Search Results (67)

For 40 years, Intron Retention (IR) was dismissed as splicing noise and is now recognized as a dynamic and evolutionarily conserved mechanism of post-transcriptional gene regulation. Unlike canonical splicing, which excises all introns from pre-mRNAs, IR selectively retains intronic sequences, albeit at seemingly random places; however, current research now reveals that this process is strategic in its retention. IR influences mRNA stability, localization, and translational potential. Retained introns can lead to nonsense-mediated decay, promote nuclear retention, or give rise to novel protein isoforms that contribute to expanding proteomic and transcriptomic profiles. IR is finely regulated by splice site strength, splicing regulatory elements, chromatin structure, methylation patterns, RNA polymerase II elongation rates, and the availability of co-transcriptional splicing factors. IR plays critical roles in cell-type and tissue-specific gene expression with observed patterns, particularly during neuronal, cardiac, hematopoietic, and immune development. It also functions as a molecular switch during cellular responses to environmental and physiological stressors such as hypoxia, heat shock, and infection. Dysregulated IR is increasingly associated with cancer, neurodegeneration, aging, and immune dysfunction, where it may alter protein function, suppress tumor suppressor genes, or generate immunogenic neoepitopes. Experimental and computational tools like RNA-seq, RT-PCR, IRFinder, and IntEREst have enabled transcriptome-wide detection and validation of IR events, uncovering their widespread functional roles. This review will examine current knowledge on the function, regulation, and detection of IR, and also summarize recent advances in understanding its role in both normal and pathophysiological settings. Full article

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease predominantly of the small airways and parenchyma. COPD lungs exhibit an influx of circulating innate immune cells, which, when isolated, display impaired functions, including imbalanced protease secretion. In addition to immune cells, the extracellular matrix (ECM) plays a crucial role in COPD pathology. Remodeling of the ECM can generate ECM fragments, which can be released into circulation and subsequently induce pro-inflammatory responses. COPD is a heterogeneous disease, and serological biomarkers can be used to sub-categorize COPD patients for targeted treatments and optimal recruitment in clinical trials. This study evaluated fragments of calprotectin, collagen type VI, and versican, generated by neutrophil elastase and matrix metalloproteinases (MMP-) 2 and 12, respectively, as potential biomarkers of COPD disease, severity, and endotypes. Lower plasma levels of a neoepitope marker of calprotectin, indicative of activated neutrophils (nordicCPa9-HNETM), were detected in COPD donors compared to controls. CPa9-HNE was associated with milder disease, higher degree of air-trapping, and higher serum levels of MMP-2. Deposition of CPa9-HNE levels in lung tissue revealed no differences between groups. Taken together, CPa9-HNE was found to be a potential marker of mild COPD, but further studies are warranted to validate our findings. Full article

Peptide vaccines possess several advantages over mRNA vaccines but are generally less effective at inducing antitumor immunity. The bottlenecks limiting peptide vaccine efficacy could be elucidated by tracking and comparing vaccine-induced T-lymphocytes in successful and unsuccessful cases. Here we have applied our recent database of neoantigen-specific T cell receptors (TCRs) to profile tumor-specific T cells following vaccination with a neoantigen peptide vaccine and to correlate this with the response. Mice were vaccinated prophylactically with p30 peptide encoding B16 melanoma neoantigen (K739N mutation in Kif18b gene). The B16F0 melanoma in the vaccinated mice was additionally treated by a CTLA-4 checkpoint blockade. T cells from the tumors, tumor-draining lymph nodes (tdLNs) and vaccine depots were isolated, phenotyped, sorted by subsets and sequenced for TCR repertoires. The vaccine induced the accumulation of tumor-specific CD4+ Th cells in the tdLNs, while in the tumors these cells were present and their frequencies were not changed by the vaccine. These cells also accumulated at the vaccine depots, where they were phenotypically skewed by the vaccine components; however, these effects were minor due to approximately 50-fold lower cell quantities compared to the tdLNs. Only some of the p30-specific Th cells showed tumoricidal activity, as revealed by the reverse correlation of their frequencies in the tdLNs with the tumor size. The CTLA-4 blockade did not affect the tumor growth or the frequencies of tumor-specific cells but did stimulate Th cell motility. Thus, we have shown that tumor-specific Th clones accumulate and/or expand in the tdLNs, which correlates with tumor suppression but only for some of these clones. Tumor infiltration by these clones is not correlated with the growth rate. Full article

The increasing incidence of cutaneous squamous cell carcinoma (cSCC), together with the ominous risks of metastasis and recurrence, underscores the importance of identifying novel therapies and validated biomarkers to augment patient management, particularly in the context of well-established and advanced disease. Following a brief overview of the well-recognized epidemiology, clinical features, and diagnosis of cSCC, the current review is focused on risk factors, most prominently excessive exposure to ultraviolet radiation (UVR) as a cause of persistent, pro-tumorigenic mutagenesis, and immune suppression. The next phase of the review encompasses an evaluation of the search for key driver mutations in the pathogenesis of cSCC, including the role of these and other mutations in the formation of immunologically reactive neoepitopes. With respect to additional mechanisms of tumorigenesis, immune evasion is prioritized, specifically the involvement of cell-free and infiltrating cellular mediators of immune suppression. Prominent amongst the former are the cytokine, transforming growth factor-β1 (TGF-β1), the prostanoid, prostaglandin E2, and the emerging immune suppressive nucleoside adenosine. In the case of the latter, tumor-infiltrating and circulating regulatory T cells have been implicated as being key players. The final sections of the review are focused on an update of the immunotherapy of established and advanced disease, as well as on the search for novel, reliable lesional and systemic biomarkers with the potential to guide patient management. Full article

Interferon-gamma (IFN-γ) is a critical cytokine that plays a pivotal role in immune system regulation. It is a key mediator of both cellular defense mechanisms and antitumor immunity. As the sole member of the type II interferon family, IFN-γ modulates immune responses by activating macrophages, enhancing natural killer cell function, and regulating gene expression across multiple cellular processes. Alternative splicing is a post-transcriptional gene expression regulatory mechanism that generates multiple mature messenger RNAs from a single gene, dramatically increasing proteome diversity without the need of a proportional genome expansion. This process occurs in 90–95% of human genes, with alternative splicing events allowing for the production of diverse protein isoforms that can have distinct—or even opposing—functional properties. Alternative splicing plays a crucial role in cancer immunology, potentially generating tumor neoepitopes and modulating immune responses. However, how alternative splicing affects IFN-γ’s activity is still poorly understood. This review explores how alternative splicing regulates the expression and function of both upstream regulators and downstream effectors of IFN-γ, revealing complex mechanisms of gene expression and immune response modulation. Key transcription factors and signaling molecules of the IFN-γ pathway are alternatively spliced, and alternative splicing can dramatically alter IFN-γ signaling, immune cell function, and response to environmental cues. Specific splice variants can enhance or inhibit IFN-γ-mediated immune responses, potentially influencing cancer immunotherapy, autoimmune conditions, and infectious disease outcomes. The emerging understanding of these splicing events offers promising therapeutic strategies for manipulating immune responses through targeted molecular interventions. Full article

Hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer, presents significant therapeutic challenges due to its molecular complexity, late-stage diagnosis, and inherent resistance to conventional treatments. The intermediate to low mutational burden in HCC and its ability to evade the immune system through multiple mechanisms complicate the development of effective therapies. Recent advancements in immunotherapy, particularly neoantigen-based vaccines, offer a promising, personalized approach to HCC treatment. Neoantigens are tumor-specific peptides derived from somatic mutations in tumor cells. Unlike normal cellular antigens, neoantigens are foreign to the immune system, making them highly specific targets for immunotherapy. Neoantigens arise from genetic alterations such as point mutations, insertions, deletions, and gene fusions, which are expressed as neoepitopes that are not present in healthy tissues, thus evading the immune tolerance mechanisms that typically protect normal cells. Preclinical and early-phase clinical studies of neoantigen-based vaccines have shown promising results, demonstrating the ability of these vaccines to elicit robust T cell responses against HCC. The aim of the current review is to provide an in-depth exploration of the therapeutic potential of neoantigen-based vaccines in HCC, focusing on neoantigen identification, vaccine platforms, and their integration with immune checkpoint inhibitors to enhance immunogenicity. It also evaluates preclinical and clinical data on efficacy and safety while addressing challenges in clinical translation. By taking advantage of the unique antigenic profile of each patient’s tumor, neoantigen-based vaccines represent a promising approach in the treatment of HCC, offering the potential for improved patient outcomes, long-term remission, and a shift towards personalized, precision medicine in liver cancer therapy. Full article

Recombinant antibodies and, more recently, T cell receptor (TCR)-engineered T cell therapies represent two immunological strategies that have come to the forefront of clinical interest for targeting intracellular neoantigens in benign and malignant diseases. T cell-based therapies targeting neoantigens use T cells expressing a recombinant complete TCR (TCR-T cell), a chimeric antigen receptor (CAR) with the variable domains of a neoepitope-reactive TCR as a binding domain (TCR-CAR-T cell) or a TCR-like antibody as a binding domain (TCR-like CAR-T cell). Furthermore, the synthetic T cell receptor and antigen receptor (STAR) and heterodimeric TCR-like CAR (T-CAR) are designed as a double-chain TCRαβ-based receptor with variable regions of immunoglobulin heavy and light chains (VH and VL) fused to TCR-Cα and TCR-Cβ, respectively, resulting in TCR signaling. In contrast to the use of recombinant T cells, anti-neopeptide MHC (pMHC) antibodies and intrabodies neutralizing intracellular neoantigens can be more easily applied to cancer patients. However, different limitations should be considered, such as the loss of neoantigens, the modification of antigen peptide presentation, tumor heterogenicity, and the immunosuppressive activity of the tumor environment. The simultaneous application of immune checkpoint blocking antibodies and of CRISPR/Cas9-based genome editing tools to engineer different recombinant T cells with enhanced therapeutic functions could make T cell therapies more efficient and could pave the way for its routine clinical application. Full article

The field of cancer immunotherapy has seen incredible advancements in the past decades. mRNA-based cancer vaccines generating de novo T cell responses, particularly against tumor-specific antigens (TSAs), have demonstrated promising clinical outcomes and overcome diverse challenges. Despite the high potential of neoantigens to provide personalized immunotherapies through their tumor specificity and immunogenicity, challenges related to the scarcity of immunogenic neoepitopes have prompted continuous research towards finding new tumor-associated antigens (TAAs) and broader therapeutic frameworks, which may now learn from the genuine successes obtained with neoantigens. As an example, human endogenous retroviruses (HERVs) have emerged as potential alternatives to tumor neoantigens due to their high tumoral expression and ability to elicit both T cell reactivity and B cell responses associated with the efficacy of existing immunotherapies. This review aims to assess the status and limitations of TSA-directed mRNA cancer vaccines and the lessons that can be derived from these and checkpoint inhibitor studies to guide TAA vaccine development. We expect that shared B cell, CD4 and CD8 T cell antigen presentation will be key to stimulate continuous T cell expansion and efficacy for tumors that do not contain pre-existing tertiary lymphoid structures. When these structures are present in highly mutated tumors, the current checkpoint-based immunotherapies show efficacy even in immune privileged sites, and vaccines may hold the key to broaden efficacy to more tumor types and stages. Full article

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies. Full article

Therapeutic cancer vaccines have been considered in recent decades as important immunotherapeutic strategies capable of leading to tumor regression. In the development of these vaccines, the identification of neoepitopes plays a critical role, and different computational methods have been proposed and employed to direct and accelerate this process. In this context, this review identified and systematically analyzed the most recent studies published in the literature on the computational prediction of epitopes for the development of therapeutic vaccines, outlining critical steps, along with the associated program’s strengths and limitations. A scoping review was conducted following the PRISMA extension (PRISMA-ScR). Searches were performed in databases (Scopus, PubMed, Web of Science, Science Direct) using the keywords: neoepitope, epitope, vaccine, prediction, algorithm, cancer, and tumor. Forty-nine articles published from 2012 to 2024 were synthesized and analyzed. Most of the identified studies focus on the prediction of epitopes with an affinity for MHC I molecules in solid tumors, such as lung carcinoma. Predicting epitopes with class II MHC affinity has been relatively underexplored. Besides neoepitope prediction from high-throughput sequencing data, additional steps were identified, such as the prioritization of neoepitopes and validation. Mutect2 is the most used tool for variant calling, while NetMHCpan is favored for neoepitope prediction. Artificial/convolutional neural networks are the preferred methods for neoepitope prediction. For prioritizing immunogenic epitopes, the random forest algorithm is the most used for classification. The performance values related to the computational models for the prediction and prioritization of neoepitopes are high; however, a large part of the studies still use microbiome databases for training. The in vitro/in vivo validations of the predicted neoepitopes were verified in 55% of the analyzed studies. Clinical trials that led to successful tumor remission were identified, highlighting that this immunotherapeutic approach can benefit these patients. Integrating high-throughput sequencing, sophisticated bioinformatics tools, and rigorous validation methods through in vitro/in vivo assays as well as clinical trials, the tumor neoepitope-based vaccine approach holds promise for developing personalized therapeutic vaccines that target specific tumor cancers. Full article

Pathological cleavage of type II collagen (Col2) and generation of Col2 neoepitopes can serve as useful molecular markers of the progression of osteoarthritis (OA). One of such potential biomarkers is type II collagen neoepitope C2C. The aim of this study was to correlate the degree of articular cartilage damage in OA patients with C2C expression in histological samples of tissues removed during total knee replacement. Cartilage samples were obtained from 27 patients ranging in age from 55 to 66 years. In each patient, medial and lateral tibia plateau samples were analyzed according to the OARSI histopathology grading system. The C2C expression was evaluated on histological slides by semi-quantitative analysis using ImageJ Fiji 2.14.0 software. Spearman’s rank correlation analysis revealed a positive weak correlation (rho = 0.289, p = 0.0356) between the histological grade of tissue damage and the percentage of C2C staining. In addition, a highly significant positive correlation (rho = 0.388, p = 0.0041) was discovered between the osteoarthritis score (combining the histological grade of damage with the OA macroscopic stage) and the percentage of C2C staining in the samples. The C2C expression was detected in all the regions of the articular cartilage (i.e., the superficial zone, mid zone, deep zone and tidemark area, and the zone of calcified cartilage). Our findings imply that local expression of C2C correlates with the articular cartilage damage in OA-affected knees. This confirms that C2C can be used as a prospective marker for assessing pathological changes in the OA course and OA clinical trials. Full article

With the advent of immunotherapeutics, a new era in the combat against cancer has begun. Particularly promising are neo-epitope-targeted therapies as the expression of neo-antigens is tumor-specific. In turn, this allows the selective targeting and killing of cancer cells whilst healthy cells remain largely unaffected. So far, many advances have been made in the development of treatment options which are tailored to the individual neo-epitope repertoire. The next big step is the achievement of efficacious “off-the-shelf” immunotherapies. For this, shared neo-epitopes propose an optimal target. Given the tremendous potential, a thorough understanding of the underlying mechanisms which lead to the formation of neo-antigens is of fundamental importance. Here, we review the various processes which result in the formation of neo-epitopes. Broadly, the origin of neo-epitopes can be categorized into three groups: canonical, noncanonical, and viral neo-epitopes. For the canonical neo-antigens that arise in direct consequence of somatic mutations, we summarize past and recent findings. Beyond that, our main focus is put on the discussion of noncanonical and viral neo-epitopes as we believe that targeting those provides an encouraging perspective to shape the future of cancer immunotherapeutics. Full article

Ovarian cancer is one of the most common cancers among women and the most lethal malignancy of all gynecological cancers. Surgery is promising in the early stages; however, most patients are first diagnosed in the advanced stages, where treatment options are limited. Here, we present a 49-year-old patient who was first diagnosed with stage III ovarian cancer. After the tumor progressed several times under guideline therapies with no more treatment options available at that time, the patient received a fully individualized neoantigen-derived peptide vaccine in the setting of an individual healing attempt. The tumor was analyzed for somatic mutations via whole exome sequencing and potential neoepitopes were vaccinated over a period of 50 months. During vaccination, the patient additionally received anti-PD-1 therapy to prevent further disease progression. Vaccine-induced T-cell responses were detected using intracellular cytokine staining. After eleven days of in vitro expansion, four T-cell activation markers (namely IFN-ɣ, TNF-α, IL-2, and CD154) were measured. The proliferation capacity of neoantigen-specific T-cells was determined using a CFSE proliferation assay. Immune monitoring revealed a very strong CD4+ T-cell response against one of the vaccinated peptides. The vaccine-induced T-cells simultaneously expressed CD154, TNF, IL-2, and IFN-ɣ and showed a strong proliferation capacity upon neoantigen stimulation. Next-generation sequencing, as well as immunohistochemical analysis, revealed a loss of Beta-2 microglobulin (B2M), which is essential for MHC class I presentation. The results presented here implicate that the application of neoantigen-derived peptide vaccines might be considered for those cancer stages, where promising therapeutic options are lacking. Furthermore, we provide more data that endorse the intensive investigation of B2M loss as a tumor escape mechanism in clinical trials using anti-cancer vaccines together with immune-checkpoint inhibitors. Full article

Fibroblast activation protein (FAP) is a known promoter of tumor development and is associated with poor clinical outcome for various cancer types. Being specifically expressed in pathological conditions including multiple types of fibrosis and cancers, FAP is an optimal target for diagnostics and treatment. Treatment strategies utilizing the unique proteolytic activity of FAP are emerging, thus emphasizing the importance of biomarkers to directly assess FAP activity. FAP is a type II transmembrane serine protease that has been shown to cleave collagens and other ECM components. In this study, we developed an ELISA assay (C3F) targeting a circulating type III collagen fragment derived from FAP cleavage to reflect FAP activity. We demonstrated that C3F was specific to the neoepitope of the cleavage site and that the fragment was generated through FAP cleavage of type III collagen. We measured C3F in serum from a cohort of patients with non-small cell lung cancer (NSCLC) (n = 109) matched to healthy subjects (n = 42) and a cohort of patients with spondyloarthritis (SpA) (n = 17) matched to healthy subjects (n = 19). We found that C3F was significantly elevated in patients with NSCLC and in patients with SpA compared to healthy controls (p < 0.0001 and p = 0.0015, respectively). These findings suggest that C3F is a promising non-invasive biomarker reflecting FAP activity, which may aid in understanding tumor heterogeneity and potentially FAP-targeted therapies. Full article

The formation of neutralizing antibodies is a growing concern in the use of botulinum neurotoxin A (BoNT/A) as it may result in secondary treatment failure. Differences in the immunogenicity of BoNT/A formulations have been attributed to the presence of pharmacologically unnecessary bacterial components. Reportedly, the rate of antibody-mediated secondary non-response is lowest in complexing protein-free (CF) IncobotulinumtoxinA (INCO). Here, the published data and literature on the composition and properties of the three commercially available CF-BoNT/A formulations, namely, INCO, Coretox^® (CORE), and DaxibotulinumtoxinA (DAXI), are reviewed to elucidate the implications for their potential immunogenicity. While all three BoNT/A formulations are free of complexing proteins and contain the core BoNT/A molecule as the active pharmaceutical ingredient, they differ in their production protocols and excipients, which may affect their immunogenicity. INCO contains only two immunologically inconspicuous excipients, namely, human serum albumin and sucrose, and has demonstrated low immunogenicity in daily practice and clinical studies for more than ten years. DAXI contains four excipients, namely, L-histidine, trehalosedihydrate, polysorbate 20, and the highly charged RTP004 peptide, of which the latter two may increase the immunogenicity of BoNT/A by introducing neo-epitopes. In early clinical studies with DAXI, antibodies against BoNT/A and RTP004 were found at low frequencies; however, the follow-up period was critically short, with a maximum of three injections. CORE contains four excipients: L-methionine, sucrose, NaCl, and polysorbate 20. Presently, no data are available on the immunogenicity of CORE in human beings. It remains to be seen whether all three CF BoNT/A formulations demonstrate the same low immunogenicity in patients over a long period of time. Full article