Role of the afferent lymph as an immunological conduit to analyze tissue antigenic and inflammatory load.

The lymphatic fluid is the conduit by which part of the tissue "omics" is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance.

CXCL16-dependent scavenging of oxidized lipids by islet macrophages promotes differentiation of pathogenic CD8+ T cells in diabetic autoimmunity.

The pancreatic islet microenvironment is highly oxidative, rendering ß cells vulnerable to autoinflammatory insults. Here, we examined the role of islet resident macrophages in the autoimmune attack that initiates type 1 diabetes. Islet macrophages highly expressed CXCL16, a chemokine and scavenger receptor for oxidized low-density lipoproteins (OxLDLs), regardless of autoimmune predisposition. Deletion of Cxcl16 in nonobese diabetic (NOD) mice suppressed the development of autoimmune diabetes. Mechanistically, Cxcl16 deficiency impaired clearance of OxLDL by islet macrophages, leading to OxLDL accumulation in pancreatic islets and a substantial reduction in intra-islet transitory (Texint) CD8+ T cells displaying proliferative and effector signatures. Texint cells were vulnerable to oxidative stress and diminished by ferroptosis; PD-1 blockade rescued this population and reversed diabetes resistance in NOD.Cxcl16-/- mice. Thus, OxLDL scavenging in pancreatic islets inadvertently promotes differentiation of pathogenic CD8+ T cells, presenting a paradigm wherein tissue homeostasis processes can facilitate autoimmune pathogenesis in predisposed individuals.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

Chromogranin A Deficiency Confers Protection From Autoimmune Diabetes via Multiple Mechanisms.

Recognition of ß-cell antigens by autoreactive T cells is a critical step in the initiation of autoimmune type1 diabetes. A complete protection from diabetes development in NOD mice harboring a point mutation in the insulin B-chain 9-23 epitope points to a dominant role of insulin in diabetogenesis. Generation of NOD mice lacking the chromogranin A protein (NOD.ChgA-/-) completely nullified the autoreactivity of the BDC2.5 T cell and conferred protection from diabetes onset. These results raised the issue concerning the dominant antigen that drives the autoimmune process. Here we revisited the NOD.ChgA-/- mice and found that their lack of diabetes development may not be solely explained by the absence of chromogranin A reactivity. NOD.ChgA-/- mice displayed reduced presentation of insulin peptides in the islets and periphery, which corresponded to impaired T-cell priming. Diabetes development in these mice was restored by antibody treatment targeting regulatory T cells or inhibiting transforming growth factor-ß and programmed death-1 pathways. Therefore, the global deficiency of chromogranin A impairs recognition of the major diabetogenic antigen insulin, leading to broadly impaired autoimmune responses controlled by multiple regulatory mechanisms.

Blood leukocytes recapitulate diabetogenic peptide-MHC-II complexes displayed in the pancreatic islets.

Assessing the self-peptides presented by susceptible major histocompatibility complex (MHC) molecules is crucial for evaluating the pathogenesis and therapeutics of tissue-specific autoimmune diseases. However, direct examination of such MHC-bound peptides displayed in the target organ remains largely impractical. Here, we demonstrate that the blood leukocytes from the nonobese diabetic (NOD) mice presented peptide epitopes to autoreactive CD4 T cells. These peptides were bound to the autoimmune class II MHC molecule (MHC-II) I-Ag7 and originated from insulin B-chain and C-peptide. The presentation required a glucose challenge, which stimulated the release of the insulin peptides from the pancreatic islets. The circulating leukocytes, especially the B cells, promptly captured and presented these peptides. Mass spectrometry analysis of the leukocyte MHC-II peptidome revealed a series of ß cell-derived peptides, with identical sequences to those previously identified in the islet MHC-II peptidome. Thus, the blood leukocyte peptidome echoes that found in islets and serves to identify immunogenic peptides in an otherwise inaccessible tissue.

Cytocidal macrophages in symbiosis with CD4 and CD8 T cells cause acute diabetes following checkpoint blockade of PD-1 in NOD mice.

Autoimmune diabetes is one of the complications resulting from checkpoint blockade immunotherapy in cancer patients, yet the underlying mechanisms for such an adverse effect are not well understood. Leveraging the diabetes-susceptible nonobese diabetic (NOD) mouse model, we phenocopy the diabetes progression induced by programmed death 1 (PD-1)/PD-L1 blockade and identify a cascade of highly interdependent cellular interactions involving diabetogenic CD4 and CD8 T cells and macrophages. We demonstrate that exhausted CD8 T cells are the major cells that respond to PD-1 blockade producing high levels of IFN-γ. Most importantly, the activated T cells lead to the recruitment of monocyte-derived macrophages that become highly activated when responding to IFN-γ. These macrophages acquire cytocidal activity against ß-cells via nitric oxide and induce autoimmune diabetes. Collectively, the data in this study reveal a critical role of macrophages in the PD-1 blockade-induced diabetogenesis, providing new insights for the understanding of checkpoint blockade immunotherapy in cancer and infectious diseases.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Single-cell RNA sequencing of murine islets shows high cellular complexity at all stages of autoimmune diabetes.

Tissue-specific autoimmune diseases are driven by activation of diverse immune cells in the target organs. However, the molecular signatures of immune cell populations over time in an autoimmune process remain poorly defined. Using single-cell RNA sequencing, we performed an unbiased examination of diverse islet-infiltrating cells during autoimmune diabetes in the nonobese diabetic mouse. The data revealed a landscape of transcriptional heterogeneity across the lymphoid and myeloid compartments. Memory CD4 and cytotoxic CD8 T cells appeared early in islets, accompanied by regulatory cells with distinct phenotypes. Surprisingly, we observed a dramatic remodeling in the islet microenvironment, in which the resident macrophages underwent a stepwise activation program. This process resulted in polarization of the macrophage subpopulations into a terminal proinflammatory state. This study provides a single-cell atlas defining the staging of autoimmune diabetes and reveals that diabetic autoimmunity is driven by transcriptionally distinct cell populations specialized in divergent biological functions.

Publisher Correction: The MHC-II peptidome of pancreatic islets identifies key features of autoimmune peptides.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The MHC-II peptidome of pancreatic islets identifies key features of autoimmune peptides.

The nature of autoantigens that trigger autoimmune diseases has been much discussed, but direct biochemical identification is lacking for most. Addressing this question demands unbiased examination of the self-peptides displayed by a defined autoimmune major histocompatibility complex class II (MHC-II) molecule. Here, we examined the immunopeptidome of the pancreatic islets in non-obese diabetic mice, which spontaneously develop autoimmune diabetes based on the I-Ag7 variant of MHC-II. The relevant peptides that induced pathogenic CD4+ T cells at the initiation of diabetes derived from proinsulin. These peptides were also found in the MHC-II peptidome of the pancreatic lymph nodes and spleen. The proinsulin-derived peptides followed a trajectory from their generation and exocytosis in ß cells to uptake and presentation in islets and peripheral sites. Such a pathway generated conventional epitopes but also resulted in the presentation of post-translationally modified peptides, including deamidated sequences. These analyses reveal the key features of a restricted component in the self-MHC-II peptidome that caused autoreactivity.

MHC-II neoantigens shape tumour immunity and response to immunotherapy.

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.

Position ß57 of I-Ag7 controls early anti-insulin responses in NOD mice, linking an MHC susceptibility allele to type 1 diabetes onset.

The class II region of the major histocompatibility complex (MHC) locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQß chains supports this association; this single amino acid change influences how TCRs recognize peptides in the context of HLA-DQ8 and I-Ag7 using a mechanism termed the P9 switch. Here, we built register-specific insulin peptide MHC tetramers to examine CD4+ T cell responses to Ins12-20 and Ins13-21 peptides during the early prediabetic phase of disease in nonobese diabetic (NOD) mice. A single-cell analysis of anti-insulin CD4+ T cells performed in 6- and 12-week-old NOD mice revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans and produced either classical TH1 differentiation or an unusual Treg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by T cells specific for Ins12-20, the register that supports a P9 switch mode of recognition. The presence of the P9 switch was demonstrated by TCR sequencing, reexpression, mutagenesis, and functional testing of TCRαß pairs in vitro. Genetic correction of the I-Aß57 mutation in NOD mice resulted in the disappearance of D/E residues in the CDR3ß of anti-Ins12-20 T cells. These results provide a mechanistic molecular explanation that links the characteristic MHC class II polymorphism of T1D with the recognition of islet autoantigens and disease onset.

The Immunoreactive Platform of the Pancreatic Islets Influences the Development of Autoreactivity.

Tissue homeostasis is maintained through a finely tuned balance between the immune system and the organ-resident cells. Disruption of this process not only results in organ dysfunction but also may trigger detrimental autoimmune responses. The islet of Langerhans consists of the insulin-producing ß-cells essential for proper control of body metabolism, but less appreciated is that these cells naturally interact with the immune system, forming a platform by which the ß-cell products are sensed, processed, and responded to by the local immune cells, particularly the islet-resident macrophages. Although its physiological outcomes are not completely understood, this immunoreactive platform is crucial for precipitating islet autoreactivity in individuals carrying genetic risks, leading to the development of type 1 diabetes. In this Perspective, we summarize recent studies that examine the cross talk between the ß-cells and various immune components, with a primary focus on discussing how antigenic information generated during normal ß-cell catabolism can be delivered to the resident macrophage and further recognized by the adaptive CD4 T-cell system, a critical step to initiate autoimmune diabetes. The core nature of the islet immune platform can be extrapolated to other endocrine tissues and may represent a common mechanism underlying the development of autoimmune syndromes influencing multiple endocrine organs.

Type I and II Interferon Receptors Differentially Regulate Type 1 Diabetes Susceptibility in Male Versus Female NOD Mice.

The role of interferons, either pathogenic or protective, during autoimmune diabetes remains controversial. Herein, we examine the progression of diabetes in NOD mice lacking the type I (IFNAR) or type II (IFNGR) interferon receptor and, for the first time, in mice deficient in both receptors (double knockout [DKO]). All mice were bred, maintained, and monitored in a single specific pathogen-free facility with high female and low male diabetes incidence. Our expectation was that removal of interferon signaling would reduce autoimmune destruction. However, examination of diabetes incidence in the IFNAR- and IFNGR-deficient NOD mice showed a reduction in females and an increase in males. In DKO mice, diabetes occurred only in female mice, at decreased incidence and with delayed kinetics. These results show that interferons act as both positive and negative modulators of type 1 diabetes disease risk dependent on sex.

Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides.

Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-214. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity3-5. Although presentation of B:12-20 is evident in the islets3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in ß-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.

The resident macrophages in murine pancreatic islets are constantly probing their local environment, capturing beta cell granules and blood particles.

AIMS/HYPOTHESIS: We studied here the interactions between the resident macrophages of pancreatic islets with beta cells and the blood vasculature. We also examined the immunological consequences of such interactions. METHODS: Islets were isolated from C57BL/6 mice expressing CX3C motif chemokine receptor 1-green fluorescent protein (CX3CR-GFP) and examined live by two-photon microscopy. Islets were also examined by electron microscopy to study the relationship of the intra-islet macrophages with the beta cells. In NOD.Rag1-/- mice and young (non-diabetic) male mice, the acquisition of beta cell granules was tested functionally by probing with CD4+ T cells directed against insulin epitopes. RESULTS: Two-photon microscopy showed that the islet resident macrophages were in close contact with blood vessels and had extensive filopodial activity. Some filopodia had direct access to the vessel lumen and captured microparticles. Addition of glucose at high concentration reduced the degree of filopodia sampling of islets. This finding applied to in vivo injection of glucose or to in vitro cultures. Ultrastructural examination showed the close contacts of macrophages with beta cells. Such macrophages contained intact dense core granules. Functional studies in NOD mice indicated that the macrophages presented insulin peptides to insulin-reactive T cells. Presentation was increased after glucose challenge either ex vivo or after an in vivo pulse. In agreement with the morphological findings, presentation was not affected by insulin receptor blockade. CONCLUSIONS/INTERPRETATION: Islet resident macrophages are highly active, sampling large areas of the islets and blood contents and capturing beta cell granules. After such interactions, macrophages present immunogenic insulin to specific autoreactive T cells.

Antigen recognition in autoimmune diabetes: a novel pathway underlying disease initiation.

Development of human autoimmune disorders results from complex interplay among genetic, environmental, and immunological risk factors. Despite much heterogeneity in environmental triggers, the leading genes that give the propensity for tissue-specific autoimmune diseases, such as type 1 diabetes, are those associated with particular class II major histocompatibility complex alleles. Such genetic predisposition precipitates presentation of tissue antigens to MHC-II-restricted CD4 T cells. When properly activated, these self-reactive CD4 T cells migrate to the target tissue and trigger the initial immune attack. Using the non-obese diabetic mouse model of spontaneous autoimmune diabetes, much insight has been gained in understanding how presentation of physiological levels of self-antigens translates into pathological outcomes. In this review, we summarize recent advances illustrating the features of the antigen presenting cells, the sites of the antigen recognition, and the nature of the consequent T cell responses. We emphasize emerging evidence that highlights the importance of systemic presentation of catabolized tissue antigens in mobilization of pathogenic T cells. The implication of these studies in therapeutic perspectives is also discussed.

Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice.

Treatment of C57BL/6 or NOD mice with a monoclonal antibody to the CSF-1 receptor resulted in depletion of the resident macrophages of pancreatic islets of Langerhans that lasted for several weeks. Depletion of macrophages in C57BL/6 mice did not affect multiple parameters of islet function, including glucose response, insulin content, and transcriptional profile. In NOD mice depleted of islet-resident macrophages starting at 3 wk of age, several changes occurred: (i) the early entrance of CD4 T cells and dendritic cells into pancreatic islets was reduced, (ii) presentation of insulin epitopes by dispersed islet cells to T cells was impaired, and (iii) the development of autoimmune diabetes was significantly reduced. Treatment of NOD mice starting at 10 wk of age, when the autoimmune process has progressed, also significantly reduced the incidence of diabetes. Despite the absence of diabetes, NOD mice treated with anti-CSF-1 receptor starting at 3 or 10 wk of age still contained variably elevated leukocytic infiltrates in their islets when examined at 20-40 wk of age. Diabetes occurred in the anti-CSF-1 receptor protected mice after treatment with a blocking antibody directed against PD-1. We conclude that treatment of NOD mice with an antibody against CSF-1 receptor reduced diabetes incidence and led to the development of a regulatory pathway that controlled autoimmune progression.

TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease.

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-ß pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

The islet-resident macrophage is in an inflammatory state and senses microbial products in blood.

We examined the transcriptional profiles of macrophages that reside in the islets of Langerhans of 3-wk-old non-obese diabetic (NOD), NOD.Rag1-/-, and B6.g7 mice. Islet macrophages expressed an activation signature with high expression of Tnf, Il1b, and MHC-II at both the transcript and protein levels. These features are common with barrier macrophages of the lung and gastrointestinal tract. Moreover, injection of lipopolysaccharide induced rapid inflammatory gene expression, indicating that blood stimulants are accessible to the macrophages and that these macrophages can sense them. In NOD mice, the autoimmune process imparted an increased inflammatory signature, including elevated expression of chemokines and chemokine receptors and an oxidative response. The elevated inflammatory signature indicates that the autoimmune program was active at the time of weaning. Thus, the macrophages of the islets of Langerhans are poised to mount an immune response even at steady state, while the presence of the adaptive immune system elevates their activation state.