the Tip2 interval. This 15bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 and highlights the multigenic nature of TB control in the Collaborative Cross.\n\nImportance statementThe variable outcome of Mycobacterium tuberculosis infection observed natural populations is difficult to model in genetically homogenous small animal models. The newly-developed Collaborative Cross (CC) represents a reproducible panel of genetically-diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene that is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.

microbiology↗

bioRxiv2019

Limited recognition of Mycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice

Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination are frequently assessed by measuring T cell recognition of crude Mtb antigens, recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-specific T cells recognize Mtb-infected macrophages. Thus, an important question is what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. We answer this question by developing a modified elispot assay using viable Mtb-infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of T cells that recognize Mtb-infected macrophages were determined using interferon-{gamma} elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the numbers of Mtb antigen-specific T cells for antigens such as ESAT-6 and TB10.4, low frequencies of pulmonary CD4 and CD8 T cells elicited by aerosolized Mtb infection recognize Mtb-infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that recognize Mtb-infected macrophages. We propose that the frequency of T cells that recognize infected macrophages could correlate with protective immunity and may be an alternative approach to measuring T cell responses to Mtb antigens.

immunology↗

bioRxiv2018

Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages

Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages.\n\nWe derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. Importantly, CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner.\n\nAs TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.

immunology↗