Antigen cross-presentation is a functional feature of some specialized antigen-presenting cells, allowing them to internalize and process antigens from extracellular sources and present them via major histocompatibility class I (MHC-I) molecules to CD8+ T lymphocytes. This process is critical for the mounting of protective immunity to tumors and intracellular pathogens. Antigen cross-presentation can be monitored through in vivo and in vitro assays that are key to evaluate the efficiency and the molecular and cellular mechanisms of cross-presentation by a given cell type. This chapter intends to complement published protocols by describing how to use MutuDC, a murine immortalized type 1 conventional dendritic cell-like line, and a "nonprofessional" human antigen-presenting cell, the human embryonic kidney HEK 293T cell line, endowed with the ability to cross-present immune complexes through overexpression of an Fc receptor, for in vitro antigen cross-presentation assays.

Polymorphism in endoplasmic reticulum antigen processing peptidases (ERAAP) that trim MHC class I ligands is associated with autoimmune diseases, but the mechanism is unknown. We analyzed the effect of Eraap deficiency in the non-obese diabetic (NOD) mouse model of type 1 diabetes. Eraap -/- NOD mice displayed reduced and delayed diabetes, harbored splenic effector T cells unable to transfer diabetes, and exhibited a strong shift from effector to central memory T cells in attenuated islet infiltrates. Eraap deficiency increased presentation of the immunodominant epitope insulin B 15-23 by beta cells but at the same time provided complete protection from diabetes to chimeras reconstituted with bone marrow encoding a CD8 + T cell recognizing this epitope. These results underline the strong impact of self-antigen presentation to CD8 + T cells in diabetes. At the same time, they highlight the complex consequences of interfering with MHC-I antigen presentation in autoimmunity and advise caution in therapeutic modulation of ERAP activity in this context.

The antigen presentation machinery processes proteins for presentation to T cells, thereby controlling activation of the adaptive cellular immune response. Perturbation of this machinery has been linked to the development of various diseases. This review describes the function of the Major Histocompatibility Complex class I antigen presentation machinery and highlights how its perturbation can lead to compromised immune function and disease progression in the context of cancer. We categorize these perturbations into four distinct mechanistic levels: peptide generation, peptide loading, MHC class I integrity, and epigenetic regulation. This enables an integrated view of their functional impact on immune recognition, supporting therapeutic efforts to target antigen presentation or exploit these alterations in cancer.

The autonomic nervous system (ANS) regulates myeloid cell biology across lymphoid and non-lymphoid tissues. Sympathetic, parasympathetic, and enteric circuits shape myeloid cell development, positioning or activation, and antigen presentation through norepinephrine, acetylcholine, dopamine and neuropeptides. In primary lymphoid organs, autonomic signals regulate myeloid progenitors and thymic macrophages, shaping the output and functional competence of innate immune compartments. In secondary lymphoid organs, most extensively characterized in the spleen, neural efferent pathways control macrophages, dendritic cells, monocytes and neutrophils, influencing phagocytosis, cytokine production and lymphocyte priming. Distinct neuro-myeloid interactions occur in the gut, where the enteric nervous system is a mandatory relay for parasympathetic signals to intestinal myeloid cells, and in the tumor microenvironment, where sympathetic and dopaminergic signals regulate tumor-associated myeloid subsets in tumor type-specific ways. The functional output of ANS-myeloid communication is guided by tissue context, myeloid cell identity, receptor type, and signal dynamics. The systemic characterization of these neuroimmune variables will be critical to design therapeutic strategies targeting the ANS-myeloid axis in infection, inflammation, autoimmunity and cancer.

BACKGROUND AND PURPOSE:Multiple myeloma (MM) patients frequently develop resistance to proteasome inhibitors (PIs). Identifying novel, targetable pathways to restore drug potency is of utmost importance. EXPERIMENTAL APPROACH:Insulin-degrading enzyme (IDE) regulates the activity of the proteasome. We hypothesised that targeting IDE could boost MM response to PIs. We have interrogated the transcriptomic data of a cohort of MM patients and developed novel IDE inhibitors. X-ray crystallography, enzymatic assays and engagement studies showed that these inhibitors bind IDE with high affinity. We treated MM cell lines, and primary MM cells from newly diagnosed or relapsing patients, with IDE inhibitors and PIs to measure their synergy in inducing apoptosis. We used transcriptomic analysis to describe the underlying molecular mechanism. We confirmed these results in a mouse model of MM. KEY RESULTS:Low expression of IDE is associated with a higher overall survival in MM patients. When MM cells are treated with IDE inhibitors at a concentration that fully engages IDE, the chymotrypsin activity of the proteasome is more sensitive to PI. Their combination reduces c-Myc and induces integrated stress response and DNA damage. Whilst sparing the tumour micro-environment, they enhanced the cytotoxicity of PI in resistant MM cell lines and cells from relapsing patients. Finally, an optimised IDE inhibitor boosts bortezomib in a syngeneic, immunocompetent MM mouse model. CONCLUSIONS AND IMPLICATIONS:IDE emerges as a novel biomarker and therapeutic vulnerability in MM. This study demonstrates the relevance of IDE inhibitors as boosters of PIs to better treat patients with resistant MM.

The islet immune microenvironment contributes critically to β cell dysfunction in type 2 diabetes (T2D), but its regulatory mechanisms remain unclear. We show that β cell dysfunction in T2D patients and diabetic mice correlates with elevated nucleolar stress and reduced expression of BAF60C, a switching defective/sucrose nonfermenting (SWI/SNF) chromatin-remodeling factor. β cell-specific BAF60C deletion aggravates high-fat diet (HFD)-induced hyperglycemia, nucleolar stress, and islet inflammation, whereas BAF60C overexpression displays protection. BAF60C suppresses islet inflammation by promoting REG3B expression and secretion, thereby modulating β cell-macrophage crosstalk. Mechanistically, BAF60C forms an RNA-protein complex with nucleophosmin (NPM1) and Reg3b mRNA to modulate Reg3b mRNA decay. Restoration of the BAF60C-REG3B axis through REG3B supplementation or exercise alleviates inflammation and improves glucose homeostasis in obese and T2D mice, revealing a non-canonical role for BAF60C in linking nucleolar stress to β cell failure.

Autoimmune type 1 diabetes (T1D) results from the failure of the physiologic regulatory mechanisms that are designed to maintain immune tolerance to pancreatic beta cells. Consequently, the design of strategies to restore tolerance to beta cell antigens is an attractive objective of translational research. We have designed ultrasmall nanoparticles (NPs) loaded with a proinsulin (PI) fusion protein and an agonist for the aryl hydrocarbon receptor (AhR), a transcription factor promoting tolerance induction by different immune cells. We report that a 4 week-treatment with these NPs in non-obese diabetic (NOD) mice starting at disease onset induces temporary and sometimes durable disease remission. Mechanistically, short-term NP treatment induces a rapid depletion of islet infiltrates with a dramatic reduction in the number of CD8+ T cells and dendritic cells. This is accompanied by the emergence of B lymphocytes producing IL-10. In the rare mice that undergo durable disease remission, the disappearance of islet infiltrates is associated with the emergence of Foxp3+ CD4+ regulatory T cells, IFN-γ-producing memory T cells in the spleen, and draining lymph nodes (LNs). We conclude that treatment with these NPs could be of interest in the treatment of recent-onset autoimmune diabetes, but is unlikely to be sufficient for the induction of long-term remission as a stand-alone therapy.

MHC class I (MHC-I) cross-presentation involves final proteolytic peptide processing by the endosomal insulin-regulated aminopeptidase (IRAP). Reasoning that analysis of the IRAP-proximal proteome may inform about dynamic remodeling of a key cross-presentation compartment during antigen uptake, we developed a proximity biotinylation system by expressing an IRAP-TurboID fusion protein in MuTuDCs, a cell line resembling murine type 1 conventional dendritic cells. Analysis of luminal proteins associated with IRAP at steady state and during phagocytosis revealed a massive shift upon uptake of yeast but not apoptotic cells, favoring enrichment of antigen-processing machinery, MHC-I molecules, and proteins involved in ER-associated folding and trafficking. Importantly, Sec22b modulated this proteomic landscape, promoting the localization of MHC-I cross-presentation proteins (e.g., MHC-I, Tap1, Wdfy4, transferrin receptor) to the IRAP environment, while its absence favored MHC-II and ER-related proteins, suggesting a Sec22b-dependent dichotomy between pathways favoring cross-presentation versus antigen degradation. Intriguingly, uptake of apoptotic cells failed to promote cross-presentation but induced two proteins related to immune tolerance, suggesting potential adaptation of proteome modulation to the outcome of antigen presentation. These data suggest that IRAP(+) endosomes serve as adaptive hubs integrating secretory and endocytic pathways, with Sec22b acting as a key determinant in tailoring this compartment for MHC-I-mediated cross-presentation.

Peptide presentation by major histocompatibility complex (MHC) class I molecules enables CD8+ T lymphocytes to monitor the intracellular proteome of tissue cells. CD8+ T cell priming and acquisition of effector functions is affected by cognate peptide-MHC-I complex density on the cell surface, which partly depends on the efficacy of intracellular proteolytic peptide generation. Peptide generation frequently requires final trimming by the human aminopeptidases ERAP1, ERAP2, and IRAP. All display genetic polymorphism associated with the risk of multiple autoimmune diseases but also some cancers. This finding has prompted interest in the development of small molecule inhibitors to enhance antitumor or conversely attenuate autoreactive T cell responses. However, efficient assays for assessment of inhibitor effects are wanting. We describe the development of an assay for quantitative assessment by flow cytometry of selective inhibitor effects on peptide trimming both in the endogenous MHC-I processing pathway and in cross-presentation. We use the assay to identify a selective ERAP2 inhibitor and show that inhibitor effects can be read out not only through assessing a specific peptide-MHC complexes but also by measuring cell surface levels of bulk MHC-I molecules. Next to its practical interest as tool for inhibitor testing, our assay highlights how ERAP1-dependent immunodominance of a single epitope processed with exceptional efficacy can have a massive effect on the immunopeptidomic identity of cells presented to CD8+ T cells. We propose that ERAP effects on the presentation of such rare and exceptionally immunodominant epitopes may underlie the epistatic genetic associations of ERAP polymorphism with HLA class I-linked autoimmune diseases.

Macrophages expressing the sialic acid-binding lectin Siglec-1 are positioned strategically for uptake and filtering of antigenic material circulating through the lymphatic system into the subcapsular sinus of lymph nodes and through the blood into the marginal zone of the spleen. Siglec-1+ macrophages are also found in many tissues, frequently displaying an inflammatory profile. In secondary lymphoid organs, these cells are strategically positioned and play a role in adaptive immune responses by B, NK and T lymphocytes. A potential role of the cells in antitumor immunity has attracted significant scientific interest. This concept is supported by a report of dendritic cell-independent priming of a protective antitumor response by lymph node Siglec-1+ macrophages. Indeed, examination of preclinical models and human tumors has provided evidence for a dominant protective effect of lymph node Siglec-1+ macrophages in major types of solid tumors e.g. breast or colorectal cancer. In contrast, Siglec-1+ macrophages within tumor tissue have been found to be associated variably with favorable or unfavorable outcome depending on the model and tumor studied. We have recently demonstrated in mouse models that splenic Siglec-1+ macrophages in themarginal zone can prime protective tumor immunity against both tumors disseminating through the blood and tumors invading the spleen in the absence of type 1 conventional dendritic cells. Considering this, we postulate Siglec-1+ macrophages in all secondary lymphoid organs can prime potent antitumor responses. We propose that this capacity could be exploited for therapeutic stimulation of tumor immunity.

Splenic metallophilic marginal zone macrophages (MMMs) are positioned to control the dissemination of blood-borne threats. We developed a purification protocol to enable characterization of MMMs phenotypically and transcriptionally. MMM gene expression profile was enriched for pathways associated with CD8+ T cell activation and major histocompatibility complex class I (MHC class I) cross-presentation. In vitro, purified MMMs equaled conventional dendritic cells type 1 (cDC1s) in cross-priming CD8+ T cells to soluble and particulate antigens, yet MMMs employed a distinct vacuolar processing pathway. In vivo biphoton and ex vivo light-sheet imaging showed long-standing contacts with cognate T cells differentiating to effectors. MMMs cross-primed protective CD8+ T cell antitumor responses both by capturing blood-borne tumor antigens and by internalizing tumor cells seeding the spleen. This cross-priming required expression of the transcription factor Batf3 by MMMs but was independent of cDC1-mediated capture of tumor material for cross-presentation or MHC class I-dressing. Thus, MMMs combine control of the dissemination of blood-borne pathogens and tumor materials with the initiation of innate and adaptive responses.

Among the M1 family of oxytocinase aminopeptidases, insulin-regulated aminopeptidase IRAP, is an emerging drug target implicated in various biological pathways and particularly in MHC-I antigen presentation through amino-terminal trimming of exogenous cross-presented peptides. A few series of inhibitors inspired either by angiotensin IV, one of IRAP substrates, or by bestatin a pan aminopeptidase inhibitor, have been disclosed. However, the variety and number of chemotypes remains relatively limited. Here we disclose the design and optimization of a series of hydroxamic acids IRAP inhibitors bearing a 5-substituted indole. Docking studies of the best compound 43 (BDM_92499), a single-digit nanomolar and selective inhibitor of IRAP, suggest an original binding mode and highlight the substituent on the indole and a primary amide as groups driving selectivity. Several inhibitors in the series displayed IRAP-dependent inhibition of antigen cross-presentation. These results pave the way to the development of novel therapeutic agents targeting IRAP.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

Type 1 diabetes results from the destruction of pancreatic beta cells by autoreactive T cells. As an autoantigen with extremely high expression in beta cells, insulin triggers and sustains the autoimmune CD4+ and CD8+ T cell responses and islet inflammation. We have previously shown that deficiency for insulin-degrading enzyme (IDE), a ubiquitous cytosolic protease with very high affinity for insulin, induces endoplasmic reticulum (ER) stress and proliferation in islet cells and protects non-obese diabetic mice (NOD) from diabetes. Here we wondered whether IDE deficiency affects autoreactive CD8+ T cell responses to insulin and thereby immune pathogenesis in NOD mice. We find that Ide-/- NOD harbor fewer diabetogenic T cells and reduced numbers of CD8+ T cells recognizing the dominant autoantigen insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Using in vitro digestions and cellular antigen presentation assays, we show that generation of the dominant insulin epitope B15-23 involves both the proteasome and IDE. IDE deficiency attenuates MHC-I presentation of the immunodominant insulin epitope by beta cells to cognate CD8+ T cells. Consequently, Ide-/- islets display reduced susceptibility to autoimmune destruction upon grafting, and to killing by insulin-specific CD8+ T cells. Moreover, Ide-/- mice are partly resistant to disease transfer by CD8+ T cells specific for insulin but not for IGRP. Thus, IDE has a dual role in beta cells, regulating ER stress and proliferation while at the same time promoting insulin-directed autoreactive CD8+ T cell responses.

The enzyme, insulin regulated aminopeptidase (IRAP), is expressed in multiple immune cells such as macrophages, dendritic cells and T cells, where it plays a role in regulating the innate and adaptive immune response. There is a genetic association between IRAP and survival outcomes in patients with septic shock where a variant of its gene was found to be associated with increased 28-day mortality. This study investigated the role for IRAP in a lipopolysaccharide (LPS)-induced inflammatory response which is thought to model facets of the systemic inflammation observed in the early stages of human gram-negative sepsis. The frequencies and activation of splenic immune cell populations were investigated in the IRAP knockout (KO) mice compared to the wildtype controls over a period of 4-, 24-, or 48-hours following LPS stimulation. Dendritic cells isolated from the spleen of female IRAP KO mice, displayed significant increases in the activation markers CD40, CD86 and MHCII at 24 hours after LPS induction. A modest heightened pro-inflammatory response to LPS was observed with increased expression of activation marker CD40 in M1 macrophages from male IRAP knockout mice. Observations in vitro in bone marrow-derived macrophages (BMDM) revealed a heightened pro-inflammatory response to LPS with significant increases in the expression of CD40 in IRAP deficient cells compared with BMDM from WT mice. The heightened LPS-induced response was associated with increased pro-inflammatory cytokine secretion in these BMDM cells. A genotype difference was also detected in the BMDM from female mice displaying suppression of the LPS-induced increases in the activation markers CD40, CD86, CD80 and MHCII in IRAP deficient cells. Thus, this study suggests that IRAP plays specific time- and sex-dependent roles in the LPS-induced inflammatory response in dendritic cells and macrophages.

Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis episodes precipitated by fasting. Autophagy functioning was analyzed in vitro, in primary skeletal myoblasts from TANGO2 patients, in basal and fasting conditions, and TANGO2 mutations were associated with reduced LC3-II levels upon starvation. In zebrafish larvae, tango2 inhibition induced locomotor defects which were exacerbated by exposure to atorvastatin, a compound known to cause rhabdomyolysis. Importantly, rhabdomyolysis features of tango2 knockdown were associated with autophagy and mitophagy defects in zebrafish. Calpeptin treatment was sufficient to rescue the locomotor properties thanks to its beneficial effect on autophagy functioning in zebrafish and to improve LC3-II levels in starved primary muscle cells of TANGO2 patients. Overall, we demonstrated that TANGO2 plays an important role in autophagy thus giving rise to new therapeutic perspectives in the prevention of RM life-threatening episodes.

The development of the human immune system lasts for several years after birth. The impact of this maturation phase on the quality of adaptive immunity and the acquisition of immunological memory after infection at a young age remains incompletely defined. Here, using an antigen-reactive T cell (ARTE) assay and multidimensional flow cytometry, we profiled circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-reactive CD3(+)CD4(+)CD154(+) T cells in children and adults before infection, during infection, and 11 months after infection, stratifying children into separate age groups and adults according to disease severity. During SARS-CoV-2 infection, children younger than 5 years old displayed a lower antiviral CD4(+) T cell response, whereas children older than 5 years and adults with mild disease had, quantitatively and phenotypically, comparable virus-reactive CD4(+) T cell responses. Adults with severe disease mounted a response characterized by higher frequencies of virus-reactive proinflammatory and cytotoxic T cells. After SARS-CoV-2 infection, preschool-age children not only maintained neutralizing SARS-CoV-2-reactive antibodies postinfection comparable to adults but also had phenotypically distinct memory T cells displaying high inflammatory features and properties associated with migration toward inflamed sites. Moreover, preschool-age children had markedly fewer circulating virus-reactive memory B cells compared with the other cohorts. Collectively, our results reveal unique facets of antiviral immunity in humans at a young age and indicate that the maturation of adaptive responses against SARS-CoV-2 toward an adult-like profile occurs in a progressive manner.

Background and PurposeNonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin‐degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo‐endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR.Experimental ApproachWe investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress‐induced UPR and lipid accumulation in the liver.Key ResultsWe found that IDE‐deficient mice display higher hepatic triglyceride content along with higher inositol‐requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress‐induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis.Conclusion and ImplicationsThese results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.