Soluble antigen arrays provide increased efficacy and safety over free peptides for tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but clinical implementation has been challenging. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA used (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Over time, the peptides induced an IgE-independent anaphylactic reaction, the incidence of which was significantly delayed when peptides were in SAgA form rather than in free form. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Immunologic anaphylaxis occurred in NOD mice in a dose-dependent manner but not in C57BL/6 or BALB/c mice; however, its incidence did not correlate with the level of anti-peptide antibodies. We provide evidence that SAgAs significantly improve the efficacy of peptides to induce tolerance and prevent autoimmune diabetes while at the same time reducing their anaphylactogenic potential.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.

Soluble antigen arrays improve the efficacy and safety of peptide-based tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but the limited in vivo stability and uptake of peptides impedes clinical implementation. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Both SAgA variants significantly delayed anaphylaxis compared to their respective free peptides. The anaphylaxis, which occurred in NOD mice but not in C57BL/6 mice, was dose-dependent but did not correlate with the production of IgG1 or IgE against the peptides. We provide evidence that SAgAs significantly improve the efficacy and safety of peptide-based immunotherapy.

Notch-mediated hepatocyte MCP-1 secretion causes liver fibrosis.

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.

Epitope-based precision immunotherapy of Type 1 diabetes.

Antigen-specific immunotherapies (ASITs) address important clinical needs in treating autoimmune diseases. However, Type 1 diabetes is a heterogeneous disease wherein patient characteristics influence responsiveness to ASITs. Targeting not only disease-relevant T cell populations, but also specific groups of patients using precision medicine is a new goal toward achieving effective treatment. HLA-restricted peptides provide advantages over protein as antigens, however, methods for profiling antigen-specific T cells need to improve in sensitivity, depth, and throughput to facilitate epitope selection. Delivery approaches are highly diverse, illustrating the many ways relevant antigen-presenting cell populations and anatomical locations can be targeted for tolerance induction. The role of persistence of antigen presentation in promoting durable antigen-specific tolerance requires further investigation. Based on the outcome of ASIT trials, the field is moving toward using patient-specific variations to improve efficacy, but challenges still lie on the path to delivering more effective and safer treatment to the T1D patient population.

Pharmacological conversion of gut epithelial cells into insulin-producing cells lowers glycemia in diabetic animals.

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic ß cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate ß-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-ß that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.

Preclinical evaluation of a precision medicine approach to DNA vaccination in type 1 diabetes.

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject's MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.

Rapid video-based deep learning of cognate versus non-cognate T cell-dendritic cell interactions.

Identification of cognate interactions between antigen-specific T cells and dendritic cells (DCs) is essential to understanding immunity and tolerance, and for developing therapies for cancer and autoimmune diseases. Conventional techniques for selecting antigen-specific T cells are time-consuming and limited to pre-defined antigenic peptide sequences. Here, we demonstrate the ability to use deep learning to rapidly classify videos of antigen-specific CD8+ T cells. The trained model distinguishes distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and DCs over 20 to 80 min. The model classified high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalized well to other types of high and low affinity CD8+ T cells. The classification accuracy achieved by the model was consistently higher than simple image analysis techniques, and conventional metrics used to differentiate between cognate and non-cognate T cells, such as speed. Also, we demonstrated that experimental addition of anti-CD40 antibodies improved model prediction. Overall, this method demonstrates the potential of video-based deep learning to rapidly classify cognate T cell-DC interactions, which may also be potentially integrated into high-throughput methods for selecting antigen-specific T cells in the future.

Modeling human T1D-associated autoimmune processes.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease characterized by impaired immune tolerance to ß-cell antigens and progressive destruction of insulin-producing ß-cells. Animal models have provided valuable insights for understanding the etiology and pathogenesis of this disease, but they fall short of reflecting the extensive heterogeneity of the disease in humans, which is contributed by various combinations of risk gene alleles and unique environmental factors. Collectively, these factors have been used to define subgroups of patients, termed endotypes, with distinct predominating disease characteristics. SCOPE OF REVIEW: Here, we review the gaps filled by these models in understanding the intricate involvement and regulation of the immune system in human T1D pathogenesis. We describe the various models developed so far and the scientific questions that have been addressed using them. Finally, we discuss the limitations of these models, primarily ascribed to hosting a human immune system (HIS) in a xenogeneic recipient, and what remains to be done to improve their physiological relevance. MAJOR CONCLUSIONS: To understand the role of genetic and environmental factors or evaluate immune-modifying therapies in humans, it is critical to develop and apply models in which human cells can be manipulated and their functions studied under conditions that recapitulate as closely as possible the physiological conditions of the human body. While microphysiological systems and living tissue slices provide some of these conditions, HIS mice enable more extensive analyses using in vivo systems.

Orchestrating multiplexity in polychromatic science through OMIPs: A decade-old resource to empower biomedical research.

As the optimized multicolor immunofluorescence panel (OMIP) platform entered its 10th anniversary since its launch, the multicolor flow cytometry landscape has changed significantly. Likewise, OMIPs have continuously evolved to cover larger panel sizes, increasing number of subpopulations profiled in a single panel, and new species. After a decade of contributions to the OMIP platform, a review of this collection, summarizing its content and purpose for the research community, is timely and due. This review provides an overview of OMIPs and a presentation of the depth and diversity of this collection of validated panels, with the expectation that readers will take advantage of them to empower and accelerate their research.

Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes.

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address the limited clinical efficacy of ASIT. Soluble antigen arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using either hydrolysable linkers (hSAgAs) or stable click chemistry linkers (cSAgAs). They were evaluated for the ability to block spontaneous development of disease in a nonobese diabetic mouse model of type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgAs) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers, such as CD73, interleukin-10, programmed death-1, and KLRG-1. Anaphylaxis caused by free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely nonoverlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to free peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

Tissue-Engineered Stromal Reticula to Study Lymph Node Fibroblastic Reticular Cells in Type I Diabetes.

INTRODUCTION: Fibroblastic reticular cells (FRCs) support and remodel the lymph node (LN), express and present self-antigens to T cells to promote tolerance. In Type 1 diabetes (T1D), decrease in FRC frequency and in their expression of T1D-related self-antigens may hinder tolerogenic engagement of autoreactive T cells. FRC reticular organization in LNs is critical for adaptive immunity. Thus, we engineered LN-like FRC reticula to determine if FRC reticular properties were altered in T1D and to study engagement of autoreactive T cells in vitro. METHODS: We characterized FRC networks in pancreatic and skin-draining LNs of 4- and 12-week old non-obese diabetic (NOD) and diabetes resistant NOR mice by immunofluorescence. Murine FRCs isolated from NOR, NOD or human pancreatic LNs were cultured in collagen sponges for up to 21 days before immunofluorescence and flow cytometry analysis. NOD FRCs expressing T1D antigens were co-cultured with CellTrace-labeled specific T cells in 2D or in scaffolds. T cell engagement was quantified by CD25 upregulation, CellTrace dilution and by T cell tracking. RESULTS: FRC networks in both 4- and 12-week old NOD LNs displayed larger reticular pores than NOR controls. NOD FRCs had delayed scaffold remodeling compared to NOR FRCs. Expression of the gp38 FRC marker in NOD FRCs was lower than in NOR but improved in 3D. FRC reticula expressing T1D antigens promoted higher engagement of specific T cells than 2D. CONCLUSION: We engineered LN-like FRC reticula that recapitulate FRC organization and phenotype of T1D LNs for studying tolerogenic autoreactive T cell engagement in T1D.

Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus.

During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9-23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8+ HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases.

Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations.

Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.

Nanoparticles versus Dendritic Cells as Vehicles to Deliver mRNA Encoding Multiple Epitopes for Immunotherapy.

The efficacy of antigen-specific immunotherapy relies heavily on efficient antigen delivery to antigen-presenting cells and engagement of as many disease-relevant T cells as possible in various lymphoid tissues, which are challenging to achieve. Here, we compared two approaches to deliver mRNA encoding multiple epitopes targeting both CD4+ and CD8+ T cells: a lipid-based nanoparticle platform to target endogenous antigen-presenting cells in vivo versus ex vivo mRNA-electroporated dendritic cells. After intraperitoneal injection, the nanoparticle platform facilitated efficient entry of mRNA into various endogenous antigen-presenting cells, including lymph node stromal cells, and elicited robust T cell responses within a wider network of lymphoid tissues compared with dendritic cells. Following intravenous injection, mRNA-electroporated dendritic cells and the nanoparticle platform localized primarily in lung and spleen, respectively. When administered locally via an intradermal route, both platforms resulted in mRNA expression at the injection site and in robust T cell responses in draining lymph nodes. This study indicates that multiple epitopes, customizable for specific patient populations and encoded by mRNA, can be targeted to different lymphoid tissues based on delivery vehicle and route, and constitute the groundwork for future studies using mRNA to reprogram exogenous or endogenous APCs for immunotherapy.

Phenotypic alterations in pancreatic lymph node stromal cells from human donors with type 1 diabetes and NOD mice.

AIMS/HYPOTHESIS: Tolerance induction in lymph nodes can be mediated by both haematopoietic cells (e.g. specific dendritic cells subsets) and by non-haematopoietic cells (e.g. lymph node stromal cells [LNSCs]) when they present peripheral tissue antigens to autoreactive T cells. LNSCs normally regulate T cell trafficking and survival and help to maintain peripheral tolerance by exerting immunosuppressive effects. However, whether autoimmunity can be associated with defective tolerogenic functions of LNSCs is unknown and studies aimed at characterising LNSCs in humans are lacking. We hypothesised that dysregulated T cell responses in pancreatic lymph nodes (PLNs) from donors with type 1 diabetes and from NOD mice may be associated with altered LNSC function. METHODS: We analysed PLNs from donors with type 1 diabetes and NOD mice for LNSC distribution and phenotype using flow cytometry. We assessed the expression of tolerance-related genes in different subsets of LNSCs from human donors, as well as in a population of dendritic cells enriched in autoimmune regulator (AIRE)+ cells and identified as HLA-DRhigh CD45low. RESULTS: The relative frequency of different LNSC subsets was altered in both donors with type 1 diabetes and NOD mice, and both MHC class II and programmed death-ligand 1 (PD-L1) expression were upregulated in human type 1 diabetes. Tolerance-related genes showed similar expression profiles between mouse and human LNSCs at steady state but were generally upregulated in the context of human type 1 diabetes, while, at the same time, many such genes were downregulated in the AIRE-enriched dendritic cell population. CONCLUSION/INTERPRETATION: Our study shows that LNSCs are substantially altered in type 1 diabetes, but, surprisingly, they exhibit an enhanced tolerogenic phenotype along with increased antigen-presenting potential, which may indicate an attempt to offset dendritic cell-related tolerogenic defects in tolerance. Thus, LNSCs could constitute alternative therapeutic targets in which to deliver antigens to help re-establish tolerance and prevent or treat type 1 diabetes. DATA AVAILABILITY: All data generated or analysed during this study are included in the published article (and its online supplementary files). Biomark gene expression data were deposited on the Mendeley repository at https://data.mendeley.com/datasets/d9rdzdmvyf/1 . Any other raw datasets are available from the corresponding author on reasonable request. No applicable resources were generated or analysed during the current study.

Soluble Antigen Arrays Displaying Mimotopes Direct the Response of Diabetogenic T Cells.

Type 1 diabetes (T1D) is an autoimmune disorder which develops when insulin-producing, pancreatic beta cells are destroyed by an aberrant immune response. Current therapies for T1D either treat symptoms or cause global immunosuppression, which leave patients at risk of developing long-term complications or vulnerable to foreign pathogens. Antigen-specific immunotherapies have emerged as a selective approach for autoimmune diseases by inducing tolerance while mitigating global immunosuppression. We previously reported SAgAs with multiple copies of a multiple sclerosis (MS) autoantigen grafted onto hyaluronic acid (HA) as an efficacious therapy in experimental autoimmune encephalomyelitis. While the immune response of MS is distinct from that of T1D, the mechanism of SAgAs was hypothesized to be similar and via induction of immune tolerance to diabetes antigens. We synthesized SAgAs composed of HA polymer backbone conjugated with multiple copies of the T1D autoantigen mimotope p79 using aminooxy chemistry (SAgAp79) or using copper-catalyzed alkyne-azide cycloaddition (cSAgAp79) chemistry. SAgAs constructed using the hydrolyzable aminooxy linkage, thus capable of releasing p79, exhibited physicochemical properties similar to the triazole linkage. Both SAgAp79 versions showed high specificity and efficacy in stimulating epitope-specific T cells. SAgAs can be taken up by most immune cell populations but do not induce their maturation, and conventional dendritic cells are responsible for the brunt of antigen presentation within splenocytes. cSAgAp79 was more stimulatory than SAgAp79 both in vitro and in vivo, an effect that was ascribed to the peptide modification rather than the type of linkage. In summary, we provide here the first proof-of-principle that SAgA therapy could also be applicable to T1D.