Endogenous self-peptides guard immune privilege of the central nervous system.

The central nervous system (CNS), despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system1,2. In fact, it remains physically connected to and can be influenced by the peripheral immune system1. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding conundrum. In searching for molecular cues that derive from the CNS and allow its direct communication with the immune system, we discovered an endogenous repertoire of CNS-derived regulatory self-peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders. During homeostasis, these regulatory self-peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of regulatory self-peptides diminished. Upon boosting the presentation of these regulatory self-peptides, a population of suppressor CD4+ T cells was expanded, controlling CNS autoimmunity in a CTLA-4 and TGFß dependent manner. This unexpected discovery of CNS-derived autoimmune self-peptides may be the molecular key adapting the CNS to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.

Engineered T cell therapy for central nervous system injury.

Traumatic injuries to the central nervous system (CNS) afflict millions of individuals worldwide1, yet an effective treatment remains elusive. Following such injuries, the site is populated by a multitude of peripheral immune cells, including T cells, but a comprehensive understanding of the roles and antigen specificity of these endogenous T cells at the injury site has been lacking. This gap has impeded the development of immune-mediated cellular therapies for CNS injuries. Here, using single-cell RNA sequencing, we demonstrated the clonal expansion of mouse and human spinal cord injury-associated T cells and identified that CD4+ T cell clones in mice exhibit antigen specificity towards self-peptides of myelin and neuronal proteins. Leveraging mRNA-based T cell receptor (TCR) reconstitution, a strategy aimed to minimize potential adverse effects from prolonged activation of self-reactive T cells, we generated engineered transiently autoimmune T cells. These cells demonstrated notable neuroprotective efficacy in CNS injury models, in part by modulating myeloid cells via IFNγ. Our findings elucidate mechanistic insight underlying the neuroprotective function of injury-responsive T cells and pave the way for the future development of T cell therapies for CNS injuries.

Crinophagic granules in pancreatic ß cells contribute to mouse autoimmune diabetes by diversifying pathogenic epitope repertoire.

Autoimmune attack toward pancreatic ß cells causes permanent loss of glucose homeostasis in type 1 diabetes (T1D). Insulin secretory granules store and secrete insulin but are also thought to be tissue messengers for T1D. Here, we show that the crinophagic granules (crinosome), a minor set of vesicles formed by fusing lysosomes with the conventional insulin dense-core granules (DCG), are pathogenic in T1D development in mouse models. Pharmacological inhibition of crinosome formation in ß cells delays T1D progression without affecting the dominant DCGs. Mechanistically, crinophagy inhibition diminishes the epitope repertoire in pancreatic islets, including cryptic, modified and disease-relevant epitopes derived from insulin. These unconventional insulin epitopes are largely undetectable in the MHC-II epitope repertoire of the thymus, where only canonical insulin epitopes are presented. CD4+ T cells targeting unconventional insulin epitopes display autoreactive phenotypes, unlike tolerized T cells recognizing epitopes presented in the thymus. Thus, the crinophagic pathway emerges as a tissue-intrinsic mechanism that transforms insulin from a signature thymic self-protein to a critical autoantigen by creating a peripheral-thymic mismatch in the epitope repertoire.

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.

Improvement of Tumor Neoantigen Detection by High-Field Asymmetric Waveform Ion Mobility Mass Spectrometry.

Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.

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.

Recent advances in immunopeptidomic-based tumor neoantigen discovery.

The role of aberrantly expressed proteins in tumors in driving immune-mediated control of cancer has been well documented for more than five decades. Today, we know that both aberrantly expressed normal proteins as well as mutant proteins (neoantigens) can function as tumor antigens in both humans and mice. Next-generation sequencing (NGS) and high-resolution mass spectrometry (MS) technologies have made significant advances since the early 2010s, enabling detection of rare but clinically relevant neoantigens recognized by T cells. MS profiling of tumor-specific immunopeptidomes remains the most direct method to identify mutant peptides bound to cellular MHC. However, the need for use of large numbers of cells or significant amounts of tumor tissue to achieve neoantigen detection has historically limited the application of MS. Newer, more sensitive MS technologies have recently demonstrated the capacities to detect neoantigens from fewer cells. Here, we highlight recent advancements in immunopeptidomics-based characterization of tumor-specific neoantigens. Various tumor antigen categories and neoantigen identification approaches are also discussed. Furthermore, we summarize recent reports that achieved successful tumor neoantigen detection by MS using a variety of starting materials, MS acquisition modes, and novel ion mobility devices.

Using mass spectrometry to identify neoantigens in autoimmune diseases: The type 1 diabetes example.

In autoimmune diseases, recognition of self-antigens presented by major histocompatibility complex (MHC) molecules elicits unexpected attack of tissue by autoantibodies and/or autoreactive T cells. Post-translational modification (PTM) may alter the MHC-binding motif or TCR contact residues in a peptide antigen, transforming the tolerance to self to autoreactivity. Mass spectrometry-based immunopeptidomics provides a valuable mechanism for identifying MHC ligands that contain PTMs and can thus provide valuable insights into pathogenesis and therapeutics of autoimmune diseases. A plethora of PTMs have been implicated in this process, and this review highlights their formation and identification.

Navigating Critical Challenges Associated with Immunopeptidomics-Based Detection of Proteasomal Spliced Peptide Candidates.

Within the tumor immunology community, the topic of proteasomal spliced peptides (PSP) has generated a great deal of controversy. In the earliest reports, careful biological validation led to the conclusion that proteasome-catalyzed peptide splicing was a rare event. To date, six PSPs have been validated biologically. However, the advent of algorithms to identify candidate PSPs in mass spectrometry data challenged this notion, with several studies concluding that the frequency of spliced peptides binding to MHC class I was quite high. Since this time, much debate has centered around the methodologies used in these studies. Several reanalyses of data from these studies have led to questions about the validity of the conclusions. Furthermore, the biological and technical validation that should be necessary for verifying PSP assignments was often lacking. It has been suggested therefore that the research community should unite around a common set of standards for validating candidate PSPs. In this review, we propose and highlight the necessary steps for validation of proteasomal splicing at both the mass spectrometry and biological levels. We hope that these guidelines will serve as a foundation for critical assessment of results from proteasomal splicing studies.

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.

Identification of spliced peptides in pancreatic islets uncovers errors leading to false assignments.

Proteasomal spliced peptides (PSPs) have been identified in the class I major histocompatibility complex (MHC) peptidomes of several tumors and have emerged as novel neoantigens that can stimulate highly specific T cells. Much debate has surrounded the percentage of PSPs in the immunopeptidome; reported numbers have ranged from <1-5% to 12-45%. Recently, our laboratory demonstrated in nonobese diabetic (NOD) mice that hybrid insulin peptides (HIPs), a special class of spliced peptides, are formed during insulin granule degradation in crinosomes of the pancreatic ß cells and that modified peptides comprised a significant source of false positive HIP assignments. Herein, this study is extended to crinosomes isolated from other mouse strains and to two recent MHC class I studies, to see if modified peptides explained discrepancies in reported percentages of PSPs. This analysis revealed that both MHC-I peptidomes contained many spectra erroneously assigned as PSPs. While many false positive PSPs did arise from modified peptides, others arose from probable data processing errors. Thus, the reported numbers of PSPs in the literature are likely elevated due to errors associated with data processing and analysis.

Dopamine-induced interactions of female mouse hypothalamic proteins with progestin receptor-A in the absence of hormone.

Neural progestin receptors (PR) function in reproduction, neural development, neuroprotection, learning, memory and the anxiety response. In the absence of progestins, PR can be activated by dopamine (DA) in the rodent hypothalamus to elicit female sexual behaviour. The present study investigated mechanisms of DA activation of PR by testing the hypothesis that proteins from DA-treated hypothalami interact with PR in the absence of progestins. Ovariectomised, oestradiol-primed mice were infused with a D1-receptor agonist, SKF38393 (SKF), into the third ventricle 30 minutes prior to death. Proteins from SKF-treated hypothalami were pulled-down with glutathione S-transferase-tagged mouse PR-A or PR-B and the interactomes were analysed by mass spectrometry. The largest functional group to interact with PR-A in a DA-dependent manner was synaptic proteins. To test the hypothesis that DA activation of PR regulates synaptic proteins, we developed oestradiol-induced PR-expressing hypothalamic-like neurones derived from human-induced pluripotent stem cells (hiPSCs). Similar to progesterone (P4), SKF treatment of hiPSCs increased synapsin1/2 expression. This SKF-dependent effect was blocked by the PR antagonist RU486, suggesting that PR are necessary for this DA-induced increase. The second largest DA-dependent PR-A protein interactome comprised metabolic regulators involved in glucose metabolism, lipid synthesis and mitochondrial energy production. Interestingly, hypothalamic proteins interacted with PR-A, but not PR-B, in an SKF-dependent manner, suggesting that DA promotes the interaction of multiple hypothalamic proteins with PR-A. These in vivo and in vitro results indicate novel mechanisms by which DA can differentially activate PR isoforms in the absence of P4 and provide a better understanding of ligand-independent PR activation in reproductive, metabolic and mental health disorders in women.

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.

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.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway.

Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3ß (GSK3ß) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3ß and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3ß and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3ß prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3ß with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3ß. A GSK3ß-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3ß regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.

Large Scale Identification of Variant Proteins in Glioma Stem Cells.

Glioblastoma (GBM), the most malignant of primary brain tumors, is a devastating and deadly disease, with a median survival of 14 months from diagnosis, despite standard regimens of radical brain tumor surgery, maximal safe radiation, and concomitant chemotherapy. GBM tumors nearly always re-emerge after initial treatment and frequently display resistance to current treatments. One theory that may explain GBM re-emergence is the existence of glioma stemlike cells (GSCs). We sought to identify variant protein features expressed in low passage GSCs derived from patient tumors. To this end, we developed a proteomic database that reflected variant and nonvariant sequences in the human proteome, and applied a novel retrograde proteomic workflow, to identify and validate the expression of 126 protein variants in 33 glioma stem cell strains. These newly identified proteins may harbor a subset of novel protein targets for future development of GBM therapy.

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase-tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I-positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.