Cell-mediated Research Articles

A self-amplifying RNA RSV prefusion-F vaccine elicits potent immunity in pre-exposed and naïve non-human primates

AbstractNewly approved subunit and mRNA vaccines for respiratory syncytial virus (RSV) demonstrate effectiveness in preventing severe disease, with protection exceeding 80% primarily through the generation of antibodies. An alternative vaccine platform called self-amplifying RNA (saRNA) holds promise in eliciting humoral and cellular immune responses. We evaluate the immunogenicity of a lipid nanoparticle (LNP)-formulated saRNA vaccine called SMARRT.RSV.preF, encoding a stabilized form of the RSV fusion protein, in female mice and in non-human primates (NHPs) that are either RSV-naïve or previously infected. Intramuscular vaccination with SMARRT.RSV.preF vaccine induces RSV neutralizing antibodies and cellular responses in naïve mice and NHPs. Importantly, a single dose of the vaccine in RSV pre-exposed NHPs elicits a dose-dependent anamnestic humoral immune response comparable to a subunit RSV preF vaccine. Notably, SMARRT.RSV.preF immunization significantly increases polyfunctional RSV.F specific memory CD4+ and CD8+ T-cells compared to RSV.preF protein vaccine. Twenty-four hours post immunization with SMARRT.RSV.preF, there is a dose-dependent increase in the systemic levels of inflammatory and chemotactic cytokines associated with the type I interferon response in NHPs, which is not observed with the protein vaccine. We identify a cluster of analytes including IL-15, TNFα, CCL4, and CXCL10, whose levels are significantly correlated with each other after SMARRT.RSV.preF immunization. These findings suggest saRNA vaccines have the potential to be developed as a prophylactic RSV vaccine based on innate, cellular, and humoral immune profiles they elicit.

Astragalus polysaccharide enhances maternal mucosal immunity against PEDV

ABSTRACT Porcine epidemic diarrhea virus (PEDV), the major causative pathogen of porcine epidemic diarrhea, poses a severe threat to the swine industry, particularly affecting neonatal piglets. Maternal milk-derived IgA antibody is crucial for protecting piglets from PEDV infection. Despite the effectiveness of current intramuscularly administered PEDV vaccines in inducing strong systemic immune responses, their ability to generate high levels of maternal milk IgA is limited. This study explores the potential of Astragalus polysaccharide (APS) to enhance PEDV vaccine efficacy, specifically focusing on maternal milk IgA levels. We first evaluated anti-PEDV antibody levels in the blood and colostrum of sows vaccinated with PEDV or subjected to feedback feeding. Our results indicated that while vaccination induced robust serum PEDV-specific IgG and IgA, milk IgA levels were lower compared to the feedback group. To address this limitation, APS was administered orally to sows before PEDV vaccination. APS supplementation significantly increased both serum and milk PEDV-specific IgA levels and enhanced cellular immune responses, as evidenced by elevated cytokine levels. Further analysis demonstrated that APS improved intestinal immune function and homeostasis in piglets. Overall, APS supplementation proved to be an effective immune booster, enhancing PEDV vaccine-induced mucosal immunity and providing a promising strategy for improving maternal immunity and piglet protection against PEDV. IMPORTANCE This study highlights the limitations of current porcine epidemic diarrhea virus (PEDV) vaccines in inducing sufficient maternal milk IgA, which is crucial for protecting neonatal piglets. By supplementing Astragalus polysaccharide (APS) into the vaccination regimen, we demonstrated a significant enhancement in milk PEDV-specific IgA levels, as well as improved cellular immune responses. APS also bolstered intestinal immune function and homeostasis in piglets. These findings suggest that APS supplementation could serve as an immune booster to enhance maternal immunity, offering a promising approach to better protect piglets against PEDV.

Neoantigen DNA vaccines are safe, feasible, and induce neoantigen-specific immune responses in triple-negative breast cancer patients

Abstract Background Neoantigen vaccines can induce or enhance highly specific antitumor immune responses with minimal risk of autoimmunity. We have developed a neoantigen DNA vaccine platform capable of efficiently presenting both HLA class I and II epitopes and performed a phase 1 clinical trial in triple-negative breast cancer patients with persistent disease on surgical pathology following neoadjuvant chemotherapy, a patient population at high risk of disease recurrence. Methods Expressed somatic mutations were identified by tumor/normal exome sequencing and tumor RNA sequencing. The pVACtools software suite of neoantigen prediction algorithms was used to identify and prioritize cancer neoantigens and facilitate vaccine design for manufacture in an academic GMP facility. Neoantigen DNA vaccines were administered via electroporation in the adjuvant setting (i.e., following surgical removal of the primary tumor and completion of standard of care therapy). Vaccines were monitored for safety and immune responses via ELISpot, intracellular cytokine production via flow cytometry, and TCR sequencing. Results Eighteen subjects received three doses of a neoantigen DNA vaccine encoding on average 11 neoantigens per patient (range 4–20). The vaccinations were well tolerated with relatively few adverse events. Neoantigen-specific T cell responses were induced in 14/18 patients as measured by ELISpot and flow cytometry. At a median follow-up of 36 months, recurrence-free survival was 87.5% (95% CI: 72.7–100%) in the cohort of vaccinated patients. Conclusion Our study demonstrates neoantigen DNA vaccines are safe, feasible, and capable of inducing neoantigen-specific immune responses. Clinical trial registration number NCT02348320.

Abstract PR005: Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, with a mere 13% average 5-year survival rate. The hallmark desmoplastic stromal response in PDAC leads to poor vascularization, drug delivery challenges, and impaired cytotoxic immune cell infiltration, contributing to de novo therapy resistance. Our previous research has shown therapeutic induction of cellular senescence with RAS pathway targeting therapies can induce cytokine and chemokine production through the senescence-associated secretory phenotype (SASP) that can effectively activate anti-tumor T cell immunity and responses to anti-PD-1 immune checkpoint blockade. To build on these findings as well as overcome limitations associated with senescence-inducing therapy toxicity and induction of pro-tumorigenic SASP cytokines, here we leveraged mRNA technology to deliver specific SASP cytokines and chemokines we have found to stimulate immune responses directly into the suppressive PDAC TME as an immune oncology platform. We created an in vitro transcription pipeline for mRNA production and multiplexing, and demonstrate we can intratumorally deliver multiple mRNAs simultaneously into the TME of transplanted and autochthonous PDAC mouse models, stimulating the local production of cytokines normally absent in PDAC. We further characterized a novel combination of five cytokines and chemokines that can effectively recruit and activate both innate and adaptive immune responses against PDAC. Through repeated bi-weekly dosing, this mRNA combination also promotes tumor destruction and improves survival outcomes. Considering the recent success of off-the-shelf neoantigen mRNA vaccines in early human PDAC patient trials, we have now developed mRNAs that encode PDAC-specific antigens. By integrating our cytokine mRNA platform with antigen mRNAs, we observe a significant increase in intratumoral antigen-presenting dendritic cell populations and a substantially enhanced T-cell mediated anti-tumor immune response in orthotopic transplant models of PDAC. Overall, this study not only unveils pivotal insights into the cytokines absent in PDAC that are crucial for promoting anti-tumor immunity, but also pioneers an innovative immunotherapy approach. This platform has the potential to be integrated with existing immunotherapy modalities, addressing the longstanding challenge of therapy resistance in PDAC. Citation Format: Chaitanya Naimesh Parikh, Kelly De Marco, Boyang Ma, Katherine Murphy, Loretah Chibaya, Lin Zhou, Youwei Qiao, Griffin Kane, Li Li, Wen Xue, Marcus Ruscetti. Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR005.

Abstract A004: RNA splicing alterations in the development of acquired MAPKi resistance in melanoma

Abstract The standard treatment for melanoma with a BRAF mutation involves Mitogen-Activated Protein Kinase inhibitors (MAPKi), which target the aberrant MAPK signaling pathway. Although patients initially respond well to this therapy, many develop acquired resistance over time. This resistance can be attributed to various altered signaling pathways, including the reactivation of the MAPK pathway, activation of alternative survival pathways like PI3K/PTEN/AKT, engagement of receptor tyrosine kinases (RTKs) such as PDGFRβ and EGFR, and developmental pathways. Despite extensive omics studies aimed at deciphering the mechanisms behind acquired resistance, the specific alterations within these pathways remain poorly understood. To gain further insights into acquired MAPKi resistance in melanoma, we explored the role of RNA splicing events by analyzing publicly available datasets of pre- and post-MAPKi treated melanoma from patient-derived cell lines and in vitro studies using PDX models. We investigated differential transcript usage (DTU) to detect specific splice variants altered during the development of resistance. Our analysis revealed significant transcript alterations during on-treatment that reverted to baseline states once resistance was established, underscoring the dynamic and adaptive nature of these changes. Genes with DTU were enriched in pathways related to MAPKi resistance, such as the MAPKi signaling pathway, PI3K/AKT pathway, and signaling by RTKs. Furthermore, the DTU-centered analysis provided better insights into MAPKi resistance mechanisms compared to standard differential gene expression (DEG) analysis, with DTUs showing higher enrichment scores in MAPKi resistance-related pathways. Further, identifying developmental splicing signatures highlights the complexity of MAPKi resistance, as it reveals significant upregulation of transcripts associated with embryonic melanoblast stem cells (MSCs) among the altered transcripts in on-treatment cell lines. This reprogramming through RNA splicing may confer an adaptive advantage, enabling melanoma cells to revert to a more plastic, stem-like state, with developmental splicing events playing a crucial role in the adaptive response to MAPKi therapy. Next, using a regulatory model based on the expression of splicing factors (SFs), we accurately predicted transcript alterations in on-treatment samples and identified key SFs. Our study highlights the crucial role of RNA splicing in the adaptive response of melanoma cells to MAPKi treatment. These insights pave the way for future research and therapeutic strategies focusing on RNA splicing to combat drug resistance in cancer. Citation Format: Sumit Mukherjee, Arashdeep Singh, Hyunjeong Joo, Sumeet Patiyal, Hyungsoo Kim, Lipika R. Pal, Kun Wang, Chi-Ping Day, Ze’ev A Ronai, Eytan Ruppin, Sridhar Hannenhalli. RNA splicing alterations in the development of acquired MAPKi resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A004.

Abstract A030: Beyond the LNP: Self-amplifying RNA with LION nanoformulation technology for development of safe and effective cancer therapeutics

Abstract HDT Bio, a Seattle biotech company, has developed a platform technology based on self-amplifying RNA and a proprietary nanoparticle delivery system. mRNA have been increasingly used in the development of vaccines in both infectious diseases and oncology. Especially LNP- delivered mRNA has been deployed, following the success during the COVID pandemic, However, self-ampliying RNA (repRNA) possesses several features of particular relevance to cancer medicine. HDT has developed a nanoparticle technology named LION[TM] to safely and effectively deliver RNA to patients. In vivo studies showed that repRNA not only is capable of delivering robust cell-mediated immune responses but also doing so with a significanlty improved safety profile compared to administration using LNP-based formulations. These data will be presented in the context of clinical safety data with repRNA/LION. Furthermore, repRNA/LION was siuccessfully used to induce robust T-cell responses against tumor antigens, and will be discussed. Citation Format: Peter Berglund. Beyond the LNP: Self-amplifying RNA with LION nanoformulation technology for development of safe and effective cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A030.

Staphylococcal superantigens evoke temporary and reversible T cell anergy, but fail to block the development of a bacterium specific cellular immune response

Staphylococcal superantigens evoke temporary and reversible T cell anergy, but fail to block the development of a bacterium specific cellular immune response

Abstract A024: Assessing Mitondiral Priming in Leukemia Using BH3 Complex Specific Antibodies (PRIMABS): Prediciting Patient Response to Apoptosis Inducing Drugs

Abstract Diagnostic tools for cancer therapy that enable oncologists to inform patients about the likely benefits of their treatment versus toxicity will empower patients to make evidence-based decisions about the management of their disease, and in some cases, their last phase of life. Here we provide a novel approach that measures key drug response features of individual cancer cells with the potential for providing prognostic/predictive diagnostic utility for practicing precision medicine. Most cancer treatments work though activation of the cell suicide program of apoptosis. This process is tightly regulated by the BCL-2 family of proteins whose members either prevent or promote cell death. The relative levels of, and the specific interactions between the pro-survival and pro-apoptotic family members that dictates the fate of all cells and determines their propensity to undergo apoptosis. Cells that more readily die in response to various stimuli are referred to as “primed”. Hence, the degree of priming of a tumor sample can be predictive of responses to anti-cancer drugs and can be established using a technique called “BH3 profiling”. The relevance of this method has been demonstrated but it has had only limited application. To address this limitation a unique panel of antibody-based reagents that are mitochondrial PRIMing state detecting Antibodies (PRIMABS), have been developed by our lab, as a novel approach to implementing personalized medicine and developing predicative diagnostic tests. These antibodies enable the degree of tumor cell priming to be determined directly, unlike BH3 profiling which is an indirect measure. The key innovation lies in the approach taken to determine the presence (or absence) of the critical pro-survival : pro-apoptotic protein complexes , as these are the principal determinants of cell priming.. Such PRIMAB reagents have hitherto not been available. Here we provide data outlining the methodology development as well as evidence demonstrating feasibility of PRIMABTM Dx platform for predicting AML and CLL patient response in retrospective studies. Our platform provides a PD biomarker that will become a clinical predictor of cancer cell response to treatments tat are intended to induce apopstosis in tumor cells. The PRIMABTM -DX platform will enable heretofore hard to achieve clinical applicability of priming measurements. This will be significant due to the important prognostic implications of PRIMAB-DxTM readouts and will provide insights into drug resistance and sensitivity mechanisms and lead to new treatment options for cancer patients. Citation Format: Michael Cardone. Assessing Mitondiral Priming in Leukemia Using BH3 Complex Specific Antibodies (PRIMABS): Prediciting Patient Response to Apoptosis Inducing Drugs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A024.

Type I interferon signaling in dendritic cells limits direct antigen presentation and CD8 + T cell responses against an arthritogenic alphavirus

ABSTRACT Ross River virus (RRV) and other alphaviruses cause chronic musculoskeletal syndromes that are associated with viral persistence, which suggests deficits in immune clearance mechanisms, including CD8 + T-cell responses. Here, we used a recombinant RRV-gp33 that expresses the immunodominant CD8 + T-cell epitope of lymphocytic choriomeningitis virus (LCMV) to directly compare responses with a virus, LCMV, that strongly induces antiviral CD8 + T cells. After footpad injection, we detected fewer gp33-specific CD8 + T cells in the draining lymph node (DLN) after RRV-gp33 than LCMV infection, despite similar viral RNA levels in the foot. However, less RRV RNA was detected in the DLN compared to LCMV, with RRV localizing principally to the subcapsular region and LCMV to the paracortical T-cell zones. Single-cell RNA-sequencing analysis of adoptively transferred gp33-specific transgenic CD8 + T cells showed rapid differentiation into effector cells after LCMV but not RRV infection. This defect in RRV-specific CD8 + T effector cell maturation was corrected by local blockade of type I interferon (IFN) signaling, which also resulted in increased RRV infection in the DLN. Studies in Wdfy4 −/− , CD11c-Cre B2m fl/fl , or Xcr1-Cre Ifnar1 fl/fl mice that respectively lack cross-presenting capacity, MHC-I antigen presentation by dendritic cells (DCs), or type I IFN signaling in the DC1 subset show that RRV-specific CD8 + T-cell responses can be improved by enhanced direct antigen presentation by DCs. Overall, our experiments suggest that antiviral type I IFN signaling in DCs limits direct alphavirus infection and antigen presentation, which likely delays CD8 + T-cell responses. IMPORTANCE Chronic arthritis and musculoskeletal disease are common outcomes of infections caused by arthritogenic alphaviruses, including Ross River virus (RRV), due to incomplete virus clearance. Unlike other viral infections that are efficiently cleared by cytotoxic CD8 + T cells, RRV infection is surprisingly unaffected by CD8 + T cells as mice lacking or having these cells show similar viral persistence in joint and lymphoid tissues. To elucidate the basis for this deficient response, we measured the RRV-specific CD8 + T-cell population size and activation state relative to another virus known to elicit a strong T-cell response. Our findings reveal that RRV induces fewer CD8 + T cells due to limited infection of immune cells in the draining lymph node. By increasing RRV susceptibility in antigen-presenting cells, we elicited a robust CD8 + T-cell response. These results highlight antigen availability and virus tropism as possible targets for intervention against RRV immune evasion and persistence.

Innate immunodeficiencies: a group of primary immunodeficiencies predisposing exclusively to common diseases

Abstract Background Innate immune deficiencies can impair both cellular and humoral immune responses. In contrast, other immune functions may appear normal, leading to increased susceptibility to specific pathogens, such as severe viral infections or Mendelian Susceptibility to Mycobacterial Disease (MSMD). Studying these deficiencies is essential for understanding the pathophysiology of these infectious diseases. Main body While primary immunodeficiencies (PIDs) generally cause vulnerability to multiple infections, innate immunodeficiencies increase susceptibility to specific pathogens, despite normal immune responses to others. Patients with these deficiencies show normal immunoglobulins and lymphocyte subpopulations, complicating diagnosis. This review highlights genetic susceptibility to mycobacteria, pneumococci, herpes simplex virus, and candidiasis, emphasizing recognizing this subset of PIDs. Conclusion This review highlights the diverse spectrum of genetic mutations contributing to defects in innate and intrinsic immunity, including Mendelian susceptibility to mycobacterial disease (MSMD), chronic mucocutaneous candidiasis, and predispositions to invasive bacterial and viral infections. Identifying key mutations in pathprovideh such as TLR3, IFN signaling, and IL-17A/F immunity provides valuable insights into the pathogenesis of these conditions. Our findings underscore the need for early genetic diagnosis and targeted interventions, particularly in regions with high undiagnosed cases, to reduce the morbidity and mortality associated with defects in innate and intrinsic immunity.

Dry polymer model for a string of pushers: Nontrivial scaling of tumbling time with shear rate

Understanding the dynamics of active polymers is an important component for modeling the dynamic response of biological cells and microorganisms when subjected to external forces. A minimal computational model of active filaments that captures all the essential features of activity-induced filament motion is required to study large systems of dense suspensions. In this paper we present a model of a dry active polymer, compare its steady-state static and dynamic properties with those of a polymer consisting of extensile stresslets, and show that the two models give very similar results. When subjected to shear flow, both models show two distinct regimes in the tumbling interval as a function of shear rate, with a diffuse crossover regime in between. At high shear rate, the active and passive polymers follow the same power law for the tumbling interval. However, the tumbling interval is smaller than that of the passive polymer for low shear rates and larger for the high shear rate. We show that activity-induced large bends increase the coupling of the polymer with the shear gradient in the low-shear-rate regime, leading to a decrease in the tumbling interval. However, in the high-shear-rate regime, the active forces on the folds at the end of the stretched polymer prevent it from tumbling, leading to a larger tumbling interval. We show that the critical shear rate, at which the tumbling interval versus the shear-rate curve of the active polymer crosses that of the passive polymer, is determined by the balance of bending and shear forces and increases with κ/N3, where κ is the bending coefficient and N is the length of the polymer. Published by the American Physical Society 2024

Modulators of Alpha-2 Macroglobulin Upregulation by High Glucose in Glomerular Mesangial Cells

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD.

Influence of dental prostheses materials on the condition of oral mucosa local homeostasis

Objective. To study the influence of base materials used in the manufacture of removable dentures on the immune homeostasis of the oral mucosa, and, as a consequence, on the clinical condition of the oral mucosa and periodontium of supporting teeth. To create fundamental principles for the development of a pathogenetically substantiated choice of basic dental materials of various chemical natures for removable prosthetics based on the analysis of the parameters of immune homeostasis of the oral mucosa. Materials and methods. 154 patients aged 40 to 85 using dentures based on acrylic, polyurethane plastics and thermoplastics for 6 months, participated in the study. Indicators of immune homeostasis and the identified clinical manifestations of the influence of the prosthesis material such as chronic periodontitis and gingivitis were compared using the statistical method of conjugate multifield tables. Results. The indicators CD4+/CD8+, CD11/CD303, CD68, CD204, CD163, Ki-67 phenotypic markers were determined in gingival biopsies of the prosthetic bed of patients using dentures based on acrylic, polyurethane and thermoplastic for 6 months. Analysis of the statistical significance of differences proved the influence of the prosthesis material on indicators of cellular immune homeostasis, with a shift of homeostasis towards the pro-inflammatory phenotype, and the suppression of the anti-inflammatory factor in circulating monocytes. Conclusions. The study shows that, although each type of removable dentures has its own advantages and disadvantages, the response of the cellular immunity of the oral mucosa to polyurethane dentures is less than to acrylic and thermoplastic ones in relation to the control group. The use of polyurethane prostheses is reasonable as temporary ones during the healing stage of surgical interventions in the oral cavity, in particular tooth extraction and implantation. This type of prosthesis can be recommended as a permanent one for patients with a severe allergic reaction, especially to polymer compounds and dyes.

Insulin-degrading enzyme regulates insulin-directed cellular autoimmunity in murine type 1 diabetes

Type 1 diabetes results from the destruction of pancreatic beta cells by autoreactive T cells. As an autoantigen with extremely high expression in beta cells, insulin triggers and sustains the autoimmune CD4+ and CD8+ T cell responses and islet inflammation. We have previously shown that deficiency for insulin-degrading enzyme (IDE), a ubiquitous cytosolic protease with very high affinity for insulin, induces endoplasmic reticulum (ER) stress and proliferation in islet cells and protects non-obese diabetic mice (NOD) from diabetes. Here we wondered whether IDE deficiency affects autoreactive CD8+ T cell responses to insulin and thereby immune pathogenesis in NOD mice. We find that Ide-/- NOD harbor fewer diabetogenic T cells and reduced numbers of CD8+ T cells recognizing the dominant autoantigen insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Using in vitro digestions and cellular antigen presentation assays, we show that generation of the dominant insulin epitope B15-23 involves both the proteasome and IDE. IDE deficiency attenuates MHC-I presentation of the immunodominant insulin epitope by beta cells to cognate CD8+ T cells. Consequently, Ide-/- islets display reduced susceptibility to autoimmune destruction upon grafting, and to killing by insulin-specific CD8+ T cells. Moreover, Ide-/- mice are partly resistant to disease transfer by CD8+ T cells specific for insulin but not for IGRP. Thus, IDE has a dual role in beta cells, regulating ER stress and proliferation while at the same time promoting insulin-directed autoreactive CD8+ T cell responses.

Abstract 4136692: Spatial Transcriptomics Identifies Novel Clinical Markers to Classify the Etiology of Rare and Aggressive Cardiac Sarcomas

Background: Cardiac sarcomas, such as leiomyosarcoma (LMS), synovial sarcoma (SS), and undifferentiated pleomorphic sarcoma (UPS), lack sufficient diagnostic markers for early diagnosis and tailored therapeutic interventions. Understanding their molecular signatures with spatial-temporal resolution compared to normal heart tissue is crucial. Hypothesis: Spatial transcriptomics will reveal a comprehensive set of novel clinical markers, including changes in cellular composition, stemness, and metabolic profiles unique for each cardiac sarcoma subtypes. Aims: Identify novel molecular markers for the accurate diagnosis of cardiac sarcoma subtypes. Methods: Using 10X Visium spatial transcriptomics, we analyzed three cardiac sarcoma subtypes (LMS, SS, UPS) and compared them with normal heart tissue. Differential expression analysis identified novel target genes suitable for clinical disease stratification. Subsequently, machine learning models reveal an unbiased transcriptomic profile to distinguish normal from sarcoma tissues. Finally, immunohistochemistry analysis validated the differential marker gene expression. Results: We identify several novel clinical markers, such as IGKC, PCOLCE, NET1, TLE2, MDFI, BGN, TNNC1, and CNN3, that classify normal heart tissue from sarcoma subtypes. Machine learning models and immunostaining analysis confirm the transcriptomic signature in each sarcoma subtype. Pathway enrichment analysis show LMS enriched in antigen processing and B cell-mediated immunity, SS in protein folding and response to incorrect proteins, and UPS in axon development and Wnt signaling regulation. Trajectory Reconstruction Analysis indicate higher stemness scores in all sarcomas, with SS highest, followed by UPS and LMS. Additional cell cycling analysis show LMS cells in G2M phase, SS in G1, and UPS in S phase. SS express a high number of cancer stem markers (EZH2, BMI1, KDM5B, LGR5, HIF1A), while UPS express KDM5B and CTNNB1, and LMS show fewer stem-related marker genes (EZH2, BMI1, KDM5B, CTNNB1). Both SS and LMS express CD44. Glucose and oxidative pathways are also differentially regulated in each sarcoma subtype. Conclusion: Spatial transcriptomics uncovers distinct clinical markers, stemness characteristics, and metabolic activity profiles in cardiac sarcomas, offering insights for fast, accurate and improved diagnostics and therapeutics.

Abundance of Tumor-Infiltrating B Cells in Human Epithelial Malignancies

Cancer is a major global health problem. The type of malignant neoplasm and the potency of the immune response against tumors are two of the key factors influencing the outcome of the disease. The degree of tumor infiltration by lymphocytes plays an important role in antitumor response development, generally correlating with a favorable prognosis of treatment for certain cancers. We analyzed the abundance of tumor-infiltrating B cells (TIBs) in solid tumors of different cancers. TIBs were shown to be more abundant in colon and sigmoid colon cancer samples compared with cecal, rectal, and kidney cancer samples. The median and interquartile range of the TIB fraction were 11.5% and 4–20% in colon cancer, 6% and 3–11% in sigmoid colon cancer, 2.7% and 0.7–3.7% in cecal cancer, 2.5% and 0.9–3.6% in rectal cancer, 1.4% and 1.0–2.3% in kidney cancer, and 3.0% and 1.8–12% in lung cancer, respectively. However, there were no significant differences in the abundance of TIBs among samples at different stages of the cancer. Hence, investigation of the B cell response in colon cancer is of particular interest, since increased quantities of TIBs may indicate the existence of immunogenic tumor markers or the cell-cell interactions involved in disease progression. We believe that studying the diversity of TIBs in colon cancer will increaseour understanding of the mechanisms of the disease, contributing to the identification of new molecular targets for targeted oncotherapy.

Abstract 4136901: Mechanosensitive channel protein Piezo1 synergizes with TLR4 to enhance osteogenic responses in human aortic valve interstitial cells

Background: Calcific aortic valve disease (CAVD), a common heart valve disease in the elderly, has a complex pathogenesis. Abnormal blood flow shear can be regarded as an important factor in the development of CAVD, and a key molecule that responds to mechanical stress is the mechanosensitive ion channel protein Piezo1; however, the role of Piezo1 in CAVD has not been clarified. Methods and results: We collected CAVD patients and their age- and sex-matched controls, and compared the differences in the protein expression levels of Piezo1 and TLR4 between the two using Western blot and other techniques, and found that the expression of Piezo1 and TLR4 proteins was elevated in the CAVD group. We gave LPS or Piezo1 agonist Yoda1 to stimulate aortic valve mesenchymal stromal cells, respectively, and the Western blot results showed that ALP and Runx2 expression were elevated after LPS and Yoda1 stimulation, and immunofluorescence and alizarin red staining showed similar results. Inhibition of TLR4 or Piezo1 attenuated the osteogenic differentiation of AVICs. Cellular immunofluorescence results showed that TLR4 could be transported from the Golgi to the cell membrane under Yoda stimulation, and Golgi-cell membrane transport of TLR4 was attenuated after Rab3b knockdown by siRNA. After first pre-stimulation with Yoda1 and then stimulation with LPS, Western blot and immunofluorescence results showed that the protein levels of ALP and Runx2 as well as NF-κB phosphorylation were to be significantly increased. Conclusion: Stimulation of Piezo1 and TLR4 can enhance the osteogenic response of AVICs. Stimulation of Piezo1 enhances the transport of TLR4 from the Golgi to the cell membrane via Rab3b, and Piezo1 synergistically enhances the osteogenic response of AVICs with TLR4, thus promoting the development of CAVD.

Fasting as an Adjuvant Therapy for Cancer: Mechanism of Action and Clinical Practice

The fundamental biological characteristics of tumor cells are characterized by irregularities in signaling and metabolic pathways, which are evident through increased glucose uptake, altered mitochondrial function, and the ability to evade growth signals. Interventions such as fasting or fasting-mimicking diets represent a promising strategy that can elicit distinct responses in normal cells compared to tumor cells. These dietary strategies can alter the circulating levels of various hormones and metabolites, including blood glucose, insulin, glucagon, growth hormone, insulin-like growth factor, glucocorticoids, and epinephrine, thereby potentially exerting an anticancer effect. Additionally, elevated levels of insulin-like growth factor-binding proteins and ketone bodies may increase tumor cells’ dependence on their own metabolites, ultimately leading to their apoptosis. The combination of fasting or fasting-mimicking diets with radiotherapy or chemotherapeutic agents has demonstrated enhanced anticancer efficacy. This paper aims to classify fasting, elucidate the mechanisms that underlie its effects, assess its impact on various cancer types, and discuss its clinical applications. We will underscore the differential effects of fasting on normal and cancer cells, the mechanisms responsible for these effects, and the imperative for clinical implementation.

Comparison of inflammatory mediator cytokine responses to inactivated virus platform COVID-19 vaccines between elderly and young adult populations

The coronavirus disease 2019 (COVID-19) pandemic has encouraged global vaccine research, yet vaccine effectiveness in the elderly remains a concern due to immunosenescence. The aim of this study was to compare the cytokine response elicited by an inactivated virus-based COVID-19 vaccine between elderly and young adults, focusing on key cytokines involved in cellular and humoral immunity: tumor necrosis factor-alpha (TNF-α), interleukin (IL)-2, IL-6, IL-10, and interferon-gamma (IFN-γ). A cross-sectional study was conducted in the Jakarta-Bogor region of Indonesia from January 2023 to December 2023. The study population was divided into two age cohorts: elderly (60−85 years) and younger adults (30−40 years). Blood samples were collected twice, after the first booster dose and four weeks after the second booster dose. Serum cytokine concentrations were measured using Luminex assays with microparticles conjugated to monoclonal antibodies against TNF-α, IL-2, IL-6, IL-10, and IFN-γ. Comparisons of the cytokine levels were conducted using Student’s t-tests or Mann-Whitney U tests as appropriate. A total of 74 individuals were included, with 37 each in the elderly and young adult groups. The results showed significant differences in cytokine responses between the two age groups. After the first booster, the levels of IL-6 and IFN-γ were significantly higher in young adults compared to the elderly. After the second booster, the levels of IL-6 were still significantly higher in the young adult group compared to the elderly group (p=0.001). Data indicated that after the second booster dose, the levels of TNF-α increased significantly in the young adult group only (p=0.004), while the levels of IL-2 (p=0.040) and IFN-γ (p=0.006) increased in the elderly group only compared to after the first dose. IL-10 levels increased in both groups (both had p=0.020). This study highlights that young adults had stronger pro-inflammatory responses, while the elderly relied more on IL-2 and IFN-γ for T-cell immunity, suggesting the need for vaccination strategies for the elderly to optimize immune responses.

Cytomegalovirus Infections in Hematopoietic Stem Cell Transplant: Moving Beyond Molecular Diagnostics to Immunodiagnostics

Human CMV, regularly reactivated by simple triggers, results in asymptomatic viral shedding, powerful cellular immune responses, and memory inflation. Immunocompetent individuals benefit from a robust immune response, which aids in viral management without causing clinically significant illness; however, immunodeficient individuals are always at a higher risk of CMV reactivation and disease. Hematopoietic stem cell transplant (HSCT) recipients are consistently at higher risk of CMV reactivation and clinically significant CMV illness due to primary disease, immunosuppression, and graft vs. host disease. Early recovery of CMV-CMI responses may mitigate effects of viral reactivation in HSCT recipients. Immune reconstitution following transplantation occurs spontaneously and is mediated initially by donor-derived T cells, followed by clonal growth of T cells produced from graft progenitors. CMV-specific immune reconstitution post-transplant is related to spontaneous clearance of CMV reactivation and may eliminate the need for prophylactic or pre-emptive medication, making it a potential predictive marker for monitoring CMV reactivation. This review highlights current thoughts and therapeutic options for CMV reactivation in HSCT, with focus on CMV immune reconstitution and post-HSCT monitoring. Immune monitoring aids in risk stratification of transplant recipients who may progress from CMV reactivation to clinically significant CMV infection. Implementing this approach in clinical practice reduces the need for periodic viral surveillance and antiviral therapy in recipients who have a high CMV-CMI and thus may experience self-limited reactivation. Therefore, in the age of precision medicine, it is critical to incorporate CMV-specific cellular immune surveillance into conventional procedures and algorithms for the management of transplant recipients.