Early-life microbiota-immune homeostasis.

As the prevalence of allergy and autoimmune disease in industrialized societies continues to rise, improving our understanding of the mechanistic roles behind microbiota-immune homeostasis has become critical for informing therapeutic interventions in cases of dysbiosis. Of particular importance, are alterations to intestinal microbiota occurring within the critical neonatal window, during which the immune system is highly vulnerable to environmental exposures. This review will highlight recent literature concerning mechanisms of early-life microbiota-immune homeostasis as well as discuss the potential for therapeutics in restoring dysbiosis in early life.

The amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes.

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. However, whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed that upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. From this we postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin, and that both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.

The Amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes.

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. Whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. We postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin and both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum (ER) stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.

Histopathologic and transcriptomic phenotypes of a conditional RANKL transgenic mouse thymus.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

Host-microbiota interactions shaping T-cell response and tolerance in type 1 diabetes.

Type-1 Diabetes (T1D) is a complex polygenic autoimmune disorder involving T-cell driven beta-cell destruction leading to hyperglycemia. There is no cure for T1D and patients rely on exogenous insulin administration for disease management. T1D is associated with specific disease susceptible alleles. However, the predisposition to disease development is not solely predicted by them. This is best exemplified by the observation that a monozygotic twin has just a 35% chance of developing T1D after their twin's diagnosis. This makes a strong case for environmental triggers playing an important role in T1D incidence. Multiple studies indicate that commensal gut microbiota and environmental factors that alter their composition might exacerbate or protect against T1D onset. In this review, we discuss recent literature highlighting microbial species associated with T1D. We explore mechanistic studies which propose how some of these microbial species can modulate adaptive immune responses in T1D, with an emphasis on T-cell responses. We cover topics ranging from gut-thymus and gut-pancreas communication, microbial regulation of peripheral tolerance, to molecular mimicry of islet antigens by microbial peptides. In light of the accumulating evidence on commensal influences in neonatal thymocyte development, we also speculate on the link between molecular mimicry and thymic selection in the context of T1D pathogenesis. Finally, we explore how these observations could inform future therapeutic approaches in this disease.

Antibody-Mediated Targeting of a Hybrid Insulin Peptide Toward Neonatal Thymic Langerin-Positive Cells Enhances T-Cell Central Tolerance and Delays Autoimmune Diabetes.

Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T-cell population. Hybrid peptides formed through transpeptidation within pancreatic ß-cell lysosomes have been proposed as a new class of autoantigens in type 1 diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the hybrid insulin peptide (HIP) 2.5HIP toward thymic resident Langerin-positive dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti-Langerin-2.5HIP delivery can enhance T-cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti-Langerin-2.5HIP during the neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neoantigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases.

A dormant T-cell population with autoimmune potential exhibits low self-reactivity and infiltrates islets in type 1 diabetes.

The contribution of low-affinity T cells to autoimmunity in the context of polyclonal T-cell responses is understudied due to the limitations in their capture by tetrameric reagents and low level of activation in response to antigenic stimulation. As a result, low-affinity T cells are often disregarded as nonantigen-specific cells irrelevant to the immune response. Our study aimed to assess how the level of self-antigen reactivity shapes T-cell lineage and effector responses in the context of spontaneous tissue-specific autoimmunity observed in NOD mice. Using multicolor flow cytometry in combination with Nur77GFP reporter of TCR signaling, we identified a dormant population of T cells that infiltrated the pancreatic islets of prediabetic NOD mice, which exhibited reduced levels of self-tissue reactivity based on expression of CD5 and Nur77GFP . We showed that these CD5low T cells had a unique TCR repertoire and exhibited low activation and minimal effector function; however, induced rapid diabetes upon transfer. The CD4+ CD5low T-cell population displayed transcriptional signature of central memory T cells, consistent with the ability to acquire effector function post-transfer. Transcriptional profile of CD5low T cells was similar to T cells expressing a low-affinity TCR, indicating TCR affinity to be an important factor in shaping CD5low T-cell phenotype and function at the tissue site. Overall, our study suggests that autoimmune tissue can maintain a reservoir of undifferentiated central memory-like autoreactive T cells with pathogenic effector potential that might be an important source for effector T cells during long-term chronic autoimmunity.

T-Cell Receptor/HLA Humanized Mice Reveal Reduced Tolerance and Increased Immunogenicity of Posttranslationally Modified GAD65 Epitope.

Accumulating evidence supports a critical role for posttranslationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen-reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115-127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes and reduced frequency of regulatory T cells. Using humanized HLA/T-cell receptor (TCR) mice, we observed that TCRs reactive to the native or deamidated GAD65115-127 led to efficient development of CD4+ effector T cells; however, regulatory T-cell development was reduced in mice expressing the PTM-reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native-specific and the deamidated-specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti-ß-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T-cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T-cell development is reduced in response to the deamidated epitope, pointing to regulatory T-cell development as a key mechanism for loss of tolerance to PTM antigenic targets.

Function, Failure, and the Future Potential of Tregs in Type 1 Diabetes.

Critical insights into the etiology of type 1 diabetes (T1D) came from genome-wide association studies that unequivocally connected genetic susceptibility to immune cell function. At the top of the susceptibility are genes involved in regulatory T-cell (Treg) function and development. The advances in epigenetic and transcriptional analyses have provided increasing evidence for Treg dysfunction in T1D. These are well supported by functional studies in mouse models and analysis of peripheral blood during T1D. For these reasons, Treg-based therapies are at the forefront of research and development and have a tangible probability to deliver a long-sought-after successful immune-targeted treatment for T1D. The current challenge in the field is whether we can directly assess Treg function at the tissue site or make informative interpretations based on peripheral data. Future studies focused on Treg function in pancreatic lymph nodes and pancreas could provide key insight into the ultimate mechanisms underlying Treg failure in T1D. In this Perspective we will provide an overview of current literature regarding Treg development and function in T1D and how this knowledge has been applied to Treg therapies.

Thymic development of gut-microbiota-specific T cells.

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection1. Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses1,2. Antigen-specific recognition of intestinal microorganisms by T cells has previously been described3,4. Although the local environment shapes the differentiation of effector cells3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.

Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αß T cell numbers. MPC1-deficient T cells have defective thymic development at the ß-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αß T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

A Critical Insulin TCR Contact Residue Selects High-Affinity and Pathogenic Insulin-Specific T Cells.

Type 1 diabetes is an autoimmune-mediated disease that culminates in the targeted destruction of insulin-producing ß-cells. CD4 responses in NOD mice are dominated by insulin epitope B:9-23 (InsB9-23) specificity, and mutation of the key T-cell receptor (TCR) contact residue within the epitope prevents diabetes development. However, it is not clear how insulin self-antigen controls the selection of autoimmune and regulatory T cells (Tregs). Here we demonstrate that mutation of insulin epitope results in escape of highly pathogenic T cells. We observe an increase in antigen reactivity, clonality, and pathogenicity of insulin-specific T cells that develop in the absence of cognate antigen. Using a single TCR system, we demonstrate that Treg development is greatly diminished in mice with the Y16A mutant epitope. Collectively, these results suggest that the tyrosine residue at position 16 is necessary to constrain TCR reactivity for InsB9-23 by both limiting the development of pathogenic T cells and supporting the selection of Tregs.

Consequences of early postnatal lipopolysaccharide exposure on developing lungs in mice.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of infants that is characterized by interrupted lung development. Postnatal sepsis causes BPD, yet the contributory mechanisms are unclear. To address this gap, studies have used lipopolysaccharide (LPS) during the alveolar phase of lung development. However, the lungs of infants who develop BPD are still in the saccular phase of development, and the effects of LPS during this phase are poorly characterized. We hypothesized that chronic LPS exposure during the saccular phase disrupts lung development by mechanisms that promote inflammation and prevent optimal lung development and repair. Wild-type C57BL6J mice were intraperitoneally administered 3, 6, or 10 mg/kg of LPS or a vehicle once daily on postnatal days (PNDs) 3-5. The lungs were collected for proteomic and genomic analyses and flow cytometric detection on PND6. The impact of LPS on lung development, cell proliferation, and apoptosis was determined on PND7. Finally, we determined differences in the LPS effects between the saccular and alveolar lungs. LPS decreased the survival and growth rate and lung development in a dose-dependent manner. These effects were associated with a decreased expression of proteins regulating cell proliferation and differentiation and increased expression of those mediating inflammation. While the lung macrophage population of LPS-treated mice increased, the T-regulatory cell population decreased. Furthermore, LPS-induced inflammatory and apoptotic response and interruption of cell proliferation and alveolarization was greater in alveolar than in saccular lungs. Collectively, the data support our hypothesis and reveal several potential therapeutic targets for sepsis-mediated BPD in infants.

Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis.

High self-reactivity drives T-bet and potentiates Treg function in tissue-specific autoimmunity.

T cell receptor (TCR) affinity is a critical factor of Treg lineage commitment, but whether self-reactivity is a determining factor in peripheral Treg function remains unknown. Here, we report that a high degree of self-reactivity is crucial for tissue-specific Treg function in autoimmunity. Based on high expression of CD5, we identified a subset of self-reactive Tregs expressing elevated levels of T-bet, GITR, CTLA-4, and ICOS, which imparted significant protection from autoimmune diabetes. We observed that T-bet expression in Tregs, necessary for control of Th1 autoimmunity, could be induced in an IFNγ-independent fashion and, unlike in conventional T cells (Tconv), was strongly correlated with the strength of TCR signaling. The level of CD5 similarly identified human Tregs with an increased functional profile, suggesting that CD5hi Tregs may constitute an efficacious subpopulation appropriate for use in adoptive Treg therapies for treatment of inflammatory conditions. Overall, this work establishes an instrumental role of high TCR self-reactivity in driving Treg function.

Cutting Edge: Low-Affinity TCRs Support Regulatory T Cell Function in Autoimmunity.

Regulatory T cells (Tregs) use a distinct TCR repertoire and are more self-reactive compared with conventional T cells. However, the extent to which TCR affinity regulates the function of self-reactive Tregs is largely unknown. In this study, we used a two-TCR model to assess the role of TCR affinity in Treg function during autoimmunity. We observed that high- and low-affinity Tregs were recruited to the pancreas and contributed to protection from autoimmune diabetes. Interestingly, high-affinity cells preferentially upregulated the TCR-dependent Treg functional mediators IL-10, TIGIT, GITR, and CTLA4, whereas low-affinity cells displayed increased transcripts for Areg and Ebi3, suggesting distinct functional profiles. The results of this study suggest mechanistically distinct and potentially nonredundant roles for high- and low-affinity Tregs in controlling autoimmunity.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Ectopic Expression of Self-Antigen Drives Regulatory T Cell Development and Not Deletion of Autoimmune T Cells.

Type 1 diabetes is a T cell-mediated autoimmune disease that is characterized by Ag-specific targeting and destruction of insulin-producing ß cells. Although multiple studies have characterized the pathogenic potential of ß cell-specific T cells, we have limited mechanistic insight into self-reactive autoimmune T cell development and their escape from negative selection in the thymus. In this study, we demonstrate that ectopic expression of insulin epitope B:9-23 (InsB9-23) by thymic APCs is insufficient to induce deletion of high- or low-affinity InsB9-23-reactive CD4+ T cells; however, we observe an increase in the proportion and number of thymic and peripheral Foxp3+ regulatory T cells. In contrast, the MHC stable insulin mimetope (InsB9-23 R22E) efficiently deletes insulin-specific T cells and prevents escape of high-affinity thymocytes. Collectively, these results suggest that Ag dose and peptide-MHC complex stability can lead to multiple fates of insulin-reactive CD4+ T cell development and autoimmune disease outcome.

Cutting Edge: CD3 ITAM Diversity Is Required for Optimal TCR Signaling and Thymocyte Development.

For the αß or γδTCR chains to integrate extracellular stimuli into the appropriate intracellular cellular response, they must use the 10 ITAMs found within the CD3 subunits (CD3γε, CD3δε, and ζζ) of the TCR signaling complex. However, it remains unclear whether each specific ITAM sequence of the individual subunit (γεδζ) is required for thymocyte development or whether any particular CD3 ITAM motif is sufficient. In this article, we show that mice utilizing a single ITAM sequence (γ, ε, δ, ζa, ζb, or ζc) at each of the 10 ITAM locations exhibit a substantial reduction in thymic cellularity and limited CD4-CD8- (double-negative) to CD4+CD8+ (double-positive) maturation because of low TCR expression and signaling. Together, the data suggest that ITAM sequence diversity is required for optimal TCR signal transduction and subsequent T cell maturation.

Understanding Autoimmune Diabetes through the Prism of the Tri-Molecular Complex.

The strongest susceptibility allele for Type 1 Diabetes (T1D) is human leukocyte antigen (HLA), which supports a central role for T cells as the drivers of autoimmunity. However, the precise mechanisms that allow thymic escape and peripheral activation of beta cell antigen-specific T cells are still largely unknown. Studies performed with the non-obese diabetic (NOD) mouse have challenged several immunological dogmas, and have made the NOD mouse a key experimental system to study the steps of immunodysregulation that lead to autoimmune diabetes. The structural similarities between the NOD I-Ag7 and HLA-DQ8 have revealed the stability of the T cell receptor (TCR)/HLA/peptide tri-molecular complex as an important parameter in the development of autoimmune T cells, as well as afforded insights into the key antigens targeted in T1D. In this review, we will provide a summary of the current understanding with regard to autoimmune T cell development, the significance of the antigens targeted in T1D, and the relationship between TCR affinity and immune regulation.