With inflammatory bowel disease, such as Crohn's disease and colitis, being heavily dependent on immune cell function, the mechanisms controlling aberrant immune cell activity in the gut have become an area of interest for biomedical research. Although the etiology of inflammatory bowel disease remains unknown, it is caused by a combination of genetic, environmental and microbial factors leading to altered immune cell functions in the gut.1 Developing and maintaining tolerance are necessary in preventing immune-mediated disorders including inflammatory bowel disease. Foxp3 (forkhead box P3 transcription factor)–expressing regulatory T cells (Tregs) are critical mediators of immune tolerance to self- and non-self-antigens and can arise both in the thymus (i.e. thymus-derived Tregs) and extrathymically in the periphery [i.e. peripherally induced Tregs (pTregs)]. While thymus-derived Tregs promote immune tolerance to self-antigens, pTregs play a major role in tolerance to foreign antigens. Although the developmental mechanisms promoting thymus-derived Treg development in the thymus, including the involvement of the thymic antigen-presenting cell (APC) autoimmune regulator (Aire) and medullary thymic epithelial cells (mTECs), are well established,2 the regulatory mechanisms governing pTreg development and tolerance to gut microbiota remain unclear. A recent study by Akagbosu and Tayyebi et al.3 has identified a new class of APC involved in pTreg development and establishing gut immune tolerance during early life. APCs promote central tolerance by participating in the removal of self-reactive T cells via negative selection, and by inducing thymus-derived Treg development in the thymus. Different types of APCs, including CD103+ dendritic cells (DCs), programmed death ligand 1/2(PD-L1/2)–expressing DCs, CX3CR1+ intestinal macrophages and major histocompatibility complex (MHC)II+ group 3 innate lymphoid cells (ILC3s), have been shown to promote immune tolerance in the gut by a variety of different mechanisms including pTreg development.4-6 In addition to these APCs, cells expressing retinoic acid–related orphan receptor-gamma t (RORγt+), the master transcription factor for T helper type 17 cells, were also shown to play a role in gut immune tolerance. For example, Zhou et al.7 suggested that RORγt+ cells in the intestine were a subset of ILC3s because of coexpression of the transcription factor ZBTB46, a gene highly expressed by Lti (lymphoid tissue-inducer)-like ILC3s. Interestingly, these ZBTB46+ ILC3 cells were shown to be important for gut immune tolerance, as selective depletion of these cells rendered mice susceptible to enteric infection and intestinal inflammation. However, previous work by Brown et al.8 identified a subset of RORγt-expressing cells thought to be a classical DC type 2 subset that expressed high levels of MHCII, CD86 and programmed death ligand 1 upon LPS exposure in vivo. The work by Akagbosu and Tayyebi et al.3 thus clarifies the identity of RORγt-expressing cells as a unique subset of RORγt+ MHCII+ APCs coined Thetis cells (TCs), critical for immune tolerance to the gut microbiota and prevention of intestinal inflammation during early life. Akagbosu and Tayyebi et al.3 have identified that the development of TCs coincides with pTreg expansion, suggesting the involvement of TCs in pTreg differentiation. Building on a previous finding that mice deficient in MHCII-restricted antigen presentation by RORγt+ APCs do not establish tolerance to commensal bacteria and develop severe colitis,5 the authors explored a potential connection between RORγt+ APCs and pTreg development in the gut during early life. The authors found that mice with a constitutive deletion of MHCII on RORγt+ APCs exhibited a marked reduction in pTreg cells associated with an increase in inflammatory T helper type 17 cells. However, deletion of MHCII on RORγt+ APCs in adult mice did not affect the pTreg pool in the gut, demonstrating an essential role for RORγt+ APCs in pTreg development during early life. APCs are known to play a major role in T-cell priming.6 Based on their finding that TCs coincides with pTreg expansion and that deletion of MHCII on RORγt+ APCs abrogates pTreg induction, the authors next determined whether TCs exhibited antigen-presenting characteristics including Treg promotion. For this, the authors performed coculture experiments with TCs and CD4+ T cells. These functional experiments revealed that TCs were able to present MHCII antigens and induce Treg cells ex vivo, suggesting that TCs are competent APCs with immune regulatory properties. APCs in the gut employ a variety of mechanisms to promote Treg development. For example, APCs in the gut have been shown to secrete higher levels of transforming growth factor-beta and express the transforming growth factor-beta–activating integrin Itgb8, retinoic acid and programmed death 1 ligands.6 In their effort to identify mechanisms by which TCs promote pTreg induction, the authors found that TCs were enriched for Treg-promoting molecules such as interleukin-2 and Itgb8. Subsequent in vivo experiments with TC-specific deletion of Itgb8 resulted in impaired pTreg development, suggesting transforming growth factor-beta activation by TCs contributes to pTreg development. However, it was shown that MHCII deletion in other cell types, such as ILC3s or classical DCs, did not influence pTreg generation, suggesting the importance of TCs in gut immune tolerance. Importantly, this work also highlights the transcriptional program of RORγt+ MHCII+ APCs that share similarities between hematopoietic and nonhematopoietic cells. Single-cell assay for transposase-accessible chromatin sequencing was used to show that TCs express a combination of transcription factors involved in myeloid cell differentiation, mTEC differentiation and Aire expression. Specifically, TCs express the transcription factor Spi-B, a critical regulator of mTEC differentiation as well as the core transcription factors governing myeloid cell differentiation such as PU.1, IRF8 and IRF4. TCs were also enriched for molecules associated with antigen presentation, T-cell activation and cell migration. Interestingly, several of the signature genes identified in TCs have been previously implicated in the modulation of Aire expression in mTECs,9 suggesting a common transcriptional network between mTECs and TCs. In addition, four subgroups of TCs were identified. The TC I subset shows low CD11c and RORγt as well as expression of DC-associated genes such as Ccr7, Cd83 and Dpp4, while the TC II–IV subsets are CD11chiRORγthi. TC IV also boasted high CD11b expression, further supporting the misidentification of these cells as classical DC type 2 in a previous study.8 Fate-mapping experiments using Rag1RFP-creERT2Rosa26lsl-YFP mice show that Lin−MHCIIhiCXCR6− TCs were absent for yellow fluorescent protein, and thus are separate from classical DCs, ILCs and mTECs, suggesting that the overlap between these cell subsets transcriptionally is due to shared functions rather than ontogeny. To translate their findings in humans, the authors analyzed single-cell atlas data from second trimester to adult human intestine and mesenteric lymph nodes and found a cluster of cells distinct from but closely related to CCR7+ DCs that expressed TC genes including high levels of AIRE. Interestingly, these TCs were found exclusively in the mesenteric lymph node and highly enriched in fetal samples, suggesting a conserved function for these cells in gut immune tolerance during early life in humans. Consistent with this study, another study by Kedmi et al.10 identified that RORγt+MHCII+ APCs are required for the induction of pTregs specific to gut bacterium Helicobacter hepaticus using a model of H. hepaticus–colonized mice adoptively transferred with H. hepaticus–specific T cells. The authors also found that pTreg induction is abrogated when MHCII is specifically deleted in RORγt+ cells but not in conventional DCs. Interestingly, this pTreg abrogation was associated with an expansion of inflammatory T helper type 17 cells, further confirming that RORγt+MHCII+ APCs play a critical role in establishing Treg-dependent tolerance to gut microbiota (Figure 1). Interestingly, another study by Lyu et al. found that ILC3s expressing MHCII in the gut are necessary to promote microbiota-specific Treg cells and prevent Th17 cell expansion. This Treg promoting effect of ILCs appears to be mediated via their ability to present antigen and process TGF-β by expressing the integrin αV.11 While this work reveals the influence of novel TCs in pTreg development and immune tolerance to gut microbiota (Figure 1), questions regarding the origin of such cells are not fully understood. This study shows that TCs express markers consistent with DCs and suggests that the shared phenotype may be due to collective functions such as cellular migration and antigen presentation. Does this imply that these cells also function more like DCs during an inflammatory state early in life? Ultimately, this exciting work is a vital step in our understanding of immune tolerance and questions the divide between hematopoietic and nonhematopoietic cells. Future studies should further explore the role of TCs and their utilization later in life for therapeutic benefit against inflammatory bowel disease and other inflammatory disorders. The authors have no competing financial interests in relation to the work described. Alkeiver S Cannon: Conceptualization; writing – original draft. Addelynn Sagadevan: Conceptualization; writing – original draft. Murugaiyan Gopal: Conceptualization; writing – original draft; writing – review and editing.

In vivo induction of antigen (Ag)-specific regulatory T cells (Treg) is considered the holy grail of therapeutic strategies for restoring tolerance in autoimmunity. Unfortunately, in the autoimmune disease multiple sclerosis, an effective and durable therapy targeting the diverse repertoire of emerging Ags without compromising the patient’s natural immunity has remained elusive. To address this deficiency, we have developed an Ag-specific adeno-associated virus (AAV) immunotherapy that will restore tolerance in a Treg-dependent manner. Using multiple strains of mice with different genetic and immunological backgrounds, we demonstrate that a liver directed AAV vector expressing a single transgene can prevent experimental autoimmune encephalomyelitis from developing and effectively mitigate pre-existing or established disease that was induced by one or more auto-reactive myelin oligodendrocyte glycoprotein-derived peptides. Overall, the results suggests that AAV can efficiently restore Ag-specific immune tolerance to an immunogenic protein that is neither restricted by the major histocompatibility complex haplotype, nor by the specific antigenic epitope(s) presented. These findings may pave the way for developing a comprehensive Ag-specific immunotherapy that does not require prior knowledge of the specific immunogenic epitopes and that may prove to be universally applicable to all MS patients, and adaptable for other autoimmune diseases.

Neuromyelitis optica (NMO) is an auto-inflammatory demyelinating disease that typically affects optic nerves and spinal cord that is characterized by the presence of serum aquaporin-4 immunoglobulin G antibodies (AQP4-IgG). NMO accounts for >45% of the demyelinating disease in Asians and warrants the development of a suitable therapy other than generalized immunosuppressants. It is thought that autoimmune activated AQP4-specific T cells disrupt the BBB and allow increased entry of AQP4-IgG and other immune effectors into tissues containing astrocytes expressing AQP4 in their membranes. Recently we have established an adeno-associated viral (AAV) gene immunotherapy that effectively prevents and reverses experimental autoimmune encephalomyelitis (EAE) via generation of suppressive antigen-specific regulatory T cells (Tregs). In this report, using a similar antigen-specific approach, we have demonstrated that AAV.AQP4 gene immunotherapy prevented the development of AQP4-mediated neuroinflammation and clinical neurological disability in almost 100% of C57BL/6J mice when AAV.AQP4 vector was administered to mice 2 weeks prior to immunization with an immunogenic epitope of AQP4. Moreover, in contrast to AAV.AQP4 treated mice which remained unremarkable, histological analysis of the spinal cord sections from untreated vehicle only mice showed multiple areas of significant focal inflammation within the spinal cord. Based on our clinical and pathological data, we’ve successfully demonstrated that our novel AAV.AQP4 gene immunotherapy can indeed suppress the induction of AQP4 mediated autoimmune disease. Further evaluation will determine if it can also treat preexisting disease.

Abstract To re-establish long-term immune tolerance in Multiple Sclerosis (MS), our lab had previously developed a pre-clinical Adeno-associated virus (AAV) gene immunotherapy that is capable of preventing and reversing Myelin Oligodendrocyte Glycoprotein (MOG) induced Experimental Autoimmune Encephalomyelitis (EAE). However, MS is a disease that involves multiple myelin proteins including Proteolipid Protein (PLP). Therefore we expanded the capability of our gene immunotherapy to restore tolerance and ameliorate disease to multiple major myelin proteins, simultaneously. In this report we demonstrate that a mixture of 2 individual vectors, AAV MOG & AAV.PLP, was capable of preventing, and more importantly reversing, preexisting EAE disease induced with a mixture of MOG35–55 & PLP139–151 peptides in (C57BL/6JxSJL)F1 mice. To minimize the total vector load we further developed this immunotherapy into a novel single AAV-dual transgene expressing vector (AAV.MOG.PLP) using specific gene linkers for independent expression. Following a single peripheral injection, western blot analysis confirmed stable and simultaneous hepatic expression of both neuroprotein transgenes. When injected into mice 2 weeks before induction of EAE with either MOG35–55 or PLP139–151, or combination of both, the dual transgene AAV immunotherapy significantly reduced or prevented disease in mice compared to controls. Overall, these results demonstrate proof of concept that a single AAV vector simultaneously expressing more than one transgene may be an effective therapeutic treatment for restoring tolerance in an autoimmune disease like MS. Supported by NIHSarepta Pharmaceuticals NIH R01Act AI128074Project

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is known to be associated with congenital heart disease. However, little is known about the mechanism how blood glucose impacts heart formation. Using a chemically-defined human pluripotent stem cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic and electrophysiological levels via nucleotide biosynthesis through the pentose phosphate pathway. Even though blood glucose level stays stable in utero during normal pregnancy, glucose uptake by fetal cardiac tissue was found drastically reduced at late gestational stages in the mouse. Interestingly, perturbation of glucose dynamics during gestation in a murine model of diabetic pregnancy promoted mitosis and inhibited maturation of fetal cardiomyocytes. Therefore, this observation suggests that the metabolic switch is not only to meet the energy demand but also to induce a genetic program to facilitate cardiac maturation, providing a possible mechanistic basis for the congenital heart disease in diabetic pregnancy.