TOFIMS mass spectrometry-based immunopeptidomics refines tumor antigen identification.

T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the implementation of ion mobility separation-based time-of-flight (TOFIMS) MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. Applying TOFIMS-based immunopeptidomics, a novel extensive benignTOFIMS dataset was generated from 94 primary benign samples of solid tissue and hematological origin, which enabled the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides, the refinement of previously described tumor antigens, as well as the identification of frequently presented self antigens and not yet described tumor antigens comprising low abundant mutation-derived neoepitopes that might serve as targets for future cancer immunotherapy development.

A T-cell antigen atlas for meningioma: novel options for immunotherapy.

Meningiomas are the most common primary intracranial tumors. Although most symptomatic cases can be managed by surgery and/or radiotherapy, a relevant number of patients experience an unfavorable clinical course and additional treatment options are needed. As meningiomas are often perfused by dural branches of the external carotid artery, which is located outside the blood-brain barrier, they might be an accessible target for immunotherapy. However, the landscape of naturally presented tumor antigens in meningioma is unknown. We here provide a T-cell antigen atlas for meningioma by in-depth profiling of the naturally presented immunopeptidome using LC-MS/MS. Candidate target antigens were selected based on a comparative approach using an extensive immunopeptidome data set of normal tissues. Meningioma-exclusive antigens for HLA class I and II are described here for the first time. Top-ranking targets were further functionally characterized by showing their immunogenicity through in vitro T-cell priming assays. Thus, we provide an atlas of meningioma T-cell antigens which will be publicly available for further research. In addition, we have identified novel actionable targets that warrant further investigation as an immunotherapy option for meningioma.

Microglia Transcriptional Profiling in Major Depressive Disorder Shows Inhibition of Cortical Gray Matter Microglia.

BACKGROUND: Microglia have been implicated in the pathophysiology of major depressive disorder (MDD), but information on biological mechanisms is limited. Therefore, we investigated the gene expression profile of microglial cells in relation to neuronal regulators of microglia activity in well-characterized MDD and control autopsy brains. METHODS: Pure, intact microglia were isolated at brain autopsy from occipital cortex gray matter (GM) and corpus callosum white matter of 13 donors with MDD and 10 age-matched control donors for RNA sequencing. Top differentially expressed genes were validated using immunohistochemistry staining. Because gene expression changes were only detected in GM microglia, neuronal regulators of microglia were investigated in cortical tissue and synaptosomes from the cortex by reverse transcriptase-quantitative polymerase chain reaction and Western blot. RESULTS: Transcriptome analysis revealed 92 genes differentially expressed in microglia isolated from GM, but none in microglia from white matter in donors with MDD, compared with control donors. Of these, 81 genes were less abundantly expressed in GM in MDD, including CD163, MKI67, SPP1, CD14, FCGR1A/C, and C1QA/B/C. Accordingly, pathways related to effector mechanisms, such as the complement system and phagocytosis, were differentially regulated in GM microglia in MDD. Immunohistochemistry staining revealed significantly lower expression of CD163 protein in MDD. Whole tissue analysis showed an increase in CD200 (p = .0009) and CD47 (p = .068) messenger RNA, and CD47 protein was significantly elevated (p = .0396) in synaptic fractions of MDD cases. CONCLUSIONS: Transcriptional profiling indicates an immune-suppressed microglial phenotype in MDD that is possibly caused by neuronal regulation.

The HLA ligandome of oropharyngeal squamous cell carcinomas reveals shared tumour-exclusive peptides for semi-personalised vaccination.

BACKGROUND: The immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies. METHODS: Mapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPV-positive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens. RESULTS: MS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse. CONCLUSION: Tumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi)personalised strategies.

Characterizing microglial gene expression in a model of secondary progressive multiple sclerosis.

Multiple sclerosis (MS) is the most common inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. Chronic-relapsing experimental autoimmune encephalomyelitis (crEAE) in Biozzi ABH mice is an experimental model of MS. This crEAE model is characterized by an acute phase with severe neurological disability, followed by remission of disease, relapse of neurological disease and remission that eventually results in a chronic progressive phase that mimics the secondary progressive phase (SPEAE) of MS. In both MS and SPEAE, the role of microglia is poorly defined. We used a crEAE model to characterize microglia in the different phases of crEAE phases using morphometric and RNA sequencing analyses. At the initial, acute inflammation phase, microglia acquired a pro-inflammatory phenotype. At the remission phase, expression of standard immune activation genes was decreased while expression of genes associated with lipid metabolism and tissue remodeling were increased. Chronic phase microglia partially regain inflammatory gene sets and increase expression of genes associated with proliferation. Together, the data presented here indicate that microglia obtain different features at different stages of crEAE and a particularly mixed phenotype in the chronic stage. Understanding the properties of microglia that are present at the chronic phase of EAE will help to understand the role of microglia in secondary progressive MS, to better aid the development of therapies for this phase of the disease.

Increased soluble HLA in COVID-19 present a disease-related, diverse immunopeptidome associated with T cell immunity.

HLA-presented antigenic peptides are central components of T cell-based immunity in infectious disease. Beside HLA molecules on cell surfaces, soluble HLA molecules (sHLA) are released in the blood suggested to impact cellular immune responses. We demonstrated that sHLA levels were significantly increased in COVID-19 patients and convalescent individuals compared to a control cohort and positively correlated with SARS-CoV-2-directed cellular immunity. Of note, patients with severe courses of COVID-19 showed reduced sHLA levels. Mass spectrometry-based characterization of sHLA-bound antigenic peptides, the so-called soluble immunopeptidome, revealed a COVID-19-associated increased diversity of HLA-presented peptides and identified a naturally presented SARS-CoV-2-derived peptide from the viral nucleoprotein in the plasma of COVID-19 patients. Of interest, sHLA serum levels directly correlated with the diversity of the soluble immunopeptidome. Together, these findings suggest an inflammation-driven release of sHLA in COVID-19, directly influencing the diversity of the soluble immunopeptidome with implications for SARS-CoV-2-directed T cell-based immunity and disease outcome.

Epigenetic regulation of innate immune memory in microglia.

BACKGROUND: Microglia are the tissue-resident macrophages of the CNS. They originate in the yolk sac, colonize the CNS during embryonic development and form a self-sustaining population with limited turnover. A consequence of their relative slow turnover is that microglia can serve as a long-term memory for inflammatory or neurodegenerative events. METHODS: Using ATAC-, ChIP- and RNA-sequencing, we characterized the epigenomes and transcriptomes of FACS-purified microglia from mice exposed to different stimuli. A repeated endotoxin challenge (LPS) was used to induce tolerance in microglia, while genotoxic stress (DNA repair deficiency-induced accelerated aging through Ercc1 deficiency) resulted in primed (hypersensitive) microglia. RESULTS: Whereas the enrichment of permissive epigenetic marks at enhancer regions could explain training (hyper-responsiveness) of primed microglia to an LPS challenge, the tolerized response of microglia seems to be regulated by loss of permissive epigenetic marks. We identify that inflammatory stimuli and accelerated aging as a result of genotoxic stress activate distinct gene networks. These gene networks and associated biological processes are partially overlapping, which is likely driven by specific transcription factor networks, resulting in altered epigenetic signatures and distinct functional (desensitized vs. primed) microglia phenotypes. CONCLUSION: This study provides insight into epigenetic profiles and transcription factor networks associated with transcriptional signatures of tolerized and trained microglia in vivo, leading to a better understanding of innate immune memory of microglia.

Preexisting and Post-COVID-19 Immune Responses to SARS-CoV-2 in Patients with Cancer.

Patients with cancer, in particular patients with hematologic malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4+ and CD8+ T-cell responses in unexposed and SARS-CoV-2-infected patients with cancer to characterize SARS-CoV-2 immunity and to identify immunologic parameters contributing to COVID-19 outcome. Unexposed patients with hematologic malignancies presented with reduced prevalence of preexisting SARS-CoV-2 cross-reactive CD4+ T-cell responses and signs of T-cell exhaustion compared with patients with solid tumors and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between patients with COVID-19 and cancer and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T-cell responses were profoundly reduced in patients with cancer, and the latter associated with a severe course of COVID-19. This identifies impaired SARS-CoV-2 T-cell immunity as a potential determinant for dismal outcome of COVID-19 in patients with cancer. SIGNIFICANCE: This first comprehensive analysis of SARS-CoV-2 immune responses in patients with cancer reports on the potential implications of impaired SARS-CoV-2 T-cell responses for understanding pathophysiology and predicting severity of COVID-19, which in turn might allow for the development of therapeutic measures and vaccines for this vulnerable patient population.See related commentary by Salomé and Horowitz, p. 1877.This article is highlighted in the In This Issue feature, p. 1861.

Intrinsic DNA damage repair deficiency results in progressive microglia loss and replacement.

The DNA excision repair protein Ercc1 is important for nucleotide excision, double strand DNA break, and interstrand DNA crosslink repair. In constitutive Ercc1-knockout mice, microglia display increased phagocytosis, proliferation and an enhanced responsiveness to lipopolysaccharide (LPS)-induced peripheral inflammation. However, the intrinsic effects of Ercc1-deficiency on microglia are unclear. In this study, Ercc1 was specifically deleted from Cx3cr1-expressing cells and changes in microglia morphology and immune responses at different times after deletion were determined. Microglia numbers were reduced with approximately 50% at 2-12 months after Ercc1 deletion. Larger and more ramified microglia were observed following Ercc1 deletion both in vivo and in organotypic hippocampal slice cultures. Ercc1-deficient microglia were progressively lost, and during this period, microglia proliferation was transiently increased. Ercc1-deficient microglia were gradually replaced by nondeficient microglia carrying a functional Ercc1 allele. In contrast to constitutive Ercc1-deficient mice, microglia-specific deletion of Ercc1 did not induce microglia activation or increase their responsiveness to a systemic LPS challenge. Gene expression analysis suggested that Ercc1 deletion in microglia induced a transient aging signature, which was different from a priming or disease-associated microglia gene expression profile.

The effects of postmortem delay on mouse and human microglia gene expression.

Microglia are specialized macrophages of the central nervous system (CNS) and first to react to pathogens or injury. Over the last decade, transcriptional profiling of microglia significantly contributed to our understanding of their functions. In the case of human CNS samples, either potential CNS pathology in the case of surgery samples, or a postmortem delay (PMD) due to the time needed for tissue access and collection, are potential factors that affect gene expression profiles. To determine the effect of PMD on the microglia transcriptome, we first analyzed mouse microglia, where genotype, antemortem conditions and PMD can be controlled. Microglia were isolated from mice after different PMDs (0, 4, 6, 12, and 24 hr) using fluorescence-activated cell sorting (FACS). The number of viable microglia significantly decreased with increasing PMD, but even after a 12 hr PMD, high-quality RNA could be obtained. PMD had very limited effect on mouse microglia gene expression, only 50 genes were differentially expressed between different PMDs. These genes were related to mitochondrial, ribosomal, and protein binding functions. In human microglia transcriptomes we previously generated, 31 of the 50 PMD-associated mouse genes had human homologs, and their relative expression was also affected by PMD. This study provides a set of genes that shows relative expression changes in relation to PMD, both in mouse and human microglia. Although the gene expression changes detected are subtle, these genes need to be accounted for when analyzing microglia transcriptomes generated from samples with variable PMDs.

Transcriptional heterogeneity between primary adult grey and white matter astrocytes underlie differences in modulation of in vitro myelination.

BACKGROUND: Multiple sclerosis (MS) is an inflammation-mediated demyelinating disease of the central nervous system that eventually results in secondary axonal degeneration due to remyelination failure. Successful remyelination is orchestrated by astrocytes (ASTRs) and requires sequential activation, recruitment, and maturation of oligodendrocyte progenitor cells (OPCs). In both MS and experimental models, remyelination is more robust in grey matter (GM) than white matter (WM), which is likely related to local differences between GM and WM lesions. Here, we investigated whether adult gmASTRs and wmASTRs per se and in response to MS relevant Toll-like receptor (TLR) activation differently modulate myelination. METHODS: Differences in modulation of myelination between adult gmASTRs and wmASTRs were examined using an in vitro myelinating system that relies on a feeding layer of ASTRs. Transcriptional profiling and weighted gene co-expression network analysis were used to analyze differentially expressed genes and gene networks. Potential differential modulation of OPC proliferation and maturation by untreated adult gmASTRs and wmASTRs and in response to TLR3 and TLR4 agonists were assessed. RESULTS: Our data reveal that adult wmASTRs are less supportive to in vitro myelination than gmASTRs. WmASTRs more abundantly express reactive ASTR genes and genes of a neurotoxic subtype of ASTRs, while gmASTRs have more neuro-reparative transcripts. We identified a gene network module containing cholesterol biosynthesis enzyme genes that positively correlated with gmASTRs, and a network module containing extracellular matrix-related genes that positively correlated with wmASTRs. Adult wmASTRs and gmASTRs responding to TLR3 agonist Poly(I:C) distinctly modulate OPC behavior, while exposure to TLR4 agonist LPS of both gmASTRs and wmASTRs results in a prominent decrease in myelin membrane formation. CONCLUSIONS: Primary adult gmASTRs and wmASTRs are heterogeneous at the transcriptional level, differed in their support of in vitro myelination, and their pre-existing phenotype determined TLR3 agonist responses. These findings point to a role of ASTR heterogeneity in regional differences in remyelination efficiency between GM and WM lesions.

Profiling Microglia From Alzheimer's Disease Donors and Non-demented Elderly in Acute Human Postmortem Cortical Tissue.

Microglia are the tissue-resident macrophages of the central nervous system (CNS). Recent studies based on bulk and single-cell RNA sequencing in mice indicate high relevance of microglia with respect to risk genes and neuro-inflammation in Alzheimer's disease (AD). Here, we investigated microglia transcriptomes at bulk and single-cell levels in non-demented elderly and AD donors using acute human postmortem cortical brain samples. We identified seven human microglial subpopulations with heterogeneity in gene expression. Notably, gene expression profiles and subcluster composition of microglia did not differ between AD donors and non-demented elderly in bulk RNA sequencing nor in single-cell sequencing.

Targeted Diet Modification Reduces Multiple Sclerosis-like Disease in Adult Marmoset Monkeys from an Outbred Colony.

Experimental autoimmune encephalomyelitis (EAE) in common marmosets is a translationally relevant model of the chronic neurologic disease multiple sclerosis. Following the introduction of a new dietary supplement in our purpose-bred marmoset colony, the percentage of marmosets in which clinically evident EAE could be induced by sensitization against recombinant human myelin oligodendrocyte glycoprotein in IFA decreased from 100 to 65%. The reduced EAE susceptibility after the dietary change coincided with reduced Callitrichine herpesvirus 3 expression in the colony, an EBV-related γ1-herpesvirus associated with EAE. We then investigated, in a controlled study in marmoset twins, which disease-relevant parameters were affected by the dietary change. The selected twins had been raised on the new diet for at least 12 mo prior to the study. In twin siblings reverted to the original diet 8 wk prior to EAE induction, 100% disease prevalence (eight out of eight) was restored, whereas in siblings remaining on the new diet the EAE prevalence was 75% (six out of eight). Spinal cord demyelination, a classical hallmark of the disease, was significantly lower in new-diet monkeys than in monkeys reverted to the original diet. In new-diet monkeys, the proinflammatory T cell response to recombinant human myelin oligodendrocyte glycoprotein was significantly reduced, and RNA-sequencing revealed reduced apoptosis and enhanced myelination in the brain. Systematic typing of the marmoset gut microbiota using 16S rRNA sequencing demonstrated a unique, Bifidobacteria-dominated composition, which changed after disease induction. In conclusion, targeted dietary intervention exerts positive effects on EAE-related parameters in multiple compartments of the marmoset's gut-immune-CNS axis.

The Kaleidoscope of Microglial Phenotypes.

Gene expression analyses of microglia, the tissue-resident macrophages of the central nervous system (CNS), led to the identification of homeostatic as well as neurological disease-specific gene signatures of microglial phenotypes. Upon alterations in the neural microenvironment, either caused by local insults from within the CNS (during neurodegenerative diseases) or by macroenvironmental incidents, such as social stress, microglia can switch phenotypes-generally referred to as "microglial activation." The interplay between the microenvironment and its influence on microglial phenotypes, regulated by (epi)genetic mechanisms, can be imagined as the different colorful crystal formations (microglial phenotypes) that change upon rotation (microenvironmental changes) of a kaleidoscope. In this review, we will discuss microglial phenotypes in relation to neurodevelopment, homeostasis, in vitro conditions, aging, and neurodegenerative diseases based on transcriptome studies. By overlaying these disease-specific microglial signatures, recent publications have identified a specific set of genes that is differentially expressed in all investigated diseases, called a microglial core gene signature with multiple diseases. We will conclude this review with a discussion about the complexity of this microglial core gene signature associated with multiple diseases.

Aging, microglia and cytoskeletal regulation are key factors in the pathological evolution of the APP23 mouse model for Alzheimer's disease.

Aging is the key risk factor for Alzheimer's disease (AD). In addition, the amyloid-beta (Aß) peptide is considered a critical neurotoxic agent in AD pathology. However, the connection between these factors is unclear. We aimed to provide an extensive characterization of the gene expression profiles of the amyloidosis APP23 model for AD and control mice and to evaluate the effect of aging on these profiles. We also correlated our findings to changes in soluble Aß-levels and other pathological and symptomatic features of the model. We observed a clear biphasic expression profile. The first phase displayed a maturation profile, which resembled features found in young carriers of familial AD mutations. The second phase reflected aging processes and showed similarities to the progression of human AD pathology. During this phase, the model displayed a clear upregulation of microglial activation and lysosomal pathways and downregulation of neuron differentiation and axon guidance pathways. Interestingly, the changes in expression were all correlated to aging in general, but appeared more extensive/accelerated in APP23 mice.