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.

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.

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.

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.

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.

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.

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.

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.

CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability.

Recent data shows that fibroblast growth factor 14 (FGF14) binds to and controls the function of the voltage-gated sodium (Nav) channel with phenotypic outcomes on neuronal excitability. Mutations in the FGF14 gene in humans have been associated with brain disorders that are partially recapitulated in Fgf14(-/-) mice. Thus, signaling pathways that modulate the FGF14:Nav channel interaction may be important therapeutic targets. Bioluminescence-based screening of small molecule modulators of the FGF14:Nav1.6 complex identified 4,5,6,7 -: tetrabromobenzotriazole (TBB), a potent casein kinase 2 (CK2) inhibitor, as a strong suppressor of FGF14:Nav1.6 interaction. Inhibition of CK2 through TBB reduces the interaction of FGF14 with Nav1.6 and Nav1.2 channels. Mass spectrometry confirmed direct phosphorylation of FGF14 by CK2 at S228 and S230, and mutation to alanine at these sites modified FGF14 modulation of Nav1.6-mediated currents. In 1 d in vitro hippocampal neurons, TBB induced a reduction in FGF14 expression, a decrease in transient Na(+) current amplitude, and a hyperpolarizing shift in the voltage dependence of Nav channel steady-state inactivation. In mature neurons, TBB reduces the axodendritic polarity of FGF14. In cornu ammonis area 1 hippocampal slices from wild-type mice, TBB impairs neuronal excitability by increasing action potential threshold and lowering firing frequency. Importantly, these changes in excitability are recapitulated in Fgf14(-/-) mice, and deletion of Fgf14 occludes TBB-dependent phenotypes observed in wild-type mice. These results suggest that a CK2-FGF14 axis may regulate Nav channels and neuronal excitability.-Hsu, W.-C. J., Scala, F., Nenov, M. N., Wildburger, N. C., Elferink, H., Singh, A. K., Chesson, C. B., Buzhdygan, T., Sohail, M., Shavkunov, A. S., Panova, N. I., Nilsson, C. L., Rudra, J. S., Lichti, C. F., Laezza, F. CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability.

Quantitative proteomics reveals protein-protein interactions with fibroblast growth factor 12 as a component of the voltage-gated sodium channel 1.2 (nav1.2) macromolecular complex in Mammalian brain.

Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.

Homocitrullination Is a Novel Histone H1 Epigenetic Mark Dependent on Aryl Hydrocarbon Receptor Recruitment of Carbamoyl Phosphate Synthase 1.

The aryl hydrocarbon receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the nonconsensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in concomitant recruitment of carbamoyl phosphate synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin-modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome, resulting in a specific enzyme-independent post-translational modification of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation.

The Nav1.2 channel is regulated by GSK3.

BACKGROUND: Phosphorylation plays an essential role in regulating voltage-gated sodium (Na(v)) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Na(v) channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Na(v) channels. METHODS: We used patch-clamp electrophysiology to record sodium currents from Na(v)1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. RESULTS: We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Na(v)1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3ß suppressed Na(v)1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Na(v)1.2 channel indicates that cell surface expression of CD4-Na(v)1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3ß phosphorylates T(1966) at the C-terminal tail of Na(v)1.2. CONCLUSION: These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail. GENERAL SIGNIFICANCE: These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.

ESI-MS/MS and MALDI-IMS Localization Reveal Alterations in Phosphatidic Acid, Diacylglycerol, and DHA in Glioma Stem Cell Xenografts.

Glioblastoma (GBM) is the most common adult primary brain tumor. Despite aggressive multimodal therapy, the survival of patients with GBM remains dismal. However, recent evidence has demonstrated the promise of bone marrow-derived mesenchymal stem cells (BM-hMSCs) as a therapeutic delivery vehicle for anti-glioma agents due to their ability to migrate or home to human gliomas. While several studies have demonstrated the feasibility of harnessing the homing capacity of BM-hMSCs for targeted delivery of cancer therapeutics, it is now also evident, based on clinically relevant glioma stem cell (GSC) models of GBMs, that BM-hMSCs demonstrate variable tropism toward these tumors. In this study, we compared the lipid environment of GSC xenografts that attract BM-hMSCs (N = 9) with those that do not attract (N = 9) to identify lipid modalities that are conducive to homing of BM-hMSC to GBMs. We identified lipids directly from tissue by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) of lipid extracts. Several species of signaling lipids, including phosphatidic acid (PA 36:2, PA 40:5, PA 42:5, and PA 42:7) and diacylglycerol (DAG 34:0, DAG 34:1, DAG 36:1, DAG 38:4, DAG 38:6, and DAG 40:6), were lower in attracting xenografts. Molecular lipid images showed that PA (36:2), DAG (40:6), and docosahexaenoic acid (DHA) were decreased within tumor regions of attracting xenografts. Our results provide the first evidence for lipid signaling pathways and lipid-mediated tumor inflammatory responses in the homing of BM-hMSCs to GSC xenografts. Our studies provide new fundamental knowledge on the molecular correlates of the differential homing capacity of BM-hMSCs toward GSC xenografts.

Systematic identification of single amino acid variants in glioma stem-cell-derived chromosome 19 proteins.

Novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome. We have developed an approach, searching high quality shotgun proteomic data against an extended protein database, to identify expressed mutant proteoforms in glioma stem cell (GSC) lines. The systematic search of MS/MS spectra using PEAKS 7.0 as the search engine has recognized 17 chromosome 19 proteins in GSCs with altered amino acid sequences. The results were further verified by manual spectral examination, validating 19 proteoforms. One of the novel findings, a mutant form of branched-chain aminotransferase 2 (p.Thr186Arg), was verified at the transcript level and by targeted proteomics in several glioma stem cell lines. The structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma. Based on our initial findings, we believe that our approach presented could contribute to construct a more complete map of the human functional proteome.

Use of ENCODE resources to characterize novel proteoforms and missing proteins in the human proteome.

We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.

Quest for Missing Proteins: Update 2015 on Chromosome-Centric Human Proteome Project.

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the Supporting Information.

Genomic impact of transient low-dose decitabine treatment on primary AML cells.

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

Integrated chromosome 19 transcriptomic and proteomic data sets derived from glioma cancer stem-cell lines.

One subproject within the global Chromosome 19 Consortium is to define chromosome 19 gene and protein expression in glioma-derived cancer stem cells (GSCs). Chromosome 19 is notoriously linked to glioma by 1p/19q codeletions, and clinical tests are established to detect that specific aberration. GSCs are tumor-initiating cells and are hypothesized to provide a repository of cells in tumors that can self-replicate and be refractory to radiation and chemotherapeutic agents developed for the treatment of tumors. In this pilot study, we performed RNA-Seq, label-free quantitative protein measurements in six GSC lines, and targeted transcriptomic analysis using a chromosome 19-specific microarray in an additional six GSC lines. The data have been deposited to the ProteomeXchange with identifier PXD000563. Here we present insights into differences in GSC gene and protein expression, including the identification of proteins listed as having no or low evidence at the protein level in the Human Protein Atlas, as correlated to chromosome 19 and GSC subtype. Furthermore, the upregulation of proteins downstream of adenovirus-associated viral integration site 1 (AAVS1) in GSC11 in response to oncolytic adenovirus treatment was demonstrated. Taken together, our results may indicate new roles for chromosome 19, beyond the 1p/19q codeletion, in the future of personalized medicine for glioma patients.