Engineered cell entry links receptor biology with single-cell genomics.

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections.

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

Computational detection of antigen-specific B cell receptors following immunization.

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.

Translational immunology: Applying fundamental discoveries to human health and autoimmune diseases.

Studying the human immune system is challenging. These challenges stem from the complexity of the immune system itself, the heterogeneity of the immune system between individuals, and the many factors that lead to this heterogeneity including the influence of genetics, environment, and immune experience. Studies of the human immune system in the context of disease are increased in complexity as multiple combinations and variations in immune pathways can lead to a single disease. Thus, although individuals with a disease may share clinical features, the underlying disease mechanisms and resulting pathophysiology can be diverse among individuals with the same disease diagnosis. This has consequences for the treatment of diseases, as no single therapy will work for everyone, therapeutic efficacy varies among patients, and targeting a single immune pathway is rarely 100% effective. This review discusses how to address these challenges by identifying and managing the sources of variation, improving access to high-quality, well-curated biological samples by building cohorts, applying new technologies such as single-cell omics and imaging technologies to interrogate samples, and bringing to bear computational expertise in conjunction with immunologists and clinicians to interpret those results. The review has a focus on autoimmune diseases, including rheumatoid arthritis, MS, systemic lupus erythematosus, and type 1 diabetes, but its recommendations are also applicable to studies of other immune-mediated diseases.

Human CD4+CD25+ T cells expressing a chimeric antigen receptor against aberrant superoxide dismutase 1 trigger antigen-specific immunomodulation.

BACKGROUND AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs' immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. METHODS: A CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. RESULTS: The CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. CONCLUSIONS: Taken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

Antigen-specific T cell balance reveals Why patients with atopic dermatitis fail to achieve immune tolerance.

The number of regulatory T cells (Tregs) and how they behave in the pathogenesis of atopic dermatitis (AD) are still controversial. We identified and quantified Tregs, mite-specific Tregs, and mite-specific effector T cells (Teffs) in patients with AD and healthy controls (HCs). We collected peripheral blood and analyzed the cells using flow cytometry after stimulation with mite antigens. Mite-specific Tregs and mite-specific Teffs were recognized by the expression of CD137 and CD154, respectively. Patients with AD had more Tregs than HCs; however, when focusing on a single antigen, the ratio of mite-specific Tregs/Teffs was lower in patients with AD than in HCs. Furthermore, the mite-specific Teffs in patients with AD were more likely to produce proinflammatory cytokines interleukin (IL)-4 and IL-13. This Teff-dominant imbalance is thought to be the cause of development of atopic status in patients with AD without immune tolerance.

Harnessing autoimmunity with dominant self-peptide: Modulating the sustainability of tissue-preferential antigen-specific Tregs by governing the binding stability via peptide flanking residues.

Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69+CD103+CD4+CD25high regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, "benign autoimmunity" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.

Communication Between the Microbiota and Mammalian Immunity.

Mammalian immune systems evolved within a diverse world dominated by microbes, making interactions between these two life-forms inevitable. Adaptive immunity protects against microbes through antigen-specific responses. In classical studies, these responses were investigated in the context of pathogenicity; however, we now know that they have significant effects on our resident microbes. In turn, microbes employ an arsenal of mechanisms to influence development and specificity of host immunity. Understanding these complex reactions will be necessary to develop microbiota-based strategies to prevent or treat disease. Here we review the literature detailing the cross talk between resident microbes with a focus on the specificity of host responses and the microbial molecules that influence them.

T-Helper 22 Cell Type Responses to Nickel in Contact Allergic Subjects Are Associated with T-Helper 1, T-Helper 2, and T-Helper 17 Cell Cytokine Profile Responses and Patch Test Reactivity.

INTRODUCTION: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. METHODS: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. RESULTS: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. CONCLUSION: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified.

Aspergillus fumigatus-Specific T Cells in Patients with Chronic Rhinosinusitis.

INTRODUCTION: Aspergillus fumigatus belongs to the saprophytic fungi, and its spores form a significant part of the daily load of fungal spores inhaled as particles in aerosols. A. fumigatus is a possible T-cell activator. Its contribution to the pathogenesis of chronic rhinosinusitis (CRS) is controversially discussed. The aim of this study was to detect and characterize A. fumigatus-specific CD4+ and CD8+ T cells in patients with CRS with (CRSwNP) and without (CRSsNP) nasal polyps. METHODS: Tissue and blood samples were collected from patients who underwent paranasal sinus surgery due to CRSwNP or CRSsNP. Afterward, purified CD4+ and CD8+ cells were cultured together with antigen-presenting cells. A peptide mix derived from A. fumigatus antigen was added to the cultures. After 6 days, multicolor flow cytometry was performed, and proliferation was measured using the marker Ki-67. Cytokine secretion was quantified from the supernatant of the cell culture. RESULTS: Significant differences in the proliferation of nasal CD4+ T cells to A. fumigatus antigen were observed for cells from patients with CRSwNP in comparison to CRSsNP, while no differences were found between nasal and peripheral blood T cells. The activation of tissue-derived CD4+ T cells was associated with significantly higher concentrations of IL-4, IL-5, and IL-17a in the cell culture from patients with CRSwNP in comparison to CRSsNP and/or healthy controls. CONCLUSION: Our findings indicate that patients with CRSwNP harbor a higher proportion of A. fumigatus-reactive CD4+ T cells in the nasal mucosa than patients with CRSsNP. A. fumigatus-reactive CD4+ T cells of CRSwNP patients secreted TH2 cytokines and IL-17. Our findings suggest a role for A. fumigatus in the pathogenesis of CRSwNP and provide a rationale for targeted therapies.

Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner.

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4+CD127-CD25hi Tregs and CD4+CD127-CD25+CD45RA+ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.

Neurodegeneration by α-synuclein-specific T cells in AAV-A53T-α-synuclein Parkinson's disease mice.

BACKGROUND: Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson's disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model. METHODS: We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)-/- mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4+/CD8-, CD4-/CD8+, or CD4+/CD8+ (JHD-/-) mice into the RAG-1-/- mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized. RESULTS: AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68-78) and surrounding the pathogenically relevant S129 (120-134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. CONCLUSIONS: Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.

Tolerogenic Dendritic Cells Induce Apoptosis-Independent T Cell Hyporesponsiveness of SARS-CoV-2-Specific T Cells in an Antigen-Specific Manner.

Although the global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, there are currently no specific and highly efficient drugs for COVID-19 available, particularly in severe cases. Recent findings demonstrate that severe COVID-19 disease that requires hospitalization is associated with the hyperactivation of CD4+ and CD8+ T cell subsets. In this study, we aimed to counteract this high inflammatory state by inducing T-cell hyporesponsiveness in a SARS-CoV-2-specific manner using tolerogenic dendritic cells (tolDC). In vitro-activated SARS-CoV-2-specific T cells were isolated and stimulated with SARS-CoV-2 peptide-loaded monocyte-derived tolDC or with SARS-CoV-2 peptide-loaded conventional (conv) DC. We demonstrate a significant decrease in the number of interferon (IFN)-γ spot-forming cells when SARS-CoV-2-specific T cells were stimulated with tolDC as compared to stimulation with convDC. Importantly, this IFN-γ downmodulation in SARS-CoV-2-specific T cells was antigen-specific, since T cells retain their capacity to respond to an unrelated antigen and are not mediated by T cell deletion. Altogether, we have demonstrated that SARS-CoV-2 peptide-pulsed tolDC induces SARS-CoV-2-specific T cell hyporesponsiveness in an antigen-specific manner as compared to stimulation with SARS-CoV-2-specific convDC. These observations underline the clinical potential of tolDC to correct the immunological imbalance in the critically ill.

Clustering based approach for population level identification of condition-associated T-cell receptor ß-chain CDR3 sequences.

BACKGROUND: Deep immune receptor sequencing, RepSeq, provides unprecedented opportunities for identifying and studying condition-associated T-cell clonotypes, represented by T-cell receptor (TCR) CDR3 sequences. However, due to the immense diversity of the immune repertoire, identification of condition relevant TCR CDR3s from total repertoires has mostly been limited to either "public" CDR3 sequences or to comparisons of CDR3 frequencies observed in a single individual. A methodology for the identification of condition-associated TCR CDR3s by direct population level comparison of RepSeq samples is currently lacking. RESULTS: We present a method for direct population level comparison of RepSeq samples using immune repertoire sub-units (or sub-repertoires) that are shared across individuals. The method first performs unsupervised clustering of CDR3s within each sample. It then finds matching clusters across samples, called immune sub-repertoires, and performs statistical differential abundance testing at the level of the identified sub-repertoires. It finally ranks CDR3s in differentially abundant sub-repertoires for relevance to the condition. We applied the method on total TCR CDR3ß RepSeq datasets of celiac disease patients, as well as on public datasets of yellow fever vaccination. The method successfully identified celiac disease associated CDR3ß sequences, as evidenced by considerable agreement of TRBV-gene and positional amino acid usage patterns in the detected CDR3ß sequences with previously known CDR3ßs specific to gluten in celiac disease. It also successfully recovered significantly high numbers of previously known CDR3ß sequences relevant to each condition than would be expected by chance. CONCLUSION: We conclude that immune sub-repertoires of similar immuno-genomic features shared across unrelated individuals can serve as viable units of immune repertoire comparison, serving as proxy for identification of condition-associated CDR3s.

Predicting antigen specificity of single T cells based on TCR CDR3 regions.

It has recently become possible to simultaneously assay T-cell specificity with respect to large sets of antigens and the T-cell receptor sequence in high-throughput single-cell experiments. Leveraging this new type of data, we propose and benchmark a collection of deep learning architectures to model T-cell specificity in single cells. In agreement with previous results, we found that models that treat antigens as categorical outcome variables outperform those that model the TCR and antigen sequence jointly. Moreover, we show that variability in single-cell immune repertoire screens can be mitigated by modeling cell-specific covariates. Lastly, we demonstrate that the number of bound pMHC complexes can be predicted in a continuous fashion providing a gateway to disentangle cell-to-dextramer binding strength and receptor-to-pMHC affinity. We provide these models in the Python package TcellMatch to allow imputation of antigen specificities in single-cell RNA-seq studies on T cells without the need for MHC staining.

Fine-tuning of antigen-specific immune responses by regulatory T cells activated via antigen recognition-independent and humoral factor-dependent mechanisms.

CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) are highly sensitive to IL-2, one of the many cytokines produced during immune responses, for their development, survival and functions. Although the effects of IL-2 administration on Tregs in vivo are well characterized, the effects on Tregs elicited by IL-2 produced during an immune response have not been elucidated. Hence, in this study, Treg behaviour during IL-2-producing immune responses was explored using in vivo and in vitro murine systems. The use of murine mixed lymphocyte culture (MLC) revealed that a large proportion of Tregs increased in size, accompanied by both cell death and proliferation status. Further, these large Tregs, which were found to not recognize specific antigens, were observed in MLCs as being functionally activated by various cytokines, including IL-2, produced by antigen-specific T cells. This 'bystander Treg activation' was also observed in mice with graft-versus-host reactions (GvHRs). Alternatively, effector cells from Treg-depleted MLCs exhibited lower antigen-specific responses or higher cross-reactivity as compared to control MLCs with Tregs. Taken together, these results suggest that Tregs are activated by cytokines, mainly IL-2, released from T cells that are activated by a specific antigen. Subsequently, these activated bystander Tregs contribute to the fine-tuning of highly antigen-specific immune responses.

Functional roles of graft-infiltrating lymphocytes during early-phase post-transplantation in mouse cardiac transplantation models.

Immunological behavior of graft-infiltrating lymphocytes (GILs) determines the graft fate (i.e., rejection or acceptance). Nevertheless, the functional alloreactivity and the phenotype of GILs at various times during the early post-transplantation phase have not been fully elucidated. We examined the immunological activities of early-phase GILs using a murine model of cardiac transplantation. GILs from 120-h allografts, but not 72-h allografts, showed robust activation and produced proinflammatory cytokines. In particular, a significant increase in CD69+ T-bet+ Nur77+ T cells was detected in 120-h allografts. Furthermore, isolated GILs were used to reconstitute BALB/c Rag2-/- γc-/- (BRG) mice. BRG mice reconstituted with 120-h GILs displayed donor-specific immune reactivity and rejected donor strain cardiac allografts; conversely, 72-h GILs exhibited weak anti-donor reactivity and did not reject allografts. These findings were confirmed by re-transplantation of cardiac allografts into BRG mice at 72-h post-transplantation. Re-transplanted allografts continued to function for >100 days, despite the presence of CD3+ GILs. In conclusion, the immunological behavior of GILs considerably differs over time during the early post-transplantation phase. A better understanding of the functional role of early-phase GILs may clarify the fate determination process in the graft-site microenvironment.

Revealing the specificity of regulatory T cells in murine autoimmune diabetes.

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαß pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Šresolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

CD1b presents self and Borrelia burgdorferi diacylglycerols to human T cells.

Lyme disease is a common multisystem disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here, we show that CD1b presents BbGL-II to human T cells and that the TCR mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing APCs in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells.