PAHSAs attenuate immune responses and promote ß cell survival in autoimmune diabetic mice.

Palmitic acid esters of hydroxy stearic acids (PAHSAs) are endogenous antidiabetic and antiinflammatory lipids. Here, we show that PAHSAs protect against type 1 diabetes (T1D) and promote ß cell survival and function. Daily oral PAHSA administration to nonobese diabetic (NOD) mice delayed the onset of T1D and markedly reduced the incidence of T1D, whether PAHSAs were started before or after insulitis was established. PAHSAs reduced T and B cell infiltration and CD4+ and CD8+ T cell activation, while increasing Treg activation in pancreata of NOD mice. PAHSAs promoted ß cell proliferation in both NOD mice and MIN6 cells and increased the number of ß cells in NOD mice. PAHSAs attenuated cytokine-induced apoptotic and necrotic ß cell death and increased ß cell viability. The mechanism appears to involve a reduction of ER stress and MAPK signaling, since PAHSAs lowered ER stress in NOD mice, suppressed thapsigargin-induced PARP cleavage in human islets, and attenuated ERK1/2 and JNK1/2 activation in MIN6 cells. This appeared to be mediated in part by glucagon-like peptide 1 receptor (GLP-1R) and not the G protein-coupled receptor GPR40. PAHSAs also prevented impairment of glucose-stimulated insulin secretion and improved glucose tolerance in NOD mice. Thus, PAHSAs delayed the onset of T1D and reduced its incidence by attenuating immune responses and exerting direct protective effects on ß cell survival and function.

Imaging the emergence and natural progression of spontaneous autoimmune diabetes.

Type 1 diabetes in the nonobese diabetic mouse stems from an infiltration of the pancreatic islets by a mixed population of immunocytes, which results in the impairment and eventual destruction of insulin-producing ß-cells. Little is known about the dynamics of lymphocyte movement in the pancreas during disease progression. Using advanced intravital imaging approaches and newly created reporter mice (Flt3-BFP2, Mertk-GFP-DTR, Cd4-tdTomato, Cd8a-tdTomato), we show that the autoimmune process initiates first with a T cell infiltration into the islets, where they have restricted mobility but reside and are activated in apposition to CX3CR1+ macrophages. The main expansion then occurs in the connective tissue outside the islet, which remains more or less intact. CD4+ and CD8+ T cells, Tregs, and dendritic cells (DCs) are highly mobile, going along microvascular tracks, while static macrophages (MF) form a more rigid structure, often encasing the islet cell mass. Transient cell-cell interactions are formed between T cells and both MFs and DCs, but also surprisingly between MFs and DCs themselves, possibly denoting antigen transfer. In later stages, extensive islet destruction coincides with preferential antigen presentation to, and activation of, CD8+ T cells. Throughout the process, Tregs patrol the active compartments, consistent with the notion that they control the activation of many cell types.

Radiation therapy primes tumors for nanotherapeutic delivery via macrophage-mediated vascular bursts.

Efficient delivery of therapeutic nanoparticles (TNPs) to tumors is critical in improving efficacy, yet strategies that universally maximize tumoral targeting by TNP modification have been difficult to achieve in the clinic. Instead of focusing on TNP optimization, we show that the tumor microenvironment itself can be therapeutically primed to facilitate accumulation of multiple clinically relevant TNPs. Building on the recent finding that tumor-associated macrophages (TAM) can serve as nanoparticle drug depots, we demonstrate that local tumor irradiation substantially increases TAM relative to tumor cells and, thus, TNP delivery. High-resolution intravital imaging reveals that after radiation, TAM primarily accumulate adjacent to microvasculature, elicit dynamic bursts of extravasation, and subsequently enhance drug uptake in neighboring tumor cells. TAM depletion eliminates otherwise beneficial radiation effects on TNP accumulation and efficacy, and controls with unencapsulated drug show that radiation effects are more pronounced with TNPs. Priming with combined radiation and cyclophosphamide enhances vascular bursting and tumoral TNP concentration, in some cases leading to a sixfold increase of TNP accumulation in the tumor, reaching 6% of the injected dose per gram of tissue. Radiation therapy alters tumors for enhanced TNP delivery in a TAM-dependent fashion, and these observations have implications for the design of next-generation tumor-targeted nanomaterials and clinical trials for adjuvant strategies.

An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk.

Investigation of host-environment interactions in the gut would benefit from a culture system that maintained tissue architecture yet allowed tight experimental control. We devised a microfabricated organ culture system that viably preserves the normal multicellular composition of the mouse intestine, with luminal flow to control perturbations (e.g., microbes, drugs). It enables studying short-term responses of diverse gut components (immune, neuronal, etc.). We focused on the early response to bacteria that induce either Th17 or RORg+ T-regulatory (Treg) cells in vivo. Transcriptional responses partially reproduced in vivo signatures, but these microbes elicited diametrically opposite changes in expression of a neuronal-specific gene set, notably nociceptive neuropeptides. We demonstrated activation of sensory neurons by microbes, correlating with RORg+ Treg induction. Colonic RORg+ Treg frequencies increased in mice lacking TAC1 neuropeptide precursor and decreased in capsaicin-diet fed mice. Thus, differential engagement of the enteric nervous system may partake in bifurcating pro- or anti-inflammatory responses to microbes.

Rapid, high efficiency isolation of pancreatic ß-cells.

The ability to isolate pure pancreatic ß-cells would greatly aid multiple areas of diabetes research. We developed a fluorescent exendin-4-like neopeptide conjugate for the rapid purification and isolation of functional mouse pancreatic ß-cells. By targeting the glucagon-like peptide-1 receptor with the fluorescent conjugate, ß-cells could be quickly isolated by flow cytometry and were >99% insulin positive. These studies were confirmed by immunostaining, microscopy and gene expression profiling on isolated cells. Gene expression profiling studies of cytofluorometrically sorted ß-cells from 4 and 12 week old NOD mice provided new insights into the genetic programs at play of different stages of type-1 diabetes development. The described isolation method should have broad applicability to the ß-cell field.

A minor subset of Batf3-dependent antigen-presenting cells in islets of Langerhans is essential for the development of autoimmune diabetes.

Autoimmune diabetes is characterized by inflammatory infiltration; however, the initiating events are poorly understood. We found that the islets of Langerhans in young nonobese diabetic (NOD) mice contained two antigen-presenting cell (APC) populations: a major macrophage and a minor CD103(+) dendritic cell (DC) population. By 4 weeks of age, CD4(+) T cells entered islets coincident with an increase in CD103(+) DCs. In order to examine the role of the CD103(+) DCs in diabetes, we examined Batf3-deficient NOD mice that lacked the CD103(+) DCs in islets and pancreatic lymph nodes. This led to a lack of autoreactive T cells in islets and, importantly, no incidence of diabetes. Additional examination revealed that presentation of major histocompatibility complex (MHC) class I epitopes in the pancreatic lymph nodes was absent with a partial impairment of MHC class II presentation. Altogether, this study reveals that CD103(+) DCs are essential for autoimmune diabetes development.

Fluorescent exendin-4 derivatives for pancreatic ß-cell analysis.

The ability to reliably identify pancreatic ß-cells would have far reaching implications for a greater understanding of ß-cell biology, measurement of ß-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic ß-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of ß-cells. Some of the developed compounds should be particularly useful for basic and translational ß-cell research.

Pathogenic CD4⁺ T cells recognizing an unstable peptide of insulin are directly recruited into islets bypassing local lymph nodes.

In the nonobese diabetic mouse, a predominant component of the autoreactive CD4(+) T cell repertoire is directed against the B:9-23 segment of the insulin B chain. Previous studies established that the majority of insulin-reactive T cells specifically recognize a weak peptide-MHC binding register within the B:9-23 segment, that to the 12-20 register. These T cells are uniquely stimulated when the B:9-23 peptide, but not the insulin protein, is offered to antigen presenting cells (APCs). Here, we report on a T cell receptor (TCR) transgenic mouse (8F10) that offers important new insights into the biology of these unconventional T cells. Many of the 8F10 CD4(+) T cells escaped negative selection and were highly pathogenic. The T cells were directly recruited into islets of Langerhans, where they established contact with resident intra-islet APCs. Immunogenic insulin had to be presented in order for the T cells to localize and cause disease. These T cells bypassed an initial priming stage in the pancreatic lymph node thought to precede islet T cell entry. 8F10 T cells induced the production of antiinsulin antibodies and islets contained immunoglobulin (IgG) deposited on ß cells and along the vessel walls.

A novel pathway of presentation by class II-MHC molecules involving peptides or denatured proteins important in autoimmunity.

We describe here a pathway of presentation involving peptides or denatured proteins that generate unique peptide-MHC complexes. Such complexes select for non-conventional CD4 T cells. We have examined this pathway and the corresponding CD4 T cells in diabetic autoimmunity. Autoimmunity requires both the escape of self-reactive T cells from thymic selection and, importantly, suitable conditions in peripheral tissues that allow for activation of T cells. In the autoimmune diabetes of NOD mice, insulin reactive T cells are highly focused on a peptide, encompassing the 9-23 segment of the B chain (B:9-23) bound to I-A(g7). Examination of the B:9-23 reactive T cell repertoire revealed the presence of two independent sets of T cells that recognize this epitope. One set, called type A, reacted like conventional CD4 T cells, recognizing both processed insulin protein and soluble B:9-23 peptide presented by APC. These T cells were highly deleted in the thymus and poorly represented in the periphery. The second set, called type B, did not recognize processed insulin protein presented by APC, but reacted strongly to the presentation of soluble B:9-23 peptide. Notably, this set was not deleted in the thymus, abundant in the periphery and caused diabetes. Free insulin peptides generated a unique peptide-MHC complex not found after insulin processing. These two T cell subsets discriminated between two independent, overlapping registers found within the B:9-23 peptide. In the islets of Langerhans, beta cells constitutively generated proteolyzed peptides from insulin, which were taken up by intra-islet APC and presented to peptide-specific type B T cells. Thus, self-reactive T cells that escape thymic selection can become pathogenic in the target organ where high concentrations of antigen and/or differences in intracellular processing lead to the presentation of peptides bound in distinct registers from those found in the thymus.

Unconventional recognition of peptides by T cells and the implications for autoimmunity.

The interaction of antigen-presenting cells with free peptides or a denatured protein can give rise to peptide-MHC class II complexes that are distinct from those generated after the processing of the whole protein. Such atypical peptide-MHC complexes can be recognized by unconventional 'type B' T cells that are not a component of the normal immune response to proteins. Importantly, these unconventional T cells can be found in the setting of autoimmunity. Here, we discuss unconventional peptide recognition by type B T cells and consider the implications for type 1 diabetes and other autoimmune diseases.

A broad range of self-reactivity drives thymic regulatory T cell selection to limit responses to self.

The degree of T cell self-reactivity considered dangerous by the immune system, thereby requiring thymic selection processes to prevent autoimmunity, is unknown. Here, we analyzed a panel of T cell receptors (TCRs) with a broad range of reactivity to ovalbumin (OVA(323-339)) in the rat insulin promoter (RIP)-mOVA self-antigen model for their ability to trigger thymic self-tolerance mechanisms. Thymic regulatory T (Treg) cell generation in vivo was directly correlated with in vitro TCR reactivity to OVA-peptide in a broad ~1,000-fold range. Interestingly, higher TCR affinity was associated with a larger Treg cell developmental "niche" size, even though the amount of antigen should remain constant. The TCR-reactivity threshold to elicit thymic negative selection and peripheral T cell responses was ~100-fold higher than that of Treg cell differentiation. Thus, these data suggest that the broad range of self-reactivity that elicits thymic Treg cell generation is tuned to secure peripheral tolerance to self.

Register shifting of an insulin peptide-MHC complex allows diabetogenic T cells to escape thymic deletion.

In nonobese diabetic (NOD) mice, two sets of autoreactive CD4(+) T cells recognize the B:9-23 segment of the insulin B chain. One set, type A, recognizes insulin presented by antigen-presenting cells (APCs). These T cells are highly deleted in the thymus. The second set, type B, does not recognize insulin protein but reacts with soluble B chain peptide. This set is not deleted in the thymus but is activated in the islets of Langerhans. In this study, we examine the specificity of these two types of T cells. The protein-reactive set recognizes the stretch of residues 13-21 of the insulin B chain. The set reactive to peptide only recognizes the stretch from residues 12-20. A single amino acid shift of the B chain peptide bound to I-A(g7) determines whether T cells recognize peptides generated by the processing of insulin, and consequently their escape from thymic purging. Biochemical experiments indicate that peptides bound in the 13-21 register interact more favorably with I-A(g7) than peptides that bind in the 12-20 register. Thus, self-reactive T cells can become pathogenic in the target organ where high concentrations of antigen and/or differences in intracellular processing present peptides in registers distinct from those found in the thymus.

Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes.

In addition to the genetic framework, there are two other critical requirements for the development of tissue-specific autoimmune disease. First, autoreactive T cells need to escape thymic negative selection. Second, they need to find suitable conditions for autoantigen presentation and activation in the target tissue. We show here that these two conditions are fulfilled in diabetic mice of the nonobese diabetic (NOD) strain. A set of autoreactive CD4(+) T cells specific for an insulin peptide, with the noteworthy feature of not recognizing the insulin protein when processed by antigen-presenting cells (APCs), escaped thymic control, participated in diabetes and caused disease. Moreover, APCs in close contact with beta cells in the islets of Langerhans bore vesicles with the antigenic insulin peptides and activated peptide-specific T cells. Our findings may be relevant for other cases of endocrine autoimmunity.