Immune Cell Populations Research Articles

Extracorporeal photopheresis vs. systemic immunosuppression for immune-related adverse events: Interim analysis of a prospective two-arm study

BackgroundCheckpoint inhibitor-induced steroid-refractory (sr) and steroid-dependent (sd) immune-related adverse events (irAE) account for about 11 % of irAE. Although these patients face worse outcomes due to irAE mortality and/or sustained immunosuppression, which impairs anti-tumor response, there is no established second-line treatment based on prospective trial data. MethodsThis prospective comparative study investigates outcomes of extracorporeal photopheresis (ECP), an immunomodulating therapy, versus second-line immunosuppressants (SLI) in sr/sd-irAE. The primary endpoint was longitudinal change in immunophenotype; secondary endpoints were outcome of irAE and tumor response. Patient demographics, quality of life (EORTC QLQ-C30; global health status (GHS/QoL)) and longitudinal blood samples were analyzed at baseline; in weeks 1, 4, 8, and 12. ResultsAt interim analysis, 21 patients (11 ECP, 10 SLI) with 7 different sr/sd-irAE were included. Compared with the SLI group, the ECP group demonstrated a higher clinical response rate of irAE (93 % vs. 80 %; 95 % CI 0.83–1.92; P = 0.54) and a better GHS/QoL score throughout all follow-up visits. ECP patients showed a numerically higher overall survival (23 vs. 12 months; 95 % CI 0.02–3.02; P = 0.27) and lower cancer progression rates (33 % vs. 67 %; 95 % CI 0.09–1.60; P = 0.52). Immunophenotyping revealed changes in immune cell populations and the regulation of immune checkpoints. There were no significant safety issues in either treatment group. ConclusionThis prospective comparative study supports the clinical efficacy of ECP in the treatment of sr/sd-irAE in comparison to the SLI cohort. Thus, ECP represents a potential treatment option for this indication, given its good safety profile while maintaining anti-tumor response. Trial RegistrationClinicalTrials.gov, NCT05700565, https://classic.clinicaltrials.gov/ct2/show/NCT05700565.

Effects of switching from twice-daily tacrolimus to once-daily extended-release meltdose tacrolimus on cellular immune response.

LCP-Tacro [LCPT], a novel once-daily, extended-release formulation of tacrolimus, has a reduced Cmax with comparable AUC exposure, requiring a ∼30% dose reduction in contrast to immediate-release tacrolimus (IR-Tac). Once-daily LCPT in de novo kidney transplantation has a comparable efficacy and safety profile to that of IR-Tac with advantages in bioavailability and absorption. The present investigation intends to analyze the effects of conversion from IR-Tac to LCPT on phenotype and function of T-cells and B-cells. 16 kidney transplant patients treated by triple standard immunosuppression with a stable graft function undergoing a switch from IR-Tac to LCPT were included in this observational prospective study. We measured the main immune cell types and performed an in-depth characterization of B cell, dendritic cells and T cells including regulatory T cells of the patients before, 4 and 8 weeks after IR-Tac to LCPT conversion using multi-parameter fl ow cytometry. Additionally, we analyzed T cells by assessing third-party antigens (Tetanus Diphtheria, TD)-reactive T cells, which could be analyzed by restimulation with tetanus vaccine. Overall, we found no significant alterations following LCPT conversion for the most immune cell populations with a few cell populations showing transient quantitative increase. Thus, 4 weeks after conversion, more regulatory T cells could be measured in the patients with a significant shift from memory to naïve Tregs. Furthermore, we found a transient B cell expansion 4 weeks after conversion from IR-Tac to LCPT. There were no changes in the percentage of other basic immune cell types and the antigen-reactive T cells were also not altered after changing the medication to LCP-tacrolimus. Here, we demonstrate first insights into the immune system changes occurred under IR-Tac to LCPT conversion therapy in kidney transplant patients. While phenotypic and functional characteristics of the most immune cell populations did not change, we could observe an a transient expansion of regulatory T cells in peripheral blood following IR-Tac to LCTP conversion, which might additionally contribute to the overall immunosuppressive effect.

B cells drive neuropathic pain-related behaviors in mice through IgG-Fc gamma receptor signaling.

Neuroimmune interactions are essential for the development of neuropathic pain, yet the contributions of distinct immune cell populations have not been fully unraveled. Here, we demonstrate the critical role of B cells in promoting mechanical hypersensitivity (allodynia) after peripheral nerve injury in male and female mice. Depletion of B cells with a single injection of anti-CD20 monoclonal antibody at the time of injury prevented the development of allodynia. B cell-deficient (muMT) mice were similarly spared from allodynia. Nerve injury was associated with increased immunoglobulin G (IgG) accumulation in ipsilateral lumbar dorsal root ganglia (DRGs) and dorsal spinal cords. IgG was colocalized with sensory neurons and macrophages in DRGs and microglia in spinal cords. IgG also accumulated in DRG samples from human donors with chronic pain, colocalizing with a marker for macrophages and satellite glia. RNA sequencing revealed a B cell population in naive mouse and human DRGs. A B cell transcriptional signature was enriched in DRGs from human donors with neuropathic pain. Passive transfer of IgG from injured mice induced allodynia in injured muMT recipient mice. The pronociceptive effects of IgG are likely mediated through immune complexes interacting with Fc gamma receptors (FcγRs) expressed by sensory neurons, microglia, and macrophages, given that both mechanical allodynia and hyperexcitability of dissociated DRG neurons were abolished in nerve-injured FcγR-deficient mice. Consistently, the pronociceptive effects of IgG passive transfer were lost in FcγR-deficient mice. These data reveal that a B cell-IgG-FcγR axis is required for the development of neuropathic pain in mice.

Dynamic responses of human lung innate and adaptive immune cells highlight the roles of genes at asthma risk loci.

The lung is a unique immunological niche with diverse immune cell types. The effects of stimulation through innate and adaptive immune receptors on human lung immune cells has largely been extrapolated from studies of blood immune cells. While multiple immune cell types and many genes have been implicated as contributing to asthma, the dynamics of these in human lung immune cells following activation will yield insights into asthma pathogenesis and lung immunity more broadly. Human lung immune cells from 6 donors were isolated. Mixed leukocytes were treated separately with lipopolysaccharide (LPS), F(ab)2-anti-human-IgM/IgG + IL4 and anti-CD3/CD28 for 4 and 18 hours and underwent single cell RNA sequencing (scRNAseq). Lung immune cell types were annotated, and gene expression compared across conditions. Genes at prior asthma-associated genetic loci were characterized across cell types, treatments and timepoints. Expression of non-classical class II genes associated with asthma, HLA-DQA2 and HLA-DQB2, and their protein products was characterized with immunohistochemistry. We characterized gene expression in 116,697 lung immune cells. Cell-, treatment-, and timepoint-specific effects on gene expression were detected in all lung immune cell populations. Correlation of gene expression between lung and blood lymphocyte populations decreased following stimulation. Among the genes that were differentially expressed, 97 receptor:ligand pairs had changes with treatments. 96.0% of genes at asthma risk loci demonstrated differential expression in at least one cell type and at least one treatment. B cells were the cell type with the highest expression of HLA-DQA2 and HLA-DQB2 which increased with anti-IgM/IgG treatment and the HLA-DQB2 protein was identified in lung B cells from a donor with asthma. Human lung immune activation elicits a broad range of cellular responses that deviate from those of blood immune cells and are relevant to asthma. Lung B cells expressing HLA-DQA2 and HLA-DQB2 appear to be involved in a novel antigen presentation pathway that contributes to asthma risk.

The effect of intrauterine growth restriction on the developing pancreatic immune system.

Immune cells in the pancreas are known to participate in organ development. However, the resident pancreatic immune system has yet to be fully defined. Immune cells also play a role in pathology and are implicated in diseases such as diabetes induced by intrauterine growth restriction (IUGR). We hypothesized that the resident immune system is established during neonatal development and disrupted by IUGR. Using single cell RNAseq and flow cytometry we identified many immune cell populations in the near-term fetus (at embryologic day 22) and neonatal (postnatal day 1, 7, &14) islets, non-endocrine pancreas, and the spleen in the rat. Using flow cytometry, we observed the resident immune system is established during neonatal development in the pancreas and spleen. We identified 9 distinct immune populations in the pancreatic islets and 8 distinct immune populations in the spleen by single cell RNAseq. There were no sex-specific differences in the relative proportion of immune cells in the pancreas or spleen. Finally, we tested if IUGR disrupted the neonatal immune system using bilateral uterine artery ligation. We found significant changes to the percentage of CD11B+ HIS48- and CD8+ T cells in the islets and non-endocrine pancreas and in the spleen. IUGR-induced alterations were influenced by the tissue environment and the sex of the offspring. Future research to define the role of these immune cells in pancreatic development may identify disrupted pathways that contribute to the development of diabetes following IUGR.

Immune priming and induction of tertiary lymphoid structures in a cord-blood humanized mouse model of gastrointestinal stromal tumor

ABSTRACT Gastrointestinal stromal tumors (GISTs) harbor diverse immune cell populations but so far immunotherapy in patients has been disappointing. Here, we established cord blood humanized mouse models of localized and disseminated GIST to explore the remodeling of the tumor environment for improved immunotherapy. Specifically, we assessed the ability of a cancer vascular targeting peptide (VTP) to bind to mouse and patient GIST angiogenic blood vessels and deliver the TNF superfamily member LIGHT (TNFS14) into tumors. LIGHT-VTP treatment of GIST in humanized mice improved vascular function and tumor oxygenation, which correlated with an overall increase in intratumoral human effector T cells. Concomitant with LIGHT-mediated vascular remodeling, we observed intratumoral high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), which resemble spontaneous TLS found in GIST patients. Thus, by overcoming the limitations of immunodeficient xenograft models, we demonstrate the therapeutic feasibility of vascular targeting and immune priming in human GIST. Since TLS positively correlate with patient prognosis and improved response to immune checkpoint inhibition, vascular LIGHT targeting in GIST is a highly translatable approach to improve immunotherapeutic outcomes.

Early markers of induced pluripotent stem cell hematopoietic development

The generation of immune cell populations from induced pluripotent stem cells (iPSCs) is a valuable model to study mechanisms that control hematopoietic development; it also is a promising approach to develop immunotherapeutic strategies to treat various diseases, including hereditary, oncological and infectious ones. To date, it has been demonstrated that iPSCs can differentiate into different immune cells, including macrophages, neutrophils, natural killer cells and T cells. However, the protocols suggested so far are experimental, and they require optimization, standardization and scaling. Solution to these tasks requires methods allowing to predict the efficacy of ongoing differentiation at early differentiation stages. Here, we evaluated whether iPSC hematopoietic/myeloid differentiation can be monitored by means of flow cytometry. Human iPSCs were differentiated into hematopoietic/myeloid cells using two protocols previously suggested for the generation of macrophages from iPSCs. The protocols differed by methods used to induce early and late stages of cell differentiation. At early differentiation stages, the protocols differed by approaches used to induce the generation of mesoderm and hemogenic endothelium, i.e., 2D differentiation in the presence of exogenous factors known to promote mesoderm and hemogenic endothelium development (“factor-dependent” protocol) or 3D differentiation in the absence of exogenous cytokines and growth factors (“spontaneous” protocol). At late differentiation stages, the protocols differed by factors added to the cultures to promote hematopoietic/myeloid specification (i.e., SCF, FGF2, IL-6, IL-3 and M-CSF or only IL-3 and M-CSF). At different stages of differentiation, the expressions of antigens known to be expressed by mesoderm, hemogenic endothelium and hematopoietic cells (i.e., CD309, CD34, CD31, CD43 and CD45) were evaluated. At early differentiation stages, the main phenotypic changes observed in cell cultures were an upregulation of the expression of CD309 (a receptor for vascular endothelial growth factor), the appearance of the expression of sialophorin CD43 and the expression of CD34 antigen. In cells cultured in 2D factor-dependent conditions, these changes appeared earlier and were more pronounced as compared with cells cultured in 3D “spontaneous” conditions. The results suggest that CD309 and/or CD43 are valuable markers for an early prediction of the effectiveness of iPSC differentiation into hematopoietic/ myeloid progeny.

Characterization of Immune Cell Populations and Acid-Sensing Receptors in the Human Esophagus

Characterization of Immune Cell Populations and Acid-Sensing Receptors in the Human Esophagus

A Study of Disease Prognosis in Lung Adenocarcinoma Using Single-Cell Decomposition and Immune Signature Analysis.

Background: The development of tumors is a highly complex process that entails numerous interactions and intricate relationships between the host immune system and cancer cells. It has been demonstrated in studies that the treatment response of patients can be correlated with the tumor microenvironment (TME). Consequently, the examination of diverse immune profiles within the TME can facilitate the elucidation of tumor development and the development of advantageous models for diagnoses and prognoses. Methods: In this study, we utilized a single-cell decomposition method to analyze the relationships between cell proportions and immune signatures in lung adenocarcinoma (LUAD) patients. Results: Our findings indicate that specific immune cell populations and immune signatures are significantly associated with patient prognosis. By identifying poor prognosis signatures (PPS), we reveal the critical role of immune profiles and cellular composition in disease outcomes, emphasizing their diagnostic potential for predicting patient prognosis. Conclusions: This study highlights the importance of immune signatures and cellular composition, which may serve as valuable biomarkers for disease prognosis in LUAD patients.

Immune and inflammatory insights in atherosclerosis: development of a risk prediction model through single-cell and bulk transcriptomic analyses.

Investigation into the immune heterogeneity linked with atherosclerosis remains understudied. This knowledge gap hinders the creation of a robust theoretical framework essential for devising personalized immunotherapies aimed at combating this disease. Single-cell RNA sequencing (scRNA-seq) analysis was employed to delineate the immune cell-type landscape within atherosclerotic plaques, followed by assessments of cell-cell interactions and phenotype characteristics using scRNA-seq datasets. Subsequently, pseudotime trajectory analysis was utilized to elucidate the heterogeneity in cell fate and differentiation among macrophages. Through integrated approaches, including single-cell sequencing, Weighted Gene Co-expression Network Analysis (WGCNA), and machine learning techniques, we identified hallmark genes. A risk score model and a corresponding nomogram were developed and validated using these genes, confirmed through Receiver Operating Characteristic (ROC) curve analysis. Additionally, enrichment and immune characteristic analyses were conducted based on the risk score model. The model's applicability was further corroborated by in vitro and in vivo validation of specific genes implicated in atherosclerosis. This comprehensive scRNA-seq analysis has shed new light on the intricate immune landscape and the role of macrophages in atherosclerotic plaques. The presence of diverse immune cell populations, with a particularly enriched macrophage population, was highlighted by the results. Macrophage heterogeneity was intricately characterized, revealing four distinct subtypes with varying functional attributes that underscore their complex roles in atherosclerotic pathology. Intercellular communication analysis revealed robust macrophage interactions with multiple cell types and detailed pathways differing between proximal adjacent and atherosclerotic core groups. Furthermore, pseudotime trajectories charted the developmental course of macrophage subpopulations, offering insights into their differentiation fates within the plaque microenvironment. The use of machine learning identified potential diagnostic markers, culminating in the identification of RNASE1 and CD14. The risk score model based on these biomarkers exhibited high accuracy in diagnosing atherosclerosis. Immune characteristic analysis validated the risk score model's efficacy in defining patient profiles, distinguishing high-risk individuals with pronounced immune cell activities. Finally, experimental validation affirmed RNASE1's involvement in atherosclerotic progression, suggesting its potential as a therapeutic target. Our findings have advanced our understanding of atherosclerosis immunopathology and paved the way for novel diagnostic and therapeutic strategies.

Lipid-Laden Macrophages Recycle Myelin to Feed Glioblastoma.

Tumor-associated microglia and macrophages (TAM) make up the largest immune cell population in the glioblastoma (GBM) tumor microenvironment. Given the heterogeneity and plasticity of TAMs in the GBM tumor microenvironment, understanding the context-dependent cancer cell-TAM symbiotic interaction is crucial for understanding GBM biology and developing effective therapies. In a recent issue of Cell, Kloosterman and colleagues identified a subpopulation of glycoprotein nonmetastatic melanoma protein Bhigh lipid-laden microglia and macrophages (LLM) in GBM. Mesenchymal-like GBM cells help generate the LLM phenotype. Reciprocally, LLMs are epigenetically rewired to recycle myelin and transfer the lipid from myelin to cancer cells, fueling mesenchymal-like GBM progression in a liver X receptor/ABCA1-dependent manner. Together, leveraging LLMs opens new therapeutic possibilities for rewiring the metabolism-mediated tumor-TAM interaction during GBM progression.

Targeting Myeloid-Derived Suppressor Cells via Dual-Antibody Fluorescent Nanodiamond Conjugate.

Fluorescent nanodiamonds (FNDs) are carbon-based nanomaterials that emit bright, photostable fluorescence and exhibit a modifiable surface chemistry. Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive cell population known to expand in cancer patients and contribute to worse patient outcomes. To target MDSC, glycidol-coated FND were conjugated with antibodies against the murine MDSC markers, CD11b and GR1 (dual-Ab FND). In vitro, dual-Ab FND uptake by murine MDSC was significantly higher than IgG-coated FND (94.7% vs. 69.0%, p < 0.05). In vivo, intra-tumorally injected dual-Ab FND primarily localized to the tumor 2 and 24 h post-injection, as measured by in vivo fluorescence imaging and flow cytometry analysis of the spleen and tumor. Dual-Ab FND were preferentially taken up by intra-tumoral MDSC, representing 87.1% and 83.0% of FND+ cells in the tumor 2 and 24 h post-injection, respectively. Treatment of mice with anti-PD-L1 immunotherapy prior to intra-tumoral injection of dual-Ab FND did not significantly alter the uptake of FND by MDSC. These results demonstrate the ability of our novel dual-antibody conjugated FND to target MDSC and reveal a potential strategy for targeted delivery to other specific immune cell populations in future cancer research.

Impact of Comorbid Pathology and Severity of Clinical Course on Anti-infective Protection in Patients with COVID-19

Objective — to establish and evaluate the impact of comorbid pathology and the severity of the clinical course on the activity of anti-infective protection in patients with coronavirus disease 2019 (COVID-19), based on the analysis of the absolute and relative counts of major immune cell populations in peripheral blood. Materials and methods. A cross-sectional prospective study was conducted involving 204 patients with COVID-19-associated pneumonia of mild, moderate and severe stages. The cohort included 106 (51.97 %) women and 98 (48.03 %) men, with an average age of (55.93 ± 8.75) years. Anti-infective ­protection was studied using comprehensive clinical blood analysis, including the count of major immune-competent cells. Patients were categorised into groups based on comorbi­dity, taking into account the predominant concurrent pathology at the time of examination (endocrine, cardiovascular or other). Results and discussion. More severe COVID-19 cases were characterised by leukopenia, relative and absolute granulocytopenia, neutropenia, a slight hyporegenerative nuclear shift to the right and a reduction in the number of segmented neutrophils by 24.63—34.72 % (p ≤ 0.047—0.009) and eosinophilic granulocytes by 52.94—70.59 % (p ≤ 0.004—0.001). This was accompanied by relative and absolute agranulocytosis due to lymphocytosis (by 20.10—63.33 %; p ≤ 0.05—0.003) and monocytosis (by 40.0—74.12 %; p ≤ 0.046—0.049), indicating an active inflammatory infectious process of viral etiology against a background of decreased resistance and increased activity of the lymphocyte-macrophage link with the beginning of the formation of specific immune protection (cellular and humoral response to coronavirus intervention). Mild cases of COVID-19 showed a higher absolute count of peripheral blood granulocytes (by 48.25 %; p = 0.018), neutrophils, particularly segmented neutrophils and eosinophilic granulocytes, compared to moderate and severe cases (by 32.36—53.18 % (p ≤ 0.049—0.01) and 3.4 times (p ≤ 0.005—0.002)) and the lowest content of monocytes (by 35.41—42.57 %; p ≤ 0.049—0.046). In cases with concurrent endocrine and cardiological pathologies against a background of a more severe clinical course of COVID-19, immunoinflammatory changes intensified due to absolute and relative leukopenia, neutrophilic granulocytopenia, neutropenia, with the presence of relative lymphocytosis and monocytosis, and normal ESR indicators, confirming the inflammatory process of viral etiology, which, in the absence of treatment, may tend to form post-COVID and/or long-term effects of COVID-19 with multisystem involvement. Conclusions. The study found an enhancement of the immunoinflammatory response and an increase in the depletion of cellular factors of non-specific anti-infective protection with the progression of the clinical manifestations of COVID-19, particularly against the backdrop of comorbid endocrine and/or cardiological pathology.

Skull bone marrow-derived immune cells infiltrate the injured cerebral cortex and exhibit anti-inflammatory properties

Identifying the origins and contributions of peripheral-derived immune cell populations following brain injury is crucial for understanding their roles in neuroinflammation and tissue repair. This study investigated the infiltration and phenotypic characteristics of skull bone marrow-derived immune cells in the murine brain after traumatic brain injury (TBI). We performed calvarium transplantation from GFP donor mice and subjected the recipients to controlled cortical impact (CCI) injury 14 days post-transplant. Confocal imaging at 3 days post-CCI revealed GFP+ calvarium-derived cells were present in the ipsilateral injured cortex, expressing CD45 and CD11b immune markers. These cells included Ly6G-positive neutrophil or Ccr2-positive monocyte identities. Calvarium-derived GFP+/Iba1+ monocyte/macrophages expressed the efferocytosis receptor MERTK and displayed engulfment of NeuN+ and cleaved caspase 3+ apoptotic cells. Phenotypic analysis showed that greater calvarium-derived monocytes/macrophages disproportionately express the anti-inflammatory arginase-1 marker than pro-inflammatory CD86. To differentiate the responses of blood- and calvarium-derived macrophages, we transplanted GFP calvarium skull bone into tdTomato bone marrow chimeric mice, then performed CCI injury 14 days post-transplant. Calvarium-derived GFP+cells predominantly infiltrated the lesion boundary, while blood-derived tdTomato+ cells dispersed throughout the lesion and peri-lesion. Compared to calvarium-derived cells, more blood-derived cells expressed pro-inflammatory CD86 and displayed altered 3D morphologic traits. These findings uniquely demonstrate that skull bone marrow-derived immune cells infiltrate the brain after injury and contribute to the neuroinflammatory milieu, representing a novel immune cell source that may be further investigated for their causal role in functional outcomes.

Disruption of the PGE2 synthesis / response pathway restrains atherogenesis in programmed cell death-1 (Pd-1) deficient hyperlipidemic mice.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs still limits their clinical utility. Combinatorial strategies enhance the antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E2, formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE2 synthesis /response pathway- global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4- on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr KO) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE2 biosynthesis, reflected by its major urinary metabolite, PGE2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4 KO mice as impaired. in Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.

Abstract B091: Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics

Abstract Intraductal papillary mucinous neoplasms (IPMNs), a common precursor to pancreatic ductal adenocarcinoma, present as a spectrum of benign indolent behavior and progression to invasive carcinoma, which highlights the importance of identifying biomarkers of IPMN progression. This study utilized whole transcriptome spatial transcriptomics to define the molecular characteristics of Epithelial, Immune, and Fibroblast cell populations of the three distinct histological sub-types of IPMN to understand the dynamics between the cell types and identify biomarkers of neoplastic progression. Fifty-two surgically resected IPMN cores from 21 patients with resected IPMNs comprising 24 Gastric, 14 Intestinal, and 14 Pancreaticobiliary with a distribution of low-grade dysplasia (LGD), high-grade dysplasia (HGD), and Invasive carcinoma (IC) were analyzed. Epithelial, Immune, and Fibroblast components were analyzed using the GeoMx spatial profiler. Unsupervised hierarchical clustering of epithelial areas of interest (AOI) identified 3 distinct clusters demonstrating a spectrum of molecular phenotypes in IPMN that were correlated with the three histological subtypes. Comparing the gene expression profile based on the shared genes expressed on all three subtypes, the Intestinal type tends to have an overexpression of genes whereas the Pancreaticobiliary represents a repressed phenotype relative to the Gastric phenotype. Supervised clustering using Moffitt’s classifier to identify the Epithelial-Mesenchymal transition demonstrates the Pancreaticobiliary subtype to be more Basal-like versus the Gastric and Intestinal inclined towards the classical phenotype. Supervised clustering based on the degree of dysplasia within the epithelial compartments of each of these subtypes, demonstrated a spectrum of molecular heterogeneity and identified biomarkers specific to High-Grade dysplasia in the Gastric and Intestinal subtype. Immune AOI deconvolution analysis demonstrated that Tregs were associated with Pancreaticobiliary, Macrophage enrichment associated with the Intestinal phenotype, while there is enrichment of CD8 T-cell and CD4 T-cell gene signatures associated with the Gastric subtype. Fibroblast AOI analyses demonstrated heterogenous myCAF- iCAF signatures between the subtypes. In summary, Spatial whole transcriptome analysis of IPMN identifies molecular signatures in epithelial and immune compartments that differentiate between the three histological subtypes of IPMN and helps understand their progression from Low-grade dysplasia to High-grade dysplasia. Citation Format: Amaya Pankaj, Michael J Raabe, Yuhui Song, Bidish K Patel, Katherine Xu, Joshua R Kocher, Nicholas J Caldwell, Maria L Ganci, Linda T Nieman, Mari Mino-Kenudson, Vikram Deshpande, Nabeel Bardeesy, Carlos Fernandez-Del Castillo, Martin J Aryee, David T Ting, Yasmin G Hernandez-Barco. Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B091.

Abstract C045: Targeting metabolic heterogeneity in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the US. This poor prognosis is related to the complex tumor microenvironment and tumor heterogeneity that drives resistance to chemotherapy. Here the heterogeneity of the stromal, immune, and malignant epithelial cell populations enables signaling and metabolic crosstalk mechanisms that drive immune suppression and resistance to therapy. Recently, our group unveiled that multiple distinct metabolic subclasses of cancer cells coexist within individual PDAC tumors. Among these, two PDAC clonal populations can engage in metabolic symbiosis that supports mitochondrial metabolism. One subtype has a constitutively high integrated stress response (ISR), enabling resistance to mitochondrial inhibitors. Further, these cells release asparagine that supports the mitochondrial metabolism of the other subtype that are more sensitive to mitochondrial inhibition. Given these observations, we are developing approaches to disrupt this metabolic symbiosis. Clonal PDAC cells with constitutively active ISR exhibit increased sensitivity to inhibition of the redox activity of Redox factor 1/Apurinic-apyrimidinic endonuclease 1 (Ref-1). Importantly, this can be leveraged to disrupt the support of the mitochondrial metabolism of clonal symbiotic pairs. Moreover, polyclonal PDAC cell lines representative of the heterogeneity seen in PDAC patients show a highly synergistic response to the combination of Ref-1 redox inhibitors with mitochondrial inhibitors. These data suggest this treatment combination will be synergistic in polyclonal tumors that exist in most PDAC patients. Overall, these data have the potential to lead to new avenues to directly target heterogenous populations of cancer cells to treat pancreatic cancer- an urgent clinical need. Citation Format: Taryn Morningstar, Cecily Anaraki, Lorenzo Scipioni, Giulia Tedeschi, Michelle Digman, Aimee Edinger, Claus Jorgensen, Melissa Fishel, Christopher J Halbrook. Targeting metabolic heterogeneity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C045.

Salivary DNA methylation derived estimates of biological aging, cellular frequency and protein expression as predictors of oral mucositis severity and survival in head and neck cancer patients

BackgroundOral mucositis is a painful and debilitating condition that occurs in the majority of head and neck cancer patients receiving radiation and/or chemotherapy. While some patient and treatment related factors are known to contribute to the incidence and severity of disease, reliable biomarkers remain elusive. In the following study, we investigated the association of salivary DNA methylation derived biological aging, cellular frequency and protein concentration measures with the severity of oral mucositis and overall survival in a cohort of head and neck cancer (HNC) patients (n = 103). MethodsDNA methylation profiling was performed on saliva samples obtained prior to treatment. Biological aging measures included Horvath2, PhenoAge, FitAge and GrimAge, and cellular frequency included epithelial and specific immune cell populations. ResultsSevere mucositis (i.e. grade 3 or 4) occurred in nearly half of patients. For malignant HNC patients (n = 84), every 1-SD increase in GrimAge was associated with 2.62-times risk of severe mucositis (95 % CI: 1.38, 5.57), while a 1-SD increase in monocyte frequency was associated with a decreased risk (OR [95 %CI]: 0.40 [0.18, 0.80]). Over a median follow-up of 53 months, 39 of 103 participants died. Six protein scores (TNFSF14, GCSF, MATN3, GDF8, nCDase, TNF-β) were associated with survival at q < 0.15. ConclusionWe provide evidence that the risk-related biological aging measure GrimAge may be a useful predictor of mucositis severity in HNC patients. Salivary monocyte frequency may be protective against mucositis, and this measure could be used as a predictive biomarker while also providing clues into the pathobiology of the disease.

Cigarette smoking prolongs inflammation associated with influenza infection and delays its clearance in mice.

Epidemiological studies have shown that smoking is associated with increased incidence of severe viral infections leading to hospitalization. Moreover, studies in experimental models have identified impaired antiviral responses and altered inflammatory responses, yet it is unclear how the effects of smoke exposure and influenza A infection interact and how this varies over the course of infection. We hypothesized that smoking would exacerbate innate immune responses against influenza. To test this, female BALB/c mice were exposed to cigarette smoke or air twice a day for 24-28 days and (mock) infected with H3N2 influenza A on day 21 while smoking continued. Three and seven days after infection, changes in immune cell populations, the transcriptome, and viral clearance in lung tissue were analyzed. After influenza A infection, smoke-exposed mice lost significantly more weight than air-exposed controls, indicating that smoking resulted in more severe disease. Immune cell and lung tissue transcriptome analysis revealed that neutrophil infiltration was prolonged and macrophage activation dysregulated after infection of smoke-exposed mice compared to air-exposed controls. Expression of genes in IL-6 and interferon pathways was similarly longer active. In parallel, we observed lower clearance of influenza virus in smoke-exposed mice after infection compared to air-exposed controls, indicating ineffective antiviral responses. Altogether, the data from our mouse model indicate that cigarette smoke exposure prolongs innate immune responses against influenza A. The results from this study help to explain the susceptibility of current smokers to severe influenza A disease.

Modulation of immune responses in the liver: The role of curcumin in balancing between health and disease

The human liver, traditionally viewed as a metabolic and detoxifying organ, is now being acknowledged for its complex immunological activities involving diverse resident immune cell populations and non-hematopoietic cells. Even in the absence of disease, the liver actively engages in immune surveillance and regulatory processes to support metabolic functions and tissue remodeling, influenced by continuous exposure to dietary and microbial elements. However, maintaining a delicate balance within this dynamic microenvironment is crucial, as uncontrolled inflammation can result in pathological conditions. Effective immune activation is essential for responding to challenges such as pathogens or tissue damage, while mechanisms for resolving inflammation are necessary to maintain liver homeostasis. This review examines the diverse roles of hepatic inflammatory mechanisms in both normal and pathological liver conditions, where dysregulation contributes to chronic inflammation and diseases. A key focus of this discussion is the potential therapeutic role of curcumin, the primary curcuminoid found in turmeric. Curcumin is well known for its potent antioxidant and anti-inflammatory properties and has shown the ability to modulate various signaling pathways. Notably, curcumin acts by suppressing nuclear factor kappa B (NF-&amp;kappa;B), a crucial player in the inflammatory cascade. NF-&amp;kappa;B not only triggers inflammation but also influences cell survival, proliferation, invasion, and angiogenesis. Drawing from existing literature, we emphasize curcumin&amp;rsquo;s ability to scavenge free radicals, reduce lipid peroxidation, and target NF-&amp;kappa;B-dependent pathways. By elucidating the evolving comprehension of inflammation&amp;rsquo;s role in maintaining liver balance, we suggest that curcumin represents a promising approach for treating chronic liver inflammation, thereby preventing fibrosis and related ailments. This thorough analysis highlights the potential of curcumin in modulating immune regulatory pathways specific to the liver, providing valuable insights into its use as a targeted therapeutic intervention against chronic liver inflammation that results in fibrosis.