Citations

ABSTRACT Periprosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty that can be challenging to diagnose and treat. Neutrophils are a prominent feature in PJI, with neutrophil extracellular traps (NETs) known to be present; little is known, however, about the physiology of PJI-associated NETs. Here, the proteomes of PJI and non-infectious arthroplasty failure (NIAF), analyzed using liquid chromatography tandem mass-spectrometry (LC-MS/MS) and proximity extension assay (PEA)-based profiling of fluids derived from sonication of arthroplasty components removed from patients who had undergone revision arthroplasty, were compared, with a focus on NET formation. In addition, in vitro effects of synovial fluid components on Staphylococcus aureus- induced NET formation were characterized. LC-MS/MS (PJI n = 85, NIAF n = 26) and PEA (PJI n = 95, NIAF n = 39) showed NET formation and neutrophil degranulation to be associated with PJI, with the complement cascade and platelet activation pathways identified as potential contributors. In vitro studies showed delayed NET formation in artificial synovial fluid compared to standard cell culture media conventionally used to evaluate NETs. Together, these results demonstrate the presence of NETs in PJI, identify potential pathways involved in PJI-associated NETosis, and point to an influence of synovial components on NET formation during PJI. IMPORTANCE Periprosthetic joint infection (PJI) is a major complication of joint replacement, placing a significant burden on patients and the healthcare system. Our research reveals that neutrophils and their antimicrobial “traps” are dominant features of the immune response at the infection joint site. Importantly, we found that the unique environment of the joint delays the formation of these traps, potentially contributing to the pathogenesis of PJI. By establishing a new experimental platform tailored for the infected joint, this work provides a foundational platform for probing these mechanisms and supports the development of improved diagnostic and therapeutic strategies.

TRAIL is a TNF family ligand that trimerizes TRAIL-R1 (DR4) or TRAIL-R2 (DR5) to induce apoptosis, necroptosis, and/or NF-κB activation in receptor-bearing cells. We previously identified TRAILshort as a splice variant of TRAIL that lacks cysteine 230, cannot trimerize, and acts as a dominant-negative ligand that blocks TRAIL-mediated apoptosis. TRAILshort is expressed on cell surfaces and within extracellular vesicles, enabling it to confer TRAIL resistance to both producing and bystander cells. In this study, we showed that elevated TRAILshort levels were associated with chronic viral infections, cancer, and autoimmune diseases, suggesting a link to impaired immune regulation. Using unbiased phosphoproteomics and mechanistic studies, we demonstrated that TRAILshort binding to DR5 recruited and activated the phosphatase Src homology region 2 domain–containing phosphatase 1 (SHP-1), leading to zeta-chain-associated protein kinase 70 (ZAP-70) dephosphorylation, disruption of ZAP-70–CD3ζ interactions, and impaired T cell receptor signaling, thereby reducing T cell activation, proliferation, and cytokine production in response to antigen or CD3/CD28 ligation. Genetic or pharmacologic SHP-1 inhibition reverses these effects. In humanized mouse models, TRAILshort promoted the persistence of transformed mouse embryonic fibroblasts (MEFs) and L428 and antagonized CD19-directed CAR T cell activity, revealing TRAILshort as an immunomodulator of T cell function with therapeutic implications, including blocking TRAILshort to restore T cell immunity or delivering TRAILshort to enforce tolerance. TRAILshort, a variant of TRAIL, suppresses T cell function and immune responses in cancer and infectious diseases, highlighting its potential as a therapeutic target.

Synaptic transmission can be tuned through plasticity mechanisms that regulate synaptic strength, structure, and number. In vivo measurements demonstrate remarkable spine dynamics, with subsets of synapses persisting for months. This correlates with the longevity of certain memories, which can persist for an organism’s lifetime. The molecular basis supporting the long-term stability of specific synapses and the long-term durability of memories remains unknown. At the protein level, most proteins persist for a relatively short amount of time before they are degraded and replaced with new molecules. However, recent work has identified a population of proteins, including those present at the synapse, that are exceptionally long-lived. It has been speculated that long-lived proteins (LLPs) could contribute to long-term synapse stability, function, and memory. Here, we used stable isotope labeling in mammals to first identify LLPs in the post synaptic density (PSD) of the hippocampus and subsequently determine if protein turnover rates varied by sex or following learning. We identified synaptic LLPs and found that both sex and experience can regulate synaptic protein turnover rates. We identified sex-dependent changes in protein turnover rates in autism spectrum disorder risk genes, including increased stability of Gabrg2, a GABA-A receptor subunit, in male mice. Furthermore, we observed stabilization of a subset of PSD proteins, such as Shank3, following contextual fear conditioning. We propose that sex- and experience-dependent changes in protein turnover rates could help explain sex-differences in psychiatric risk and aid our understanding of the molecular mechanisms that support learning and memory.

Cytotoxic T cells produce the cytokine interferon-γ (IFN-γ). We sought to determine whether IFN-γ directly kills target cells and found that T cells used IFN-γ to kill murine melanoma cells lacking the kinases TBK1 and IKKε. In the absence of both kinases, IFN-γ induced the production of TNF receptor 1 (TNFR1) and the sensor Z-DNA binding protein 1 (ZBP1) in the tumor cells to stimulate receptor-interacting protein kinase 1 (RIPK1)–dependent apoptosis in a cell-autonomous manner. IFN-γ also enhanced the activation of nuclear factor κB (NF-κB) signaling in a TNFR1-dependent manner in cells deficient in both TBK1 and IKKε. Because IFN-γ–induced apoptosis occurred in a transcription-dependent manner with slow kinetics, STAT1 and NF-κB cooperated to activate the expression of inflammatory genes in the dying cells. Thus, IFN-γ–induced cell death was accompanied by an inflammatory signature in the absence of TBK1 and IKKε. TBK1 and IKKε not only mediated the induction of type I IFN but also inhibited RIPK1-dependent death and NF-κB–dependent inflammation. Therefore, we propose that these kinases may have gained these intertwined functions so that cells infected by pathogens that produce antagonists of TBK1 and IKKε are eliminated by IFN-γ–secreting T cells in an inflammatory manner to compensate for the inhibition of type I IFN production.

Abstract Background & Objectives Cholangiocarcinoma (CCA) is a highly aggressive malignancy arising from the bile duct epithelium. Despite advancements in diagnostic techniques, the diagnosis of cholangiocarcinoma (CCA) remains challenging. Currently, CA19-9 is the best available blood-based marker for detection of CCA, which has a sensitivity of 50-60% and a specificity of 80%. Moreover, individuals who are Lewis-antigen-negative (∼20% in some populations) have undetectable CA 19–9 levels resulting in false negative diagnoses. Therefore, there is a critical need for developing novel blood-based diagnostic approach for improving the sensitivity of diagnosis of CCA to improve clinical outcomes. Glycosylation, one of the most prevalent post-translational modifications, plays a crucial role in regulating biological functions such as cell-cell communication, protein folding and receptor signaling and is frequently dysregulated in cancer. Cell surface proteins, including glycoproteins, are overexpressed in CCA are likely to be shed into the blood and we investigated development of mass spectrometry-based methods to measure changes in glycosylation in patients with CCA. Methods Mass spectrometry data were acquired in parallel reaction monitoring mode using Orbitrap Eclipse Tribrid mass spectrometers. In addition, we tested the use of heavy glycopeptides for absolute quantitation of each glycopeptide. Mass spectrometry data were analyzed using Skyline to estimate glycopeptide abundance. Results First, we performed tandem mass tag-based quantitative proteomics and glycoproteomics of tumor tissues and serum collected from CCA patients using high-resolution mass spectrometry. From these experiments, a number of glycopeptides were found to be increased in abundance in CCA patients. We subsequently developed a targeted mass spectrometry–based assay to monitor four candidate glycopeptides derived from polymeric immunoglobulin receptor and aminopeptidase N to validate their performance in a large patient cohort from 269 patients with CCA and 100 healthy individuals. Conclusion We anticipate that this finding will lay the foundation for novel diagnostics that could benefit patients with this aggressive malignancy.

Abstract Introduction: Surveillance biopsies after kidney transplantation provide an opportunity to assess early renal disease processes, including diabetic nephropathy, before clinical manifestation. This study aimed to identify proteomic alterations in renal allografts at 5 years post-transplant (5yrPT) and their associations with histologic features in diabetic/obese (DM/OB) versus non-diabetic/non-obese (nDM/nOB) recipients. Methods: We studied protocol biopsies from 17 DM/OB and 14 nDM/nOB kidney transplant recipients at Mayo Clinic (2000–2012). Laser capture microdissection followed by mass spectrometry was used for proteomic profiling. Histologic assessment included Banff and Tervaert scoring and morphometric analysis. Statistical comparisons were performed using ANOVA with Tukey post-hoc testing. Results: DM/OB recipients demonstrated greater histologic and metabolic injury at 5yrPT. Proteomic analysis identified 101 glomerular and 86 non-glomerular proteins differentially expressed between groups. Significant alterations were observed in proteins associated with mitochondrial dysfunction, including VDAC1 (fold change [FC] 2.6, p = 0.03), NDUFB9 (FC 0.15, p = 0.009), and NDUFA9 (FC 1.72, p = 0.01). Extracellular matrix remodeling proteins COL6A1, COL6A2, and COL6A3 were upregulated (FC 1.6–1.8, p < 0.05). Immune and inflammatory markers C9 (FC 1.6, p < 0.03) and TGFBI (FC 1.7, p < 0.001), as well as oxidative stress–related CLU, were increased. GGACT showed the greatest decrease (FC 0.30, p < 0.001), implicating altered energy metabolism. Conclusion: Distinct proteomic signatures at 5yrPT in DM/OB recipients highlight early mitochondrial, metabolic, immune, and extracellular matrix pathways involved in diabetic kidney disease. These changes precede clinical disease and identify candidate biomarkers and therapeutic targets to improve graft longevity.

Abstract Background Mono-allelic Dehydrodolichyl Diphosphate Synthase ( DHDDS) variants are associated with juvenile Parkinsonism, developmental delay and seizures. Symptoms are progressive, and various mechanisms, such as defective glycosylation, lysosomal dysfunction and cholesterol accumulation have been hypothesized to underlie disease symptoms. There is no treatment for DHDDS-related disease. Methods Patient-derived cortical forebrain organoids were created to elucidate disease mechanisms and evaluate potential treatments. In these neuronal models, glycosylation, lipidomics, proteomics, cholesterol/ganglioside accumulation, mitochondrial function and electrophysiological activity were assessed. Finally, we investigated the effects of nicotinamide mononucleotide (NMN), identified through a yeast-based drug screen, in neuronal cell models and in six patients in an off-label, N-of-1, observational series. Results DHDDS-patient derived organoids showed visual signs of degeneration after four months of culturing. This was accompanied by significant cholesterol accumulation in astrocytes, decreased mitochondrial respiration and loss of deep-layer neurons. In addition, we identified glycosylation abnormalities, showing for the first time that glycosylation in human tissue is affected by monoallelic DHDDS variants. Proteomic analysis revealed altered protein expression of proteins involved in lipid metabolism, cytoskeletal organization and neuronal development. We found that oral Nicotinamide Mononucleotide supplementation led to significant improvement in mitochondrial respiration and electrophysiological parameters in organoids, concurring with clinical improvements in all of the treated patients, particularly regarding their ataxia and tremor. Conclusion Our findings reveal a progressive phenotype in DHDDS-patient-derived brain organoids, with mitochondrial dysfunction and astrocyte-specific metabolic alterations contributing to disease pathology. Notably, NMN treatment led to clinical improvements in patients with heterozygous DHDDS variants, highlighting its potential as a therapeutic strategy.

Tumor-associated antigens (TAAs) are non-mutated antigenic peptides expressed in cancer tissue at abnormally high levels but in normal tissue either at negligible levels, during only particular developmental stages, or in a tissue-restricted manner. The shared nature of TAAs across patients makes them attractive targets for off-the-shelf immunotherapies. However, no studies have comprehensively surveyed the TAA potential of all possible wild-type peptides originating from cancer-associated genes. In this study of 16 cancer tissue types and 39 normal tissue types, we analyzed the expression profiles of protein-coding genes to identify those with aberrantly high RNA levels across multiple solid tumor types and low RNA levels across all normal tissue types examined. We then developed a score to quantify tumor specificity of gene expression. Compared to previously reported TAA genes, those we identified exhibited substantially greater tumor specificity. Seven of these genes demonstrated consistently elevated expression in at least five tumor types and minimal expression across all non-immune-privileged normal tissues. To assess Human Leukocyte Antigen (HLA) class I presentation potential of the multi-cancer-associated genes, we computationally predicted the binding affinities between the most common class I HLA alleles and all possible wild-type TAA epitopes of 8–11 amino acids arising from those genes. We then applied rigorous filtering criteria to prioritize the most promising multi-cancer TAA peptide candidates and evaluated their HLA binding and cell-surface presentation using T2 and immunopeptidome analysis. Our results highlight new potential targets for multi-cancer immunotherapies.

ABSTRACT Interferon-γ (IFNγ) signaling is prominent in inflammatory CNS microenvironments across many neurological disorders, but the neuronal peptides presented on HLA class I under these conditions and their functional consequences for CD8 + T cells remain incompletely defined. Here we combine human iPSC-derived human neural aggregates (HNAs), HLA class I immunoprecipitation coupled to LC-MS/MS immunopeptidomics, and microfluidic co-culture assays to map IFNγ-induced neuronal antigen presentation and test antigen-specific cytotoxicity. IFNγ stimulation induced robust HLA class I expression in HNAs and enabled recovery of a canonical 8-12-mer class I ligandome enriched for 9-mers. Neuron-restricted expression of a synapsin-driven polyepitope cassette yielded presentation of defined neoantigen 9-mer peptides on donor HLA class I molecules and, in the presence of IFNγ, elicited activation of autologous antigen-specific CD8 + T cells and consequent antigen-dependent neurite injury. Across four donors, comparative immunopeptidomics identified large IFNγ-unique neural peptide repertoires distinct from matched fibroblasts and revealed a consistent enrichment of predicted high-affinity binders on HLA-B allotypes. Finally, β2-microglobulin deletion ablated peptide recovery, and neuron-restricted β2-microglobulin reconstitution enabled identification of neuron-derived peptides, including peptides derived from neurofilament light (NEFL) that were shared across donors and presented on multiple HLA allotypes. Together, these data provide an integrated platform for neuronal autoantigen discovery and functional validation and support a model in which IFNγ-driven neuronal HLA class I presentation creates an HLA-B-weighted epitope landscape that can be recognized by autoreactive cytotoxic CD8 + T cells.

Abstract Introduction: The intricate cellular structure is a constantly evolving environment where cells interact, communicate, and adapt to their surroundings. The fate, role, and actions of each cell are shaped by its specific location within the tissue. Therefore, studying the spatial proteome is crucial for gaining a deeper understanding of physiological or pathological processes. With significant advancements in microscopy and mass spectrometry, it has become feasible to explore the cellular proteome at low or even single cell numbers. However, spatial proteomics is an evolving field with scope for newer sample preparation and data analysis techniques. Our objective was to develop a sample preparation workflow for analyzing proteome from limited cells extracted from formalin fixed paraffin embedded (FFPE) tissue using laser capture microdissection (LCM). Methods: One hundred cells from the FFPE normal colon tissue were extracted by LCM and collected into an AFA-compatible 96-well plate. Decrosslinking was performed at 900C for 50 minutes followed by lysis in a buffer composed of 100 mM triethylammonium bicarbonate and 0.1% n-dodecyl-β-D-maltoside using adaptive focused acoustic (AFA) energetics in scanning mode for 5 minutes. After lysis, the proteins were digested using Trypsin/Lys-C mix using AFA energetics for 1 hour. The resulting peptides were acidified with trifluoroacetic acid and analyzed on a timsTOF Ultra 2 mass spectrometer coupled to a nanoElute 2 liquid chromatography system using a 15 cm (75 μm) IonOpticks column, in DDA-PASEF mode for a total run time of 34 minutes. The data were searched against the UniProt Human Reviewed protein database using MSFragger. Results: We utilized AFA energetics to extract and digest proteins from a limited number of cells isolated from FFPE tissue using LCM. This experiment was conducted with technical replicates to ensure accuracy and reliability. A total of 3,723 proteins were identified across the three replicates, with replicate 1 identifying 3,895 proteins, replicate 2 identifying 3,754 proteins, and replicate 3 identifying 3,519 proteins. In total, we identified 21,005 peptides, with replicate 1 identifying 22,896 peptides, replicate 2 identifying 22,377 peptides, and replicate 3 identifying 17,741 peptides from 100 colon cells. Notably, we observed a 70% overlap in the proteins identified across the three replicates, which highlights the reproducibility of the approach. Conclusion: The entire sample preparation process, after cell collection, was completed within a concise four-hour timeframe, demonstrating the efficiency of this methodology for analyzing protein profiles from small cell populations. Further, this approach is highly adaptable to automation and high-throughput applications. Its adaptability and scalability make it a promising strategy for spatial proteomics applications. Citation Format: Dong-Gi Mun, Kiran Mangalaparthi, Daigo Gunji, Amy J. French, Raghavendra Pasupuleti, Cristine Charlesworth, Sameer Vasantgadkar, DEB BHATTACHARYYA, Akhilesh Pandey. An AFA assisted workflow for mass-spectrometry based proteomics of FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7657.