Improvement in insulin sensitivity and prevention of high fat diet-induced liver pathology using a CXCR2 antagonist.

BACKGROUND: Liver pathology (LP) characteristic of non-alcoholic fatty acid disease (NAFLD)/non-alcoholic steatohepatitis (NASH) is a prevalent co-morbidity of type 2 diabetes (T2D). Accumulating evidence indicates that neutrophils driving insulin resistance (IR), including hepatic IR, precipitate T2D-associated NAFLD/NASH. We hypothesized that targeting neutrophil accumulation into insulin-sensitive tissues in mice using a CXCR2 antagonist under T2D-precipitating high fat diet (HFD) could improve insulin sensitivity and prevent the progression towards liver pathology reminiscent of NAFLD/NASH. METHODS: Mice were age-matched and on standard rodent chow prior to 1:1 randomization into control and HFD formulated with the CXCR2 antagonist AZD5069 or with biologically inactive substitute. They were monitored for metabolic changes including insulin sensitivity using the hyperinsulinemic-euglycemic clamp and hepatic histopathologic evaluation in H&E-stained sections as well as via immunofluorescence microscopy of liver sections for leukocyte markers, collagen 1A1 formation, α-smooth muscle actin (SMA), and galectin-3 expression, for 16 weeks. Statistical tests used to determine significant differences among study groups and outcomes include Student's t-test, one-way ANOVA, repeated measures two-way ANOVA, and Fisher's exact test, depending on the analytical question. RESULTS: Compared to mice on HFD, mice in the AZD5069-formulated HFD exhibited improved insulin sensitivity, a modest reduction in weight gain, and a significant improvement in LP and markers related to NAFLD/NASH. Mice in the AZD5069-formulated HFD also exhibited reduced neutrophil accumulation into the liver at the end of the 16 week study period. CONCLUSIONS: These results show, for the first time, the effectiveness of a selective CXCR2 antagonist to improve insulin sensitivity, concomitantly preventing the progression towards LP characteristic of NAFLD/NASH. This represents a novel approach to target IR and developing LP under T2D-susceptible conditions using a single agent. Furthermore, our data extend the growing evidence in support of neutrophils as a leukocyte population that imprints and maintains a chronic inflammatory state in the progression of dysregulated metabolism in liver-specific co-morbid conditions.

Neutrophils and their role in the aetiopathogenesis of type 1 and type 2 diabetes.

Multiple and complex aetiological processes underlie diabetes mellitus, which invariably result in the development of hyperglycaemia. Although there are two prevalent distinct forms of the disease, that is, type 1 and type 2 diabetes, accumulating evidence indicates that these syndromes share more aetiopathological mechanisms than originally thought. This compels a rethinking of the approaches to prevent and treat the different manifestations of what eventually becomes a hyperglycaemic state. This review aims to address the involvement of neutrophils, the most abundant type of granulocytes involved in the initiation of the acute phase of inflammation, in the aetiopathogenesis of diabetes mellitus, with a focus on type 1 and type 2 diabetes. We review the evidence that neutrophils are the first leucocytes to react to and accumulate inside target tissues of diabetes, such as the pancreas and insulin-sensitive tissues. We then review available data on the role of neutrophils and their functional alteration, with a focus on NETosis, in the progression towards clinical disease. Finally, we review potential approaches as secondary and adjunctive treatments to limit neutrophil-mediated damage in the prevention of the progression of subclinical disease to clinical hyperglycaemia.

Immune Cells Activating Biotin-Decorated PLGA Protein Carrier.

Nanoparticle formulations have long been proposed as subunit vaccine carriers owing to their ability to entrap proteins and codeliver adjuvants. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as subunit vaccine carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption and stimulate professional antigen-presenting cells (APCs). We present evidence showing bNPs are capable of retaining proteins through the biotin-avidin interaction. Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin and streptavidin horseradish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP. Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1ß, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate Th1-type helper T cell responses. Cytokine production was reduced in avidin precoated bNPs (but not with other proteins), suggesting that the proinflammatory response is due in part to exposed biotin on the surface of bNPs. bNPs injected subcutaneously were localized to draining lymph nodes detectable after 28 days and were internalized by bronchoalveolar lavage dendritic cells and macrophages in mice in a dose-dependent manner when delivered intranasally. Taken together, these data provide evidence that bNPs should be explored further as potential adjuvanting carriers for subunit vaccines.

Autoimmune Inflammation and Insulin Resistance: Hallmarks So Far and Yet So Close to Explain Diabetes Endotypes.

PURPOSE OF REVIEW: Diabetes mellitus can be categorized into two major variants, type 1 and type 2. A number of traits such as clinical phenotype, age at disease onset, genetic background, and underlying pathogenesis distinguish the two forms. RECENT FINDINGS: Recent evidence indicates that type 1 diabetes can be accompanied by insulin resistance and type 2 diabetes exhibits self-reactivity. These two previously unknown conditions can influence the progression and outcome of the disease. Unlike most conventional considerations, diabetes appears to consist of a spectrum of intermediate phenotypes that includes monogenic and polygenic loci linked to inflammatory processes including autoimmunity, beta cell impairment, and insulin resistance. Here we discuss why a shift of the classical bi-modal view of diabetes (autoimmune vs. non-autoimmune) is necessary in favor of a model of an immunological continuum of endotypes lying between the two extreme "insulin-resistant" and "autoimmune beta cell targeting," shaped by environmental and genetic factors which contribute to determine specific immune-conditioned outcomes.

A brief glimpse over the horizon for type 1 diabetes nanotherapeutics.

The pace at which nanotherapeutic technology for human disease is evolving has accelerated exponentially over the past five years. Most of the technology is centered on drug delivery which, in some instances, offers tunable control of drug release. Emerging technologies have resulted in improvements in tissue and cell targeting while others are at the initial stages of pairing drug release and drug release kinetics with microenvironmental stimuli or changes in homeostasis. Nanotherapeutics has only recently been adopted for consideration as a prophylaxis/treatment approach in autoimmunity. Herein, we summarize the current state-of-the art of nanotherapeutics specifically for type 1 diabetes mellitus and offer our view over the horizon of where we envisage this modality evolving towards.

Generation of antigen-specific Foxp3+ regulatory T-cells in vivo following administration of diabetes-reversing tolerogenic microspheres does not require provision of antigen in the formulation.

We have developed novel antisense oligonucleotide-formulated microspheres that can reverse hyperglycemia in newly-onset diabetic mice. Dendritic cells taking up the microspheres adopt a restrained co-stimulation ability and migrate to the pancreatic lymph nodes when injected into an abdominal region that is drained by those lymph nodes. Furthermore, we demonstrate that the absolute numbers of antigen-specific Foxp3+ T regulatory cells are increased only in the lymph nodes draining the site of administration and that these T-cells proliferate independently of antigen supply in the microspheres. Taken together, our data add to the emerging model where antigen supply may not be a requirement in "vaccines" for autoimmune disease, but the site of administration - subserved by lymph nodes draining the target organ - is in fact critical to foster the generation of antigen-specific regulatory cells. The implications of these observations on "vaccine" design for autoimmunity are discussed and summarized.

Cellular therapies based on stem cells and their insulin-producing surrogates: a 2015 reality check.

Stem cell technology has recently gained a substantial amount of interest as one method to create a potentially limitless supply of transplantable insulin-producing cells to treat, and possibly cure diabetes mellitus. In this review, we summarize the state-of-the art of stem cell technology and list the potential sources of stem cells that have been shown to be useful as insulin-expressing surrogates. We also discuss the milestones that have been reached and those that remain to be addressed to generate bona fide beta cell-similar, insulin-producing surrogates. The caveats, limitations, and realistic expectations are also considered for current and future technology. In spite of the tremendous technical advances realized in the past decade, especially in the field of reprogramming adult somatic cells to become stem cells, the state-of-the art still relies on lengthy and cumbersome in vitro culture methods that yield cell populations that are not particularly glucose-responsive when transplanted into diabetic hosts. Despite the current impediments toward clinical translation, including the potential for immune rejection, the availability of technology to generate patient-specific reprogrammable stem cells has, and will be critical for, important insights into the genetics, epigenetics, biology, and physiology of insulin-producing cells in normal and pathologic states. This knowledge could accelerate the time to reach the desired breakthrough for safe and efficacious beta cell surrogates.

Sequence variation in promoter of Ica1 gene, which encodes protein implicated in type 1 diabetes, causes transcription factor autoimmune regulator (AIRE) to increase its binding and down-regulate expression.

ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC(+)) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity.

Retaining antibodies in tumors with a self-assembling injectable system.

The performance of an in situ-forming injectable membrane designed to retain antibody molecules in vivo is described. The system entails an aqueous mixture of peptide amphiphiles (referred to as"EAK16-II" and "EAKH6") and intermediate proteins (anti-H6 antibody and protein A/G) through which therapeutic IgG molecules are colocalized and oriented. Scanning electron micrographs show IgG molecules localized on the EAK16-II/EAKH6 membrane. IgG were captured via specific interactions and remained biologically active in vitro. Upon administration into mice subcutaneously, the amphiphilic peptides coassembled into stable His-tags displaying materials locally. The system was shown to retain in vivo a fluorescent dye-labeled IgG in two epithelial tumor lines. IgG coadministered with the system were found to remain in 4T1 mouse mammary tumors for up to 120 h, while free antibody was cleared within the first 24 h. Decreased clearance was also found in B16 melanoma established in mouse footpads. These studies demonstrated that the immobilizing mechanism was effective in enhancing the retention of IgG locally in vivo. The injectable system may be used to enhance the delivery of immune modulatory antibodies in tumors.

Tolerogenic dendritic cells in type 1 diabetes: no longer a concept.

Tolerogenic dendritic cells (tDC) arrest the progression of autoimmune-driven dysglycemia into clinical, insulin-requiring type 1 diabetes (T1D) and preserve a critical mass of ß cells able to restore some degree of normoglycemia in new-onset clinical disease. The safety of tDC, generated ex vivo from peripheral blood leukocytes, has been demonstrated in phase I clinical studies. Accumulating evidence shows that tDC act via multiple layers of immune regulation arresting the action of pancreatic ß cell-targeting effector lymphocytes. tDC share a number of phenotypes and mechanisms of action, independent of the method by which they are generated ex vivo. In the context of safety, this yields confidence that the time has come to test the best characterized tDC in phase II clinical trials in T1D, especially given that tDC are already being tested for other autoimmune conditions. The time is also now to refine purity markers and to "universalize" the methods by which tDC are generated. This review summarizes the current state of tDC therapy for T1D, presents points of intersection of the mechanisms of action that the different embodiments use to induce tolerance, and offers insights into outstanding matters to address as phase II studies are imminent. Finally, we present a proposal for co-administration and serially-alternating administration of tDC and T-regulatory cells (Tregs) as a synergistic and complementary approach to prevent and treat T1D.

Uncoupling hepatic insulin resistance - hepatic inflammation to improve insulin sensitivity and to prevent impaired metabolism-associated fatty liver disease in type 2 diabetes.

Diabetes mellitus is a metabolic disease clinically-characterized as acute and chronic hyperglycemia. It is emerging as one of the common conditions associated with incident liver disease in the US. The mechanism by which diabetes drives liver disease has become an intense topic of discussion and a highly sought-after therapeutic target. Insulin resistance (IR) appears early in the progression of type 2 diabetes (T2D), particularly in obese individuals. One of the co-morbid conditions of obesity-associated diabetes that is on the rise globally is referred to as non-alcoholic fatty liver disease (NAFLD). IR is one of a number of known and suspected mechanism that underlie the progression of NAFLD which concurrently exhibits hepatic inflammation, particularly enriched in cells of the innate arm of the immune system. In this review we focus on the known mechanisms that are suspected to play a role in the cause-effect relationship between hepatic IR and hepatic inflammation and its role in the progression of T2D-associated NAFLD. Uncoupling hepatic IR/hepatic inflammation may break an intra-hepatic vicious cycle, facilitating the attenuation or prevention of NAFLD with a concurrent restoration of physiologic glycemic control. As part of this review, we therefore also assess the potential of a number of existing and emerging therapeutic interventions that can target both conditions simultaneously as treatment options to break this cycle.

Incidence of an Insulin-Requiring Hyperglycemic Syndrome in SARS-CoV-2-Infected Young Individuals: Is It Type 1 Diabetes?

Pancreatic ACE2 receptor expression, together with increased prevalence of insulin-requiring hyperglycemia in patients with coronavirus disease 2019 (COVID-19), suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pancreatic infection might trigger a ß-cell-selective inflammation precipitating autoimmune type 1 diabetes (T1D). We examined T1D incidence in patients with COVID-19 inside a large, global population using a "big data" approach. The incidence in 0-30-year-old patients with confirmed COVID-19 over an ∼15-month period from the beginning of the COVID-19 pandemic was compared with an age-matched population without COVID-19 inside the TriNetX COVID-19 Research Network (>80 million deidentified patient electronic medical records globally). The cohorts were used to generate outcomes of T1D postindex. In those up to 18 years of age, the incidence of insulin-requiring diabetes that could represent T1D in patients with already diagnosed, confirmed COVID-19 was statistically indistinguishable from the control population without COVID-19. In contrast, in those aged 19-30 years, the incidence was statistically greater. These data suggest that the incidence of T1D among patients with COVID-19 <30 years of age, at least up to this time since the beginning of the pandemic, is not greater when compared with an age-, sex-, and BMI-matched population without COVID-19. Nevertheless, we caution that patients with COVID-19 could be asymptomatic of a diabetic/prediabetic state and therefore would not be expected to come to medical attention, remaining undiagnosed. Hence, it is still possible that asymptomatic virus-infected individuals could acquire ß-cell autoimmunity, eventually progressing to dysglycemia and clinical T1D at higher rates.

Current state of type 1 diabetes immunotherapy: incremental advances, huge leaps, or more of the same?

Thus far, none of the preclinically successful and promising immunomodulatory agents for type 1 diabetes mellitus (T1DM) has conferred stable, long-term insulin independence to diabetic patients. The majority of these immunomodulators are humanised antibodies that target immune cells or cytokines. These as well as fusion proteins and inhibitor proteins all share varying adverse event occurrence and severity. Other approaches have included intact putative autoantigens or autoantigen peptides. Considerable logistical outlays have been deployed to develop and to translate humanised antibodies targeting immune cells, cytokines, and cytokine receptors to the clinic. Very recent phase III trials with the leading agent, a humanised anti-CD3 antibody, call into question whether further development of these biologics represents a step forward or more of the same. Combination therapies of one or more of these humanised antibodies are also being considered, and they face identical, if not more serious, impediments and safety issues. This paper will highlight the preclinical successes and the excitement generated by phase II trials while offering alternative possibilities and new translational avenues that can be explored given the very recent disappointment in leading agents in more advanced clinical trials.

Modulation of Leukocytes of the Innate Arm of the Immune System as a Potential Approach to Prevent the Onset and Progression of Type 1 Diabetes.

Type 1 diabetes (T1D) is characterized by insulin deficiency resulting from the selective destruction of pancreatic ß-cells by self-reactive T cells. Recent evidence demonstrates that innate immune responses substantially contribute to the pathogenesis of T1D, as they represent a first line of response to danger/damage signals. Here we discuss evidence on how, in a relapsing-remitting pattern, pancreas remodeling, diet, microbiota, gut permeability, and viral/bacterial infections induce the accumulation of leukocytes of the innate arm of the immune system throughout the pancreas. The subsequent acquisition and presentation of endocrine and exocrine antigens to the adaptive arm of the immune system results in a chronic progression of pancreatic damage. This process provides for the generation of self-reactive T-cell responses; however, the relative weight that genetic and environmental factors have on the etiopathogenesis of T1D is endotype imprinted and patient specific. With this Perspectives in Diabetes, our goal is to encourage the scientific community to rethink mechanisms underlying T1D pathogenesis and to consider therapeutic approaches that focus on these processes in intervention trials within new-onset disease as well as in efforts seeking the disorder's prevention in individuals at high risk.

Neutrophil-Associated Inflammatory Changes in the Pre-Diabetic Pancreas of Early-Age NOD Mice.

A growing body of evidence indicates that neutrophils are the first major leukocyte population accumulating inside the pancreas even before the onset of a lymphocytic-driven impairment of functional beta cells in type 1 diabetes mellitus (T1D). In humans, pancreata from T1D deceased donors exhibit significant neutrophil accumulation. We present a time course of previously unknown inflammatory changes that accompany neutrophil and neutrophil elastase accumulation in the pancreas of the non-obese diabetic (NOD) mouse strain as early as 2 weeks of age. We confirm earlier findings in NOD mice that neutrophils accumulate as early as 2 weeks of age. We also observe a concurrent increase in the expression of neutrophil elastase in this time period. We also detect components of neutrophil extracellular traps (NET) mainly in the exocrine tissue of the pancreas during this time as well as markers of vascular pathology as early as 2 weeks of age. Age- and sex-matched C57BL/6 mice do not exhibit these features inside the pancreas. When we treated NOD mice with inhibitors of myeloperoxidase and neutrophil elastase, two key effectors of activated neutrophil activity, alone or in combination, we were unable to prevent the progression to hyperglycemia in any manner different from untreated control mice. Our data confirm and add to the body of evidence demonstrating neutrophil accumulation inside the pancreas of mice genetically susceptible to T1D and also offer novel insights into additional pathologic mechanisms involving the pancreatic vasculature that have, until now, not been discovered inside the pancreata of these mice. However, inhibition of key neutrophil enzymes expressed in activated neutrophils could not prevent diabetes. These findings add to the body of data supporting a role for neutrophils in the establishment of early pathology inside the pancreas, independently of, and earlier from the time at onset of lymphocytic infiltration. However, they also suggest that inhibition of neutrophils alone, acting via myeloperoxidase and neutrophil elastase only, in the absence of other other effector cells, is insufficient to alter the natural course of autoimmune diabetes, at least in the NOD model of the disease.

Hydrogel Dressings for Chronic Wound Healing in Diabetes: Beyond Hydration.

Chronic wounds caused by diabetes are a significant medical challenge. Complications from non-healing can result in dire consequences for patients and cost the healthcare system billions of dollars annually. Non-healing in wounds for diabetic patient's results from a combination of factors which impair clearing of injured tissue, proliferation of healthy cell populations and increase risk of infection. Wound dressings continue to form the basis for the treatment of chronic wounds. Traditionally, these focused solely on hydration of the wound site and mitigating infection risk. Hydrogel systems are ready made to meet these basic requirements due to their intrinsic hydration properties and ability to deliver active ingredients. Flexibility in materials and methods of release allowed these systems to remain targets of research into the 21st century. Improved understanding of the wound environment and healing cascades has led to the development of more advanced systems which incorporate endogenous growth factors and living cells. Despite their promise, clinical efficacy of these systems has remained a challenge. Further, the regulatory pathways for approval add a layer of complexity to translate pre-clinical work into marketed products. In this review, we discuss systems currently in clinical use, pre-clinical directions and regulatory challenges for hydrogels in the treatment of diabetic chronic wounds.

Arrest in the Progression of Type 1 Diabetes at the Mid-Stage of Insulitic Autoimmunity Using an Autoantigen-Decorated All-trans Retinoic Acid and Transforming Growth Factor Beta-1 Single Microparticle Formulation.

Type 1 diabetes (T1D) is a disorder of impaired glucoregulation due to lymphocyte-driven pancreatic autoimmunity. Mobilizing dendritic cells (DC) in vivo to acquire tolerogenic activity is an attractive therapeutic approach as it results in multiple and overlapping immunosuppressive mechanisms. Delivery of agents that can achieve this, in the form of micro/nanoparticles, has successfully prevented a number of autoimmune conditions in vivo. Most of these formulations, however, do not establish multiple layers of immunoregulation. all-trans retinoic acid (RA) together with transforming growth factor beta 1 (TGFß1), in contrast, has been shown to promote such mechanisms. When delivered in separate nanoparticle vehicles, they successfully prevent the progression of early-onset T1D autoimmunity in vivo. Herein, we show that the approach can be simplified into a single microparticle formulation of RA + TGFß1 with surface decoration with the T1D-relevant insulin autoantigen. We show that the onset of hyperglycemia is prevented when administered into non-obese diabetic mice that are at the mid-stage of active islet-selective autoimmunity. Unexpectedly, the preventive effects do not seem to be mediated by increased numbers of regulatory T-lymphocytes inside the pancreatic lymph nodes, at least following acute administration of microparticles. Instead, we observed a mild increase in the frequency of regulatory B-lymphocytes inside the mesenteric lymph nodes. These data suggest additional and potentially-novel mechanisms that RA and TGFß1 could be modulating to prevent progression of mid-stage autoimmunity to overt T1D. Our data further strengthen the rationale to develop RA+TGFß1-based micro/nanoparticle "vaccines" as possible treatments of pre-symptomatic and new-onset T1D autoimmunity.

Human Hemangioblast-Derived Mesenchymal Stem Cells Promote Islet Engraftment in a Minimal Islet Mass Transplantation Model in Mice.

Islet transplantation can restore glycemic control in patients with type 1 diabetes. Using this procedure, the early stages of engraftment are often crucial to long-term islet function, and outcomes are not always successful. Numerous studies have shown that mesenchymal stem cells (MSCs) facilitate islet graft function. However, experimental data can be inconsistent due to variables associated with MSC generation (including donor characteristics and tissue source), thus, demonstrating the need for a well-characterized and uniform cell product before translation to the clinic. Unlike bone marrow- or adipose tissue-derived MSCs, human embryonic stem cell-derived-MSCs (hESC-MSCs) offer an unlimited source of stable and highly-characterized cells that are easily scalable. Here, we studied the effects of human hemangioblast-derived mesenchymal cells (HMCs), (i.e., MSCs differentiated from hESCs using a hemangioblast intermediate), on islet cell transplantation using a minimal islet mass model. The co-transplantation of the HMCs allowed a mass of islets that was insufficient to correct diabetes on its own to restore glycemic control in all recipients. Our in vitro studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection in vivo. Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.

Tolerogenic Dendritic Cells and T-Regulatory Cells at the Clinical Trials Crossroad for the Treatment of Autoimmune Disease; Emphasis on Type 1 Diabetes Therapy.

Tolerogenic dendritic cells and T-regulatory cells are two immune cell populations with the potential to prevent the onset of clinical stage type 1 diabetes, and manage the beginning of underlying autoimmunity, at the time-at-onset and onwards. Initial phase I trials demonstrated that the administration of a number of these cell populations, generated ex vivo from peripheral blood leukocytes, was safe. Outcomes of some of these trials also suggested some level of autoimmunity regulation, by the increase in the numbers of regulatory cells at different points in a network of immune regulation in vivo. As these cell populations come to the cusp of pivotal phase II efficacy trials, a number of questions still need to be addressed. At least one mechanism of action needs to be verified as operational, and through this mechanism biomarkers predictive of the underlying autoimmunity need to be identified. Efficacy in the regulation of the underlying autoimmunity also need to be monitored. At the same time, the absence of a common phenotype core among the different dendritic cell and T-regulatory cell populations, that have completed phase I and early phase II trials, necessitates a better understanding of what makes these cells tolerogenic, especially if a uniform phenotypic core cannot be identified. Finally, the inter-relationship of tolerogenic dendritic cells and T-regulatory cells for survival, induction, and maintenance of a tolerogenic state that manages the underlying diabetes autoimmunity, raises the possibility to co-administer, or even to serially-administer tolerogenic dendritic cells together with T-regulatory cells as a cellular co-therapy, enabling the best possible outcome. This is currently a knowledge gap that this review aims to address.