Mature Cells Research Articles

Abstract B022: Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer (BC) cases worldwide, yet due to its aggressive phenotype and lack of targeted therapies it has the worst prognosis amongst BC subtypes. Up to 40% of TNBC patients will relapse after treatment and develop distant metastasis. Metastasis is the complex process by which disseminated tumor cells (DTCs) leave the primary tumor and grow at distal sites. Since most metastases occur within 3 years of diagnosis, the rapid onset and lack of effective treatments in metastatic TNBC (mTNBC) means less than 20% of patients survive after 4 years. Even though it is the most “immune-activated” BC subtype, and immune checkpoint blockade (ICB) has shown clinical benefits in early TNBC, there is a clear need for novel immunotherapeutic approaches for patients with mTNBC. Natural killer (NK) cells are innate lymphocytes that function as the body’s first line defense against metastatic cancer cells. NK cells eliminate DTCs in the blood or at distal sites via NK cell cytotoxicity (NKCC) and this process is known to be impaired in successful metastasis. We hypothesized that other cells within the tumor microenvironment (TME) can inhibit NKCC at the earliest stages of metastatic outgrowth. We have used Cherry-niche, an in vivo labelling tool, to characterize how TME cells change in early and established lung micro-metastases by single-cell RNA sequencing (scRNA-seq) and flow cytometry. Our initial analyses showed that monocytes are highly enriched, not only in the entire metastatic lung, but specifically in the established niche surrounding DTCs. Although we detected a slight increase in total NK cells, there was a shift in maturation status with fewer mature NK cells (CD27-CD11b+) and more immature NK cells (CD27+/-CD11b-) in the metastatic lung. Furthermore, NK cells were largely absent from the metastatic niche and those that were present displayed a dysfunctional phenotype. CellChat and NicheNet analyses of our scRNA-seq data predicted that several receptor-ligand interactions, including the cytokine macrophage migration inhibitory factor (MIF), can mediate the crosstalk between NK cells and monocytes. Next, we developed an in vitro 3D co-culture system to study the effect of blood-derived monocytes on human NKCC against mTNBC cells. We found that monocytes can suppress NKCC against MDA-MB-231 cells and decrease the viability of mature NK cells. Furthermore, recombinant MIF was able to directly inhibit NKCC, whilst a selective MIF antagonist was able to rescue monocyte-induced impairment of NKCC. These data implicate MIF as a critical monocyte-derived factor in the suppression of NK cell function against mTNBC cells. Future work will aim to identify the exact mechanism by which MIF inhibits NKCC, whilst also evaluate the effect of monocyte depletion and MIF inhibition on metastatic outgrowth and NK cell activation/maturation in vivo. This study will reveal novel inter-cellular dynamics occurring in the metastatic lung, as well as immunotherapeutic targets that may restore NKCC in mTNBC. Citation Format: Christos Ermogenous, Luigi Ombrato, Gordon Beattie. Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B022.

Abstract PR012: KRAS mutation-specific effects on the tumor immune microenvironment drive tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with abysmal survival rates. The highly immunosuppressive microenvironment of PDAC poses a major barrier to effective therapy. KRAS mutations are near-ubiquitous in PDAC, and studies indicate clinical outcomes vary depending on the specific KRAS genotype. Given that tumor genotype can shape the immune cell composition of tumors, we examined the immune cell composition of pancreatic tumors with specific KRAS mutations, focusing on KRASG12D mutations (representing 40% of PDAC cases which have the worst prognosis) and KRASG12R mutations (which make up 20% of PDAC cases and have a better prognosis). Orthotopically implanted Kras G12R/+ ;Trp53 KO mouse pancreatic tumors have a distinct immune profile in comparison to Kras G12D/+ ;Trp53 KO tumors, characterized by an influx of intratumoral CD3+ T cells and mature dendritic cells. Selective depletion of CD4+ T cells induces a rapid progression in KRASG12R, but not KRASG12D mouse tumors. RNA sequencing of KRASG12R tumor cells revealed enrichment of Type I interferon (IFN) pathways accompanied by related chemokines, CXCL9 and CXCL10 in comparison to KRASG12D tumors. Analysis of publicly available and real-world human PDAC datasets and enrichment of dendritic cells and other immune related cell types in KRASG12R tumors. Collectively, these data link the KRASG12R mutation to a unique immunogenic role. Citation Format: Andrew Wenger, Tal Baron, Erika Hissong, Rohit Chandiwani, Lukas Dow, Despina Siolas, Katherine Bacchi, Huanhuan Sun, Erika Hissong, Adrian Vega, Whitney Sisso, and Kawther Abdilleh. KRAS mutation-specific effects on the tumor immune microenvironment drive tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR012.

Abstract C017: Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis

Abstract Langerhans cell histiocytosis (LCH) is a rare inflammatory hematological neoplasm, characterized by the accumulation of LCH cells in various tissues and organs. These LCH cells originate from myeloid progenitors that carry a MAPK-pathway mutation. Once these mutated cells enter the tissue from the periphery, they differentiate into so-called LCH cells. In addition to LCH cells, the tumor microenvironment (TME) consists of a wide variety of immune cells. However, the interactions between these cells within the TME and their contribution to disease progression remain poorly understood. In this study, we focus on understanding the role of lymphocytes in LCH lesions. We used single-cell RNA sequencing to comprehensively profile the immune populations in LCH lesions on the transcriptomic level, which will be supplemented with spatial profiling. Our analysis identified a wide variety of infiltrating immune cells within LCH lesions with high cellular heterogeneity among patients. LCH- and T cells are the most prevalent cell types, suggesting a key role in lesion development and maintenance. Furthermore, the immune landscape includes several dendritic cell subsets – such as plasmacytoid DCs (pDCs), cDC1, and mature regulatory dendritic cells (mregDCs) – alongside tumor-associated macrophages (TAMs), monocytes, NK cells, B cells, and plasmablasts. Among T cells, regulatory T cells (Tregs) are particularly abundant and appear to be heavily influenced by LCH cells and other myeloid cells via the TNF-TNFR2 and CD86-CTLA4 pathways. Additionally, LCH cells express high levels of TGFβ – a known regulator of T cell activity – and MMP9 – which might hamper cytotoxic T cell infiltration. This suggests that LCH cells actively contribute to creating an immunosuppressive environment. Surprisingly, pDCs seem to be a major immune regulator within lesions and are likely to interact with T cells via TNFSF9 and Granzyme B. These insights into the immunosuppressive environment within LCH lesions highlight the critical role of Tregs as central regulators within the tumor microenvironment. The implications of these findings will be further explored and validated through in-vitro experiments and high-plex spatial protein profiling. Ultimately, our work may pave the way for developing targeted therapies aimed at modulating the TME, potentially enhancing anti-tumor immunity, and offering new strategies for treating LCH. Citation Format: Wouter van Midden, Raphaela Schwentner, Sebastian Eder, Philipp Ben Soussia-Weiss, Giulio Abagnale, Caroline Hutter. Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C017.

Angiofibrolipoma in the Head and Neck Region: A Case Report and Literature Review.

Angiofibrolipomas are rare, benign soft tissue tumors composed of mature fat cells, blood vessels, and fibrous connective tissue. Typically occurring in subcutaneous locations, this case report presents a unique instance of an angiofibrolipoma arising in the pharynx. According to our review of the literature, it is the second reported case in the pharynx and the sixth in the head and neck region. A 58-year-old woman presented with a 1 year history of globus sensation and an incidentally-discovered extra-oral polyp. Endoscopic examination revealed the polyp originating from the upper esophagus and extending into the pharynx. Magnetic resonance imaging confirmed the nature and extent of the lesion. The polyp was successfully removed via endoscopic resection, and histopathological analysis confirmed the diagnosis of angiofibrolipoma. This case highlights the rarity of pharyngeal angiofibrolipomas and underscores the importance of endoscopic evaluation for patients presenting with ambiguous pharyngeal symptoms. While surgical resection is generally recommended for such tumors, endoscopic removal can be a safe and effective option for smaller pedunculated lesions.

Enduring effects of acute prenatal ischemia in rat soleus muscle, and protective role of erythropoietin.

Motor disorders are considered to originate mainly from brain lesions. Placental dysfunction or maternal exposure to a persistently hypoxic environment is a major cause of further motor disorders such as cerebral palsy. Our main goal was to determine the long-term effects of mild intrauterine acute ischemic stress on rat soleus myofibres and whether erythropoietin treatment could prevent these changes. Rat embryos were subjected to ischemic stress at embryonic day E17. They then received an intraperitoneal erythropoietin injection at postnatal days 1-5. Soleus muscles were collected at postnatal day 28. Prenatal ischemic stress durably affected muscle structure, as indicated by the greater fiber cross-sectional area (+ 18%) and the greater number of mature vessels (i.e. vessels with mature endothelial cells) per myofibres (+ 43%), and muscle biochemistry, as shown by changes in signaling pathways involved in protein synthesis/degradation balance (-81% for 4EBP1; -58% for AKT) and Hif1α expression levels (+ 95%). Erythropoietin injection in ischemic pups had a weak protective effect: it increased muscle mass (+ 25% with respect to ischemic pups) and partially prevented the increase in muscle degradation pathways and mature vascularization, whereas it exacerbated the decrease in synthesis pathways. Hence, erythropoietin treatment after acute ischemic stress contributes to muscle adaptation to ischemic conditions.

Membrane Budding Inspired Biomimetic Nanocarrier Delivers Brain‐derived Neurotrophic Factor to Improve AD Cognition

AbstractNeurotropic factors, crucial for neural cell maturation and proliferation, hold great therapeutic potential for treating neurodegenerative diseases but face challenges in brain delivery. This study introduces a novel membrane budding‐inspired lipoprotein biomimetic nanocarrier for efficient packaging and precise brain delivery of brain‐derived neurotrophic factor (BDNF). The nanocarrier is created by mixing protein‐loaded biomimetic gel with liposomes composed of lipids prone to forming liquid‐disordered and liquid‐ordered phases. This interaction triggers phase separation and lipid membrane rearrangement, enabling effective protein encapsulation. To enhance blood‐brain barrier permeability and target damaged cerebral vasculature in Alzheimer's Disease, the nanocarrier (RAP‐BHP‐rHDL) is functionalized with Apolipoprotein E3 and αRAP peptides. The obtained RAP‐BHP‐rHDL alleviates neuronal damage, promotes neurogenesis, normalizes the cerebral microvasculature, improves the function of neurovascular units, and restores memory function in 5 × FAD mice. This innovative packaging approach and biomimetic nanocarrier design offer a promising strategy for delivering neurotropic factors to the central nervous system, potentially advancing the management of neurodegenerative diseases.

Efficient Dendritic Cell Activation by a Layered Double Hydroxide‐Loaded Dual Adjuvant Cocktail

AbstractOne of the main goals of present vaccine investigations is to develop a potent and efficient adjuvant strategy. The combination of multiple agonists to different pattern recognition receptor families offers great opportunities to achieve strong immune responses in a synergy manner. Here, we employed layered double hydroxide nanomaterials with BSA coating (LB) to integrate CpG and 3pRNA dual adjuvant cocktail, together with ovalbumin antigen. With LB loading, CpG and 3pRNA led to a quick internalization and enhanced induction of dendritic cell (DC) maturation. Meanwhile, this nanoadjuvant cocktail facilitated antigen crosspresenting in activated DCs. Taken together, these results may inspire the investigation of agonists combination and LDH‐based nanoadjuvants.

A glutamine metabolic switch supports erythropoiesis.

Metabolic requirements vary during development, and our understanding of how metabolic activity influences cell specialization is incomplete. Here, we describe a switch from glutamine catabolism to synthesis required for erythroid cell maturation. Glutamine synthetase (GS), one of the oldest functioning genes in evolution, is activated during erythroid maturation to detoxify ammonium generated from heme biosynthesis, which is up-regulated to support hemoglobin production. Loss of GS in mouse erythroid precursors caused ammonium accumulation and oxidative stress, impairing erythroid maturation and recovery from anemia. In β-thalassemia, GS activity is inhibited by protein oxidation, leading to glutamate and ammonium accumulation, whereas enhancing GS activity alleviates the metabolic and pathological defects. Our findings identify an evolutionarily conserved metabolic adaptation that could potentially be leveraged to treat common red blood cell disorders.

Mechanotransduction governs CD40 function and underlies X-linked hyper-IgM syndrome.

B cell maturation depends on cognate interactions between the T and B cells. Upon interaction with CD40 ligand (CD40L) on T cells, CD40 delivers costimulatory signals alongside B cell antigen receptor (BCR) signaling to regulate affinity maturation and antibody class switch. Mutations affecting CD40-CD40L interactions cause abnormal antibody responses in immunodeficiencies known as X-linked hyper-IgM syndrome (X-HIgM). Here, we study the CD40-mediated mechanotransduction in B cells, which likely occurs during their physical contacts with T cells. We found that CD40 forms catch bond with CD40L that lasts longer at larger forces, both B and T cells exert tension on CD40-CD40L bonds, and force enhances CD40 signaling and antibody class switch. X-HIgM CD40L mutations impair catch bond formation, suppress endogenous tension, and reduce force-enhanced CD40 signaling, leading to deficiencies in antibody class switch. Our findings highlight the role of mechanotransduction in CD40 function and provide insights into the mechanisms underlying X-HIgM syndrome.

Identification of feature genes in intestinal epithelial cell types.

The intestine, is responsible for food digestion, nutrient absorption, endocrine secretion, food residue excretion, and immune defense. These function performances are based on the intricate composition of intestinal epithelial cells, encompassing differentiated mature cells, rapidly proliferative cells, and intestinal stem cells. Although the characteristics of these cell types are well-documented, in-depth exploration of their representative markers and transcription factors is critical for comprehensive cell fate trajectory analysis. Here, we unveiled the feature genes in different cell types of the human and mouse gut through single-cell RNA sequencing analysis. Further, the locations of some specific transcription factors and membrane proteins were determined by immunofluorescence staining, and their role in regulating the proliferation and differentiation of intestinal epithelial cells were explored by CRISPR/Cas9 knockout. Therefore, this study not only reports new markers for various intestinal epithelial cell types but also elucidates the involvement of relevant genes in the determination of epithelial cell fate and maintenance of stem cell homeostasis, which facilitates the tracing and functional elucidation of intestinal epithelial cells.

Abstract B001: CircRNA-derived neoantigen identification in cancer vaccine development

Abstract Immunotherapy has been a popular topic in combating cancer for its great performance in specificity, versatility and durability recently. Cancer vaccines targeting neoantigens have shown clinical efficacy against cancer, eliciting long-lasting immune response and offering a more selective therapeutic approach with less side effects. However, rare mutation rates and low expression levels of neoantigen make it challenging to identify suitable neoantigens. Traditionally, researchers focus on the canonical mRNA transcriptome and proteome to find potential targets. Recent studies suggest that exploring the non-canonical transcriptome and proteome, particularly circular RNAs (circRNAs), can expand the search. CircRNAs, endogenously formed by the unique back-splicing events during pre-RNA processing, can serve as templates for protein synthesis. Some of these non-canonical peptides are specifically expressed in cancer cells, making circRNAs a valid source for neoantigen discovery. We have developed high-throughput circRNA reporter screening methods and mass spectrometry-based peptidomic workflow to pinpoint translating circRNAs and their derived peptides. Using these techniques, we have identified circRNA-derived neoantigens in breast cancer (BC) cell lines that can be used as possible targets for cancer vaccines. These circRNA-derived neoantigens are immunogenic and can trigger dendritic cell (DC) maturation and T cell activation in vitro. Next, we will test these findings by transferring to in vivo mouse system. We will assess cancer vaccine efficacy by targeting the neoantigens using syngeneic mouse tumor models. Our long- term goal is to apply the circRNA neoantigen identification technology to BC patient samples to facilitate cancer vaccine development and improve cancer immunotherapy. Our work will demonstrate the potential of circRNA-derived non canonical neoantigens in cancer vaccines, serving as a crucial first step to prove their effectiveness in therapy. By expanding our search beyond the traditional transcriptome and proteome, we will explore new targets and transform the neoantigen-based treatment. This innovative effort shifts the current understanding of neoantigen sources and opens up new possibilities for targeted cancer therapy strategies. Citation Format: Yi-Cian Zheng, Chun-Kan Chen. CircRNA-derived neoantigen identification in cancer vaccine development [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 B001.

Combination application of Pickering emulsion and BLS protein nanoparticles enhances vaccine efficacy.

The deficiency of recombinant protein immune response could be compensated for by using nanoparticle platforms or adding immune enhancers, however existing vaccines or adjuvants struggle to elicit durable cellular immune responses. In this work, a protein nanoscaffold, lumazine synthase isolated from Brucella (BLS) was optimally designed that could facilitate cellular uptake of displayed antigens and the maturation of bone marrow-derived dendritic cells (BMDCs), and enhancing humoral immune responses. To enhance cellular immune response, chitosan hydrochloride-stabilized Pickering emulsion (CHSPE) was evaluated as an adjuvant for the BLS nanoscaffold-based vaccine. CHSPE could enhance the recruitment and activation of DCs at the injection site and the uptake of antigen and activation of DCs in the draining lymph nodes (dLNs), compared with commercial adjuvant ISA 206. In addition, CHSPE induced antigen-specific antibody levels comparable to those of ISA 206 and increased the ratio of central memory T cells (TCM) and effector memory T cells (TEM), and promoted the secretion of Th1-type cytokines. Moreover, CHSPE-formulated vaccine provided a similar level of protection to the live attenuated vaccine and was superior to that of ISA 206. Taken together, the combination of the BLS nanoscaffold and CHSPE provides a feasible strategy for recombinant protein subunit vaccine development.

Autophagy-activating aluminum hydroxide nanovaccine for enhanced antigen presentation and anti-tumor immunity.

Lymph node (LN) targeting and antigen presentation by antigen-presenting cells (APCs) are critical factors affecting the immune responses induced by tumor vaccines. Autophagy activation promotes MHC class I and II antigen presentation in APCs. To enhance antigen presentation in LNs, we developed an aluminum hydroxide nanovaccine that simultaneously incorporates the autophagy-activating peptide Beclin-1 and the antigenic protein OVA (B/O@AN nanovaccine) through layer-by-layer electrostatic interaction. B/O@AN has a particle size of approximately 80 nm and efficiently targets lymph nodes following subcutaneous administration. The combination of the Beclin-1 peptide with the aluminum hydroxide nanovaccine promotes dendritic cell (DC) maturation. More importantly, B/O@AN facilitates antigen cross-presentation by promoting lysosomal escape and autophagy induction. After immunization, compared to O/@AN without Beclin-1, B/O@AN significantly augments antigen-specific cellular immune responses, leading to substantial increases in cytotoxic T lymphocytes (CTLs), T-helper 1 (Th1) cells, as well as serum antibody levels, thereby impeding melanoma development and progression in both prophylactic and therapeutic settings. These results provide evidence that autophagy activation strengthens antigen presentation and augments the antigen-specific immune responses of the aluminum hydroxide nanovaccine.

Customizable Glycopolymers as Adjuvants for Cancer Immunotherapy: From Branching Degree Optimization to Cell Surface Engineering.

Engineering dendritic cell (DC) maturation is paramount for robust T-cell responses and immunological memory, critical for cancer immunotherapy. This work unveils a novel strategy using precisely controlled branching in synthetic glycopolymers to optimize DC activation. Using the distinct copolymerization kinetics of 2-(methacrylamido) glucopyranose (MAG) and diethylene glycol dimethacrylate (DEGDMA) in a RAFT polymerization, unique glycopolymers with varying branching degrees are created. These strategically produced gradient branched glycopolymers with sugar moieties on the outer chain potently promote DC maturation. Strikingly, low-branched glycopolymers demonstrate superior activity, both in pure form and when engineered on tumor cell surfaces. Quartz crystal microbalance and theoretical simulations elucidate the crucial role of branching in modulating glycopolymer-DC receptor interactions. Low-branched gradient glycopolymers have shown a notable advantage and are promising adjuvants in DC-based cancer immunotherapy.

A CADASIL NOTCH3 mutation leads to clonal hematopoiesis and expansion of Dnmt3a-R878H hematopoietic clones.

Clonal hematopoiesis (CH) is nearly universal in the elderly. The molecular and cellular mechanisms driving CH and the clinical consequences of carrying clonally derived mutant mature blood cells are poorly understood. We recently identified a C223Y mutation in the extracellular domain (ECD) of NOTCH3 as a putative CH driver in mice. Provocatively, germline NOTCH3 ECD mutations perturbing cysteine numbers cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), a type of vascular dementia, suggesting an unexpected link between CADASIL and CH. Here, we formally demonstrated that mouse hematopoietic stem and progenitor cells (HSPCs) expressing CADASIL-related NOTCH3C455R exhibit a proliferative advantage resulting in robust cellular expansion in vivo and in vitro. Co-expression of NOTCH3C455R and Dnmt3aR878H, homologous to a frequent human CH mutation, increased the fitness of NOTCH3C455R HSPCs, demonstrating their functional cooperation. Surprisingly, the presence of NOTCH3C455R hematopoietic cells supported the expansion of Dnmt3aR878H HSPCs in a non-cell autonomous fashion in vivo, strongly suggesting that CADASIL patients and asymptomatic carriers can be highly predisposed to DNMT3AR882H-driven CH. Considering that CADASIL-related NOTCH3 mutations are more frequent in the general population than anticipated (~1 carrier in 400 people), the effect of these NOTCH3 mutations on CH development should be considered.

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

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 B2mfl/fl, or Xcr1-Cre Ifnar1fl/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.IMPORTANCEChronic 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.

PH and Redox Dual-Responsive Nanoparticle with Enhanced Dendritic Cell Maturation for Cancer Therapy.

Type I interferons (IFNs) are essential for activating dendritic cells (DCs) and presenting tumor-associated antigens to T cells. IFNs are primarily produced from DCs among immune cells. A combination of chemotherapy and metalloimmunotherapy induces IFN production by activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. However, chemotherapeutic agents deplete DC populations, suppressing immunostimulatory activities, despite their potent anticancer activities. Furthermore, an optimal ratio between chemotherapeutic agents and metal for activating DCs at the highest level has not been reported, and evidence for ensuring DC survival is lacking. In this study, we hypothesized that there is an optimal ratio to yield the highest DC maturation and anticancer activity with minimal DC depletion. To demonstrate it, we have designed a pH and redox dual-responsive nanoparticle, MnO2@BSA@DOX (MD), to prevent DCs from depleting and activate the cGAS-STING pathway both in cancer cells and DCs, inducing considerable levels of IFNs and maturation. MD consists of a core-layer structure, a manganese dioxide (MnO2) core, and a cross-linked layer with bovine serum albumin (BSA) and doxorubicin (DOX), with a specific ratio of DOX to manganese. MD exhibits structure-based selectivity between cancer cells and DCs by targeting the extracellular pH of the tumor microenvironment and intracellular redox reactions in cancer cells. Among various formulations, the 1:1 ratio shows the highest maturation with no significant depletion. Moreover, it induces distinct cytotoxicity in cancer cells through apoptosis and cGAS-STING activation, leading to increased calreticulin expression and enhanced DC phagocytosis. Consequently, it results in superior tumor suppression and prolonged survival with the high accumulation of MD in the tumor and no observed systemic toxicities, highlighting its potential as a therapeutic agent in cancer treatments.

Single-cell Trajectories and Pseudo-time Analysis Reveal Additional Insights into Rheumatoid Arthritis Disease Progression

As the chicken embryo grows, its increasing metabolic demand drives the development of the cardiovascular system, enabling efficient nutrient delivery through complex molecular and structural formation. Key extracellular matrix (ECM) components, such as hyaluronic acid (HA) and collagen type I alpha 1 (COL1A1), play a crucial role in shaping the biomechanical environment of the heart, particularly the sinoatrial node (SAN). These ECM elements regulate cell adhesion, migration, and the maturation of cardiac progenitor cells (CPCs), essential for the heart's electrical conductivity and rhythmicity. Based on the hypothesis that ECM mechanical properties influence the spatial patterning of conduction-related genes, we analyzed spatial transcriptomics data from chicken hearts at different developmental stages. Collagen-related genes associated with a sturdy ECM showed an increasing trend over time, while soft ECM-related genes responsible for producing proteoglycans and breaking down collagen decreased. Although conduction-related genes did not fully align with ECM trends, intriguing patterns were observed, pointing to potential interactions between ECM remodeling and the construction of the cardiac conduction system. Our findings suggest significant ECM r7emodeling between days 4 and 7, which may influence the heart's structural and functional development.

Entinostat in combination with nivolumab in metastatic pancreatic ductal adenocarcinoma: a phase 2 clinical trial.

Pancreatic ductal adenocarcinoma (PDA) is characterized by low cytotoxic lymphocytes, abundant immune-suppressive cells, and resistance to immune checkpoint inhibitors (ICI). Preclinical PDA models showed the HDAC inhibitor entinostat reduced myeloid cell immunosuppression, sensitizing tumors to ICI therapy. This phase II study combined entinostat with nivolumab (PD1 inhibitor) in patients with advanced PDA (NCT03250273). Patients received entinostat 5 mg orally once weekly for 14-day lead-in, followed by entinostat and nivolumab. The primary endpoint was the objective response rate (ORR) by RECIST v1.1. Secondary endpoints included safety, duration of response, progression free-survival and overall survival. Between November 2017 and November 2020, 27 evaluable patients were enrolled. Three showed partial responses (11% ORR, 95% CI, 2.4%-29.2%) with a median response duration of 10.2 months. Median progression-free survival (PFS) and overall survival (OS) were, respectively, 1.89 (95% CI, 1.381-2.301) and 2.729 (95% CI, 1.841-5.622) months. Grade ≥3 treatment-related adverse events occurred in 19 patients (63%), including decreased lymphocyte count, anemia, hypoalbuminemia, and hyponatremia. As exploratory analysis, peripheral and tumor immune profiles changes were assessed using CyTOF, mIHC, and RNA-seq. Entinostat increased dendritic cell activation and maturation. Gene expression analysis revealed an enrichment in inflammatory response pathways with combination treatment. Although the primary endpoint was not met, entinostat and nivolumab showed durable responses in a small subset of PDA patients. Myeloid cell immunomodulation supported the preclinical hypothesis, providing a basis for future combinatorial therapies to enhance clinical benefits in PDA.

Root cap cell corpse clearance limits microbial colonization in Arabidopsis thaliana.

Programmed cell death occurring during plant development (dPCD) is a fundamental process integral for plant growth and reproduction. Here, we investigate the connection between developmentally controlled PCD and fungal accommodation in Arabidopsis thaliana roots, focusing on the root cap-specific transcription factor ANAC033/SOMBRERO (SMB) and the senescence-associated nuclease BFN1. Mutations of both dPCD regulators increase colonization by the beneficial fungus Serendipita indica, primarily in the differentiation zone. smb-3 mutants additionally exhibit hypercolonization around the meristematic zone and a delay of S. indica-induced root-growth promotion. This demonstrates that root cap dPCD and rapid post-mortem clearance of cellular corpses represent a physical defense mechanism restricting microbial invasion of the root. Additionally, reporter lines and transcriptional analysis revealed that BFN1 expression is downregulated during S. indica colonization in mature root epidermal cells, suggesting a transcriptional control mechanism that facilitates the accommodation of beneficial microbes in the roots.