Immune Cell Profiles Research Articles

Olaparib, AZD1390, ceralasertib, saruparib and consolidation durvalumab (CONCORDE) phase Ib platform study of novel DNA damage response inhibitor (DDRi) agents in combination with radiotherapy in non-small cell lung cancer (NSCLC).

TPS8119 Background: Lung cancer is the leading cause of cancer mortality globally, with cases projected to exceed 62,000 by 2035. Outcomes remain poor despite advances in radiotherapy (RT) technologies. Combining novel mechanism-based agents with RT could improve the therapeutic index. DNA inhibition of cellular response to radiation-induced DNA damage can overcome intrinsic radio resistance and poses a promising strategy. Methods: CONCORDE is an open label, randomised, multi-arm, phase Ib, clinical trial opened to accrual in 11 UK hospitals designed to assess multiple DDRi in combination with radical thoracic RT (60Gy in 30 fractions over 6 weeks). Patients are randomised 3:1 to RT+/-DDRi. The study utilises an adaptive Bayesian model-based approach to dose escalation aiming to identify the recommended phase II dose for each treatment combination. Two arms will deliver up to 12 months consolidation durvalumab+/-DDRi following RT. Patients are eligible based on key criteria: not suitable for concurrent chemotherapy RT, inoperable, stage IIB/III NSCLC, performance status (KPS≥70). The dose limiting toxicity (DLT) period for treatment-related toxicities is 13.5 months post start of RT treatment, with most toxicities expected within the first 4.5 months (short DLT period). At least one patient must complete the short DLT period before dose escalation. A RT-alone calibration arm aids toxicity attribution. This trial is in progress: CONCORDE-A (olaparib (PARP inhibitor)), CONCORDE-B (AZD1390 (ATM inhibitor)), CONCORDE-C (ceralasertib (ATR inhibitor) followed by consolidation durvalumab+/-ceralasertib) and CONCORDE-E (AZD5305/saruparib (PARP1select inhibitor) followed by consolidation durvalumab) are open to accrual as of 24/11/2023. 57 of 74 registered participants have been randomised across 4 study arms. Of those 57 randomised: 55 started treatment: 20 received RT-alone, 12 received olaparib+RT, 14 received AZD1390+RT, 6 received ceralasertib+RT, 3 received AZD5305+RT and 2 withdrew. CONCORDE continues to recruit to the target sample size of 160 (40/arm) and welcomes further UK centres. A parallel multimodality translational program to identify biomarkers of treatment response, toxicity and the impact on the immune system are in development. Biomarkers of interest include plasma toxicity markers, immune cell profiling, radiomics and ctDNA. Clinical trial information: 10142971.

Feasibility and safety of neoadjuvant nivolumab and chemotherapy for resectable diffuse pleural mesotheliomas: Results of a prospective pilot study.

8003 Background: Diffuse pleural mesotheliomas (DPM) are an aggressive malignancy where even patients with potentially resectable disease have poor outcomes. With an evolving understanding of the role of pleurectomy/decortication (P/D) as the standard of care for resectable disease, the need to improve upfront therapies is more urgent. Here we report the results of our single institution prospective pilot study of nivolumab with platinum (cisplatin/carboplatin) and pemetrexed prior to P/D for patients with resectable DPM (NCT04162015). Methods: Patients deemed operable and resectable by a multidisciplinary team received 2 cycles of neoadjuvant nivolumab, pemetrexed, and platinum followed by P/D. Subsequent therapy was at the physician's discretion. Using a minimax Simon two-stage design, the primary objective was defined as attempted P/D within 30 days of the planned surgery date. Secondary objectives included evaluating safety, overall survival (OS) from diagnosis, and progression free survival (PFS). Tumor tissue and plasma were collected for exploratory analyses such as immune cell profiling and pathologic response. Using single cell RNA sequencing (scRNAseq), we explored DPM transcriptional heterogeneity and treatment-associated tumor microenvironment (TME) changes. Results: 22 patients received neoadjuvant chemo-immunotherapy and 19 underwent attempted P/D (2 progressed and 1 declined surgery). 68% were men (13/19), 89% were epithelioid (17/19; remaining 2 were biphasic), and the median age at diagnosis was 72 (range: 33-80). No patient experienced pre-operative toxicity precluding P/D. 95% (18/19) of patients underwent P/D within 30 days of the planned date; 1 patient had biopsy-proven tumor flare after cycle 2 and had surgery at day +37. As of January 2024, median OS for the 19 patients that went for attempted P/D was 41.0 mos (median follow up: 25.3mos). OS for those who underwent R1 resections (n = 12) was not reached and for R2 (n = 7) was 31.4 mos (p = 0.67; HR 1.43, 95% CI 0.28-7.30). 26% of specimens (5/19) had ≤ 10% viable tumor cells on the resection; there was no clear correlation with outcomes. scRNAseq was performed on 15 resection specimens with available material. Comparing patients with PFS greater than or less than 12mos (PFS12), we found significantly increased abundance of CD8 cytotoxic T cells, and ISG+ (interferon stimulated) T cells. Conclusions: Neoadjuvant platinum, pemetrexed, and nivolumab was reliably able to be given to patients with DPM without impacting their ability to undergo P/D. The encouraging OS of this cohort to date, and tolerance of the regimen, highlights the importance of patient selection and preoperative treatments when considering P/D. Ongoing exploratory analyses will profile the cellular and transcriptional landscape of DPM and identify correlates of response to neoadjuvant chemo-immunotherapy. Clinical trial information: NCT04162015 .

Deep mapping of the cytokine release syndrome inflammatory continuum: Comprehensive grade-specific profiling and differentiation from sepsis in patients receiving immune checkpoint therapy.

12129 Background: The delineation of cytokine release syndrome (irCRS) from sepsis and its transition to compensatory anti-inflammatory response syndrome (irCARS) is crucial during ICI therapy for personalized care. Methods: We conducted an extensive biological and immune profiling on PBMC and serum from 38 oncology patients with various irCRS grades, including immune-related hemophagocytic lymphohistiocytosis irHLH (n=9) and sepsis during ICI treatment (n=8). Results: Our results show a dominant Th1 response in irCRS. Early markers of irCRS increased progressively with clinical severity in different grades, especially CXCL9, CXCL10 and IFN-γ levels. Starting with low-grade (G) (G1 and 2), a concomitant irCARS response was detected, characterized by an initial increase in IL-1RA and a significant upregulation of IL-10 specifically in G4. 3 subclusters with different overall survival (OS) and cytokine profiles were identified using routine biomarkers. Cluster 1 (C1, n=7) and Cluster 2 (C2, n=17) showed a 2-year OS of 100% and 48.49%, respectively (HR=4.025 [95% CI = 1.052-15.39], p=0.0419) and differed with higher levels of CCL3/11, CXCL10 and Il-1RA. Cluster 3 (C3, n=3) had the lowest OS, 6-month OS of 0%, and a distinct biological profile with highest levels of CXCL8/12/13, IL1-RA, IL-6, IL-21, HGF, significantly different from C1 (HR=111.4 [95% CI=6.63-1871], p=0.0011) and C2 (HR=30.04 [95% CI=2.686-335.9], p=0.0256). In 12 patients with severe irCRS (G3 and 4) and refractory to steroids, treatment with tocilizumab (TCZ) resulted in 100% resolution. In irHLH patients (n=9), the clusters retained their prognostic impact. Similarly, in irHLH with poor prognosis (irHLH cluster 3, n=3), high levels of CXCL8/12/13, IL-6 and IL-21 were identified. 13 patients with sepsis-like symptoms (irHLH, n=8; or sepsis during ICI, n=5) showed distinct cytokine and immune cell profiles. irHLH showed elevated IL-1-RA, IL-10, IL-18, IFN-γ, CXCL9/10, CCL2/3/4 while sepsis was characterized by increased IL-7. In irHLH, higher HLA-DR+/CD38+ populations were seen in T cells, monocytes, DCs and NK cells. There was significant CD62L downregulation in neutrophils during sepsis. Our decision tree using ferritin, EGF, IL-6 and leukocytes effectively discriminated irHLH from sepsis and G3 irCRS. High CXCL-9 levels predicted the diagnosis and severity of irHLH more accurately than the conventional HScore. Conclusions: We've identified a panel of biomarkers associated with OS. CXCL9 emerges as a superior marker, outperforming HScore. The successful treatment with TCZ underscores the importance of targeted interventions. Our decision tree enhances diagnostic accuracy for sepsis-like symptoms, aiding in the differentiation between irCRS, irHLH, and sepsis. This supports the optimal implementation of personalized strategies.

Role of plasmacytoid dendritic cells in vascular dysfunction in mice with renovascular hypertension

Endothelial dysfunction and inflammation are clinically significant risk factors for cardiovascular diseases in hypertension. Although immune cells play a role in hypertension, the impact of plasmacytoid dendritic cells in established renovascular hypertension-induced cardiovascular complications is not fully understood. We investigated plasmacytoid dendritic cells' contribution to arterial endothelial dysfunction and inflammation in renovascular hypertension.A two-kidney one-clip (2K1C) model for four weeks in both male and female mice was used to induce renovascular hypertension. We treated mice with or without anti-PDCA-1 antibodies for one week to deplete the plasmacytoid dendritic cells. Renovascular hypertension causes cardiac hypertrophy, lung edema, and microvascular endothelial dysfunction associated with inflammation induction in mice. Moreover, renovascular hypertension affects the profile of immune cells, including dendritic cells and macrophages, with variations between male and female mice. Interestingly, the depletion of plasmacytoid dendritic cells significantly reduces blood pressure, cardiac hypertrophy, lung edema, inflammation, and oxidative stress and improves microvascular endothelial function via the endoplasmic reticulum (ER) stress, autophagy, and mTOR-dependent mechanisms.Plasmacytoid dendritic cells significantly contribute to the development of cardiovascular complications in renovascular hypertension by modulating immune cells, inflammation, oxidative stress, and ER stress.

Effect of the Replacement of Wheat Straw by Spent Mushroom Substrate in the Diet of Dairy Ewes during Late Lactation on Milk Production, Composition, Oxidation Stability and Udder Health

The aim of the present study was to investigate the effects of different dietary levels of spent mushroom substrate (SMS) at the expense of wheat straw (WS) on milk characteristics in dairy sheep. Thirty ewes at their final stage of lactation (145 ± 5 days after parturition) were randomly assigned into three groups; control (C), provided with a diet consisting of concentrates, alfalfa hay and WS, and SMS1 and SMS2, where WS was replaced by SMS at 50 or 100%, respectively. The experiment lasted for 28 days, and milk yield, composition, somatic cell count (SCC) and oxidative stability were monitored weekly, while milk fatty acid and immune cell profile were also determined on day 28. No significant differences were found in produced milk quantity, fat, protein, lactose, total solids non-fat, SCC and fatty acid profile between the experimental groups. However, milk oxidative stability was significantly improved as an effect of SMS (p < 0.001). At the same time, milk polymorphonuclear leukocyte percentage was decreased in SMS2 group (p < 0.05). As indicated, SMS seems to be a promising agro-industrial by-product for ewes’ diet that could improve milk oxidative stability, without negatively affecting milk yield, composition and ewe health status, contributing in the context of sustainability, circular economy and protection of natural resources.

Exploring T-cell exhaustion features in Acute myocardial infarction for a Novel Diagnostic model and new therapeutic targets by bio-informatics and machine learning

BackgroundT-cell exhaustion (TEX), a condition characterized by impaired T-cell function, has been implicated in numerous pathological conditions, but its role in acute myocardial Infarction (AMI) remains largely unexplored. This research aims to identify and characterize all TEX-related genes for AMI diagnosis.MethodsBy integrating gene expression profiles, differential expression analysis, gene set enrichment analysis, protein-protein interaction networks, and machine learning algorithms, we were able to decipher the molecular mechanisms underlying TEX and its significant association with AMI. In addition, we investigated the diagnostic validity of the leading TEX-related genes and their interactions with immune cell profiles. Different types of candidate small molecule compounds were ultimately matched with TEX-featured genes in the “DrugBank” database to serve as potential therapeutic medications for future TEX-AMI basic research.ResultsWe screened 1725 differentially expressed genes (DEGs) from 80 AMI samples and 71 control samples, identifying 39 differential TEX-related transcripts in total. Functional enrichment analysis identified potential biological functions and signaling pathways associated with the aforementioned genes. We constructed a TEX signature containing five hub genes with favorable prognostic performance using machine learning algorithms. In addition, the prognostic performance of the nomogram of these five hub genes was adequate (AUC between 0.815 and 0.995). Several dysregulated immune cells were also observed. Finally, six small molecule compounds which could be the future therapeutic for TEX in AMI were discovered.ConclusionFive TEX diagnostic feature genes, CD48, CD247, FCER1G, TNFAIP3, and FCGRA, were screened in AMI. Combining these genes may aid in the early diagnosis and risk prediction of AMI, as well as the evaluation of immune cell infiltration and the discovery of new therapeutics.

Immune cell profiles associated with human exposure to perfluorinated compounds (PFAS) suggest changes in natural killer, T helper, and T cytotoxic cell subpopulations

Per- and polyfluoroalkyl substances (PFAS) constitutes a group of highly persistent man-made substances. Recent evidence indicates that PFAS negatively impact the immune system. However, it remains unclear how different PFAS are associated with alterations in circulating leukocyte subpopulations. More detailed knowledge of such potential associations can provide better understanding into mechanisms of PFAS immunotoxicity in humans. In this exploratory study, associations of serum levels of common PFAS (perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS)) and immune cell profiles of peripheral blood mononuclear cells, both with and without immunostimulation, were investigated. High-dimensional single cell analysis by mass cytometry was done on blood leukocytes from fifty participants in the Norwegian human biomonitoring EuroMix study.Different PFAS were associated with changes in various subpopulations of natural killer (NK), T helper (Th), and cytotoxic T (Tc) cells. Broadly, PFAS concentrations were related to increased frequencies of NK cells and activated subpopulations of NK cells. Additionally, increased levels of activated T helper memory cell subpopulations point to Th2/Th17 and Treg-like skewed profiles. Finally, PFAS concentrations were associated with decreased frequencies of T cytotoxic cell subpopulations with CXCR3+ effector memory (EM) phenotypes. Several of these observations point to biologically plausible mechanisms that may contribute to explaining the epidemiological reports of immunosuppression by PFAS. Our results suggest that PFAS exposures even at relatively low levels are associated with changes in immune cell subpopulations, a finding which should be explored more thoroughly in a larger cohort. Additionally, causal relationships should be confirmed in experimental studies. Overall, this study demonstrates the strength of profiling by mass cytometry in revealing detailed changes in immune cells at a single cell level.

Programmed cell death-1 is involved with peripheral blood immune cell profiles in patients with hepatitis C virus antiviral therapy.

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance.

Chemokine receptor 7 contributes to T- and B-cell filtering in ageing bladder, cystitis and bladder cancer

BackgroundResearch has suggested significant correlations among ageing, immune microenvironment, inflammation and tumours. However, the relationships among ageing, immune microenvironment, cystitis and bladder urothelial carcinoma (BLCA) in the bladder have rarely been reported.MethodsBladder single-cell and transcriptomic data from young and old mice were used for immune landscape analysis. Transcriptome, single-cell and The Cancer Genome Atlas Program datasets of BLCA and interstitial cystitis/bladder pain syndrome (IC/BPS) were used to analyse immune cell infiltration and molecular expression. Bladder tissues from mice, IC/BPS and BLCA were collected to validate the results.ResultsEight types of immune cells (macrophages, B-cells, dendritic cells, T-cells, monocytes, natural killer cells, γδ T-cells and ILC2) were identified in the bladder of mice. Aged mice bladder tissues had a significantly higher number of T-cells, γδ T-cells, ILC2 and B-cells than those in the young group (P < 0.05). Three types of T-cells (NK T-cells, γδ T-cells and naïve T-cells) and three types of B-cells (follicular B-cells, plasma and memory B-cells) were identified in aged mice bladder. Chemokine receptor 7 (CCR7) is highly expressed in aged bladder, IC/BPS and BLCA (P < 0.05). CCR7 is likely to be involved in T- and B-cell infiltration in aged bladder, IC/BPS and BLCA. Interestingly, the high CCR7 expression on BLCA cell membranes was a prognostic protective factor.ConclusionsIn this study, we characterised the expression profiles of immune cells in bladder tissues of aged and young mice and demonstrated that CCR7-mediated T- and B-cell filtration contributes to the development of bladder ageing, IC/BPS and BLCA.

Abstract PR003: T cell receptor repertoire and diversity are prognostic markers in bladder cancer

Abstract T cells represent essential effector cells in the intrinsic antitumor immune response. Cancer-specific T cells can recognize cancer neoantigens through their T cell receptors (TCRs). The expansion of these clones is believed to be an early response to malignancy. We hypothesized that the T cell landscape offers insights into immune competency. We aimed to characterize the TCR repertoire in patients with bladder cancer and explore correlations with disease outcomes. We analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer (MIBC) treated with chemotherapy followed by radical cystectomy. Peripheral and tumor TCR repertoires were produced using ultradeep amplicon-based sequencing of the TCR beta chain. T cell fractions were inferred from whole exome sequencing data of blood DNA using TcellExTRECT. Latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. The T cell subtype compositions in tumor and blood were investigated in four patients with bladder cancer using the Chromium Single Cell Immune profiling kit from 10X Genomics. Low pre-treatment peripheral TCR diversity was associated with worse overall survival (HR = 2.3, p = 0.024, n = 119) in patients with MIBC, particularly when combined with a low fraction of circulating T cells (HR= 4.7, p = 0.00049, n = 67). We validated these findings in two independent cohorts of patients with MIBC (p = 0.01, n = 107; p = 0.042, n = 79). The low-diversity TCR repertoires were characterized by large expanded T cell clones that persisted over time. Longitudinal analysis indicated a potential adverse impact of treatment, evidenced by a reduction in TCR diversity and circulating T cells over time in patients with initially high-diversity repertoires. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections and cytomegalovirus DNA detection was strongly associated with low TCR diversity (p = 5.3 × 10−5). We observed a notable disparity between tumor and peripheral blood TCR repertoires. Single-cell sequencing revealed that regulatory T cells were prevalent in the tumor, whereas a combination of cytotoxic and naïve T cells dominated the blood. More explicitly, expanded clones in the blood were commonly annotated as terminally differentiated effector memory T cells expressing high levels of cytotoxic and exhausted genes. Our results suggest that high peripheral TCR diversity and high T cell fraction are markers of general immune competence, reflecting the ability to eradicate cancer and other diseases. In contrast, the expanded persistent clones predominating the low peripheral TCR diversity repertoires are likely exhausted and less likely to exhibit tumor specificity or effective disease-fighting capabilities. Our findings underline the crucial role of the immune system in determining disease outcomes and highlight the potential for improving immune health as a promising approach for future treatment and prevention. Citation Format: Nanna Kristjánsdóttir, Asbjørn Kjær, Iver Nordentoft, Randi I Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Trine Strandgaard, Deema Radif, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen B Jensen, Nicolai J. Birkbak, Lars Dyrskjøt. T cell receptor repertoire and diversity are prognostic markers in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR003.

Dynamic changes of immunocyte subpopulations in thermogenic activation of adipose tissues.

The effects of cold exposure on whole-body metabolism in humans have gained increasing attention. Brown or beige adipose tissues are crucial in cold-induced thermogenesis to dissipate energy and thus have the potential to combat metabolic disorders. Despite the immune regulation of thermogenic adipose tissues, the overall changes in vital immune cells during distinct cold periods remain elusive. This study aimed to discuss the overall changes in immune cells under different cold exposure periods and to screen several potential immune cell subpopulations on thermogenic regulation. Cibersort and mMCP-counter algorithms were employed to analyze immune infiltration in two (brown and beige) thermogenic adipose tissues under distinct cold periods. Changes in some crucial immune cell populations were validated by reanalyzing the single-cell sequencing dataset (GSE207706). Flow cytometry, immunofluorescence, and quantitative real-time PCR assays were performed to detect the proportion or expression changes in mouse immune cells of thermogenic adipose tissues under cold challenge. The proportion of monocytes, naïve, and memory T cells increased, while the proportion of NK cells decreased under cold exposure in brown adipose tissues. Our study revealed dynamic changes in immune cell profiles in thermogenic adipose tissues and identified several novel immune cell subpopulations, which may contribute to thermogenic activation of adipose tissues under cold exposure.

Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon

Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.

Profiling of Tumor-Infiltrating Immune Cells and Their Impact on Survival in Glioblastoma Patients Undergoing Immunotherapy with Dendritic Cells.

Glioblastomas (GBM) are the most common primary malignant brain tumors, comprising 2% of all cancers in adults. Their location and cellular and molecular heterogeneity, along with their highly infiltrative nature, make their treatment challenging. Recently, our research group reported promising results from a prospective phase II clinical trial involving allogeneic vaccination with dendritic cells (DCs). To date, six out of the thirty-seven reported cases remain alive without tumor recurrence. In this study, we focused on the characterization of infiltrating immune cells observed at the time of surgical resection. An analytical model employing a neural network-based predictive algorithm was used to ascertain the potential prognostic implications of immunological variables on patients' overall survival. Counterintuitively, immune phenotyping of tumor-associated macrophages (TAMs) has revealed the extracellular marker PD-L1 to be a positive predictor of overall survival. In contrast, the elevated expression of CD86 within this cellular subset emerged as a negative prognostic indicator. Fundamentally, the neural network algorithm outlined here allows a prediction of the responsiveness of patients undergoing dendritic cell vaccination in terms of overall survival based on clinical parameters and the profile of infiltrated TAMs observed at the time of tumor excision.

Exploring the immune characteristions of CRKP pneumonia at single-cell level

The immune dysregulation associated with carbapenem-resistant Klebsiella pneumoniae (CRKP) severity was investigated through single-cell RNA sequencing (scRNA-seq) of 5 peripheral blood samples from 3 patients with moderate and severe CRKP pneumonia. Additionally, scRNA-seq datasets from two individuals with COVID-19 were included for comparative analysis. The dynamic characterization and functional properties of each immune cell type were examined by delineating the transcriptional profiles of immune cells throughout the transition from moderate to severe conditions. Overall, most immune cells in CRKP patients exhibited a robust interferon-α response and inflammatory reaction compared to healthy controls, mirroring observations in COVID-19 patients. Furthermore, cell signatures associated with NK cells, macrophages, and monocytes were identified in CRKP progression including PTPRCAP for NK cells, C1QB for macrophages, and S100A12 for both macrophages and monocytes. In summary, this study offers a comprehensive scRNA-seq resource for illustrating the dynamic immune response patterns during CRKP progression, thereby shedding light on the associations between CRKP and COVID-19.

Analysis and validation of biomarkers of immune cell-related genes in postmenopausal osteoporosis: An observational study.

Postmenopausal osteoporosis (PMOP) is a common metabolic inflammatory disease. In conditions of estrogen deficiency, chronic activation of the immune system leads to a hypo-inflammatory phenotype and alterations in its cytokine and immune cell profile, although immune cells play an important role in the pathology of osteoporosis, studies on this have been rare. Therefore, it is important to investigate the role of immune cell-related genes in PMOP. PMOP-related datasets were downloaded from the Gene Expression Omnibus database. Immune cells scores between high bone mineral density (BMD) and low BMD samples were assessed based on the single sample gene set enrichment analysis method. Subsequently, weighted gene co-expression network analysis was performed to identify modules highly associated with immune cells and obtain module genes. Differential analysis between high BMD and low BMD was also performed to obtain differentially expressed genes. Module genes are intersected with differentially expressed genes to obtain candidate genes, and functional enrichment analysis was performed. Machine learning methods were used to filter out the signature genes. The receiver operating characteristic (ROC) curves of the signature genes and the nomogram were plotted to determine whether the signature genes can be used as a molecular marker. Gene set enrichment analysis was also performed to explore the potential mechanism of the signature genes. Finally, RNA expression of signature genes was validated in blood samples from PMOP patients and normal control by real-time quantitative polymerase chain reaction. Our study of PMOP patients identified differences in immune cells (activated dendritic cell, CD56 bright natural killer cell, Central memory CD4 T cell, Effector memory CD4 T cell, Mast cell, Natural killer T cell, T follicular helper cell, Type 1 T-helper cell, and Type 17 T-helper cell) between high and low BMD patients. We obtained a total of 73 candidate genes based on modular genes and differential genes, and obtained 5 signature genes by least absolute shrinkage and selection operator and random forest model screening. ROC, principal component analysis, and t-distributed stochastic neighbor embedding down scaling analysis revealed that the 5 signature genes had good discriminatory ability between high and low BMD samples. A logistic regression model was constructed based on 5 signature genes, and both ROC and column line plots indicated that the model accuracy and applicability were good. Five signature genes were found to be associated with proteasome, mitochondria, and lysosome by gene set enrichment analysis. The real-time quantitative polymerase chain reaction results showed that the expression of the signature genes was significantly different between the 2 groups. HIST1H2AG, PYGM, NCKAP1, POMP, and LYPLA1 might play key roles in PMOP and be served as the biomarkers of PMOP.

Multi-Omics Characterization of Colon Mucosa and Submucosa/Wall from Crohn's Disease Patients.

Crohn's disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by transmural disease. The concept of transmural healing (TH) has been proposed as an indicator of deep clinical remission of CD and as a predictor of favorable treatment endpoints. Understanding the pathophysiology involved in transmural disease is critical to achieving these endpoints. However, most studies have focused on the intestinal mucosa, overlooking the contribution of the intestinal wall in Crohn's disease. Multi-omics approaches have provided new avenues for exploring the pathogenesis of Crohn's disease and identifying potential biomarkers. We aimed to use transcriptomic and proteomic technologies to compare immune and mesenchymal cell profiles and pathways in the mucosal and submucosa/wall compartments to better understand chronic refractory disease elements to achieve transmural healing. The results revealed similarities and differences in gene and protein expression profiles, metabolic mechanisms, and immune and non-immune pathways between these two compartments. Additionally, the identification of protein isoforms highlights the complex molecular mechanisms underlying this disease, such as decreased RTN4 isoforms (RTN4B2 and RTN4C) in the submucosa/wall, which may be related to the dysregulation of enteric neural processes. These findings have the potential to inform the development of novel therapeutic strategies to achieve TH.

Elevated neutrophils and reduced NK cells are associated with altered tear molecular signatures and clinical sequelae of chronic ocular Stevens-Johnson syndrome

PurposeStevens-Johnson syndrome (SJS) is characterised as an immuno-inflammatory condition with potentially blinding ocular sequelae. Therefore, we have investigated the ocular surface immune cell profile and correlated it with secreted tear molecular factors and clinical ocular sequelae in SJS patients. Methods21 patients (42 eyes) with chronic ocular SJS and 16 healthy controls (20 eyes) were included in the study. Severity, types of keratopathies and ocular surface (OS) manifestations were determined. OS wash samples from study subjects were used to determine the status of 13 immune cell subsets using flow cytometry. Levels of 42 secreted immuno-inflammatory factors were measured by flow cytometry-based multiplex ELISA in tear samples. ResultsNeutrophils (Total, activated), neutrophils/NK cells ratio, neutrophils/T cells ratio were significantly (p < 0.05) elevated in SJS, while, proportions of T cells and NKT cells were significantly lower in SJS patients. Positive association between neutrophils and chronic ocular surface complication score (COCS) was observed, whereas, a negative association was noted between NK cells and COCS. Tear fluid levels of IL-6, IL-8, IL-18, IFNα/β/γ, TNFα, LIF, IL-8, HGF, sTNFR-I, NGAL, Granzyme, Perforins, MMP9/TIMP1 ratio were significantly higher in SJS. Loss of Limbal niche correlated significantly with immune profile and clinical sequelae. Increased neutrophils, decreased NK cells and specific set of altered secreted immuno-inflammatory mediators including bFGF, and IL-8 were observed in SJS patients with different types of keratopathies compared to those without keratopathy. ConclusionDistinct ocular surface immune profile variations were observed to correlate with clinical stages of chronic ocular SJS. Our findings uncover novel mechanisms and potential for targeted therapy in chronic ocular SJS patients.

Abstract PO3-19-02: White Button Mushroom and Biomarkers of Immune Cell and Inflammatory Responses in Obese Postmenopausal Women at High Risk of Breast Cancer

Abstract Background: White Button Mushroom (WBM), Agaricus bisporus, has anti-tumor and anti-inflammatory effects. Oral WBM mitigated the effects of a high fat diet on hepatic steatosis and insulin resistance in ovariectomized mice. A Phase I trial of WBM in men with prostate cancer demonstrated decreased myeloid derived suppressor cells (MDSC) in those with decreased PSA; MDSC have been linked to different cancers, including breast, and conditions of chronic inflammation. Design: This is a preliminary evidence trail in postmenopausal, obese women at high risk of breast cancer with a single intervention arm of 14g of WBM taken as 28 tablets daily for 3 months to assess effects on inflammation. Participants undergo a physical exam, measuring height, weight, and waist circumference, an optional fine needle aspiration (FNA) of breast tissue, a blood draw, a food frequency questionnaire and a 24-hour food recall at baseline and final visits. Participants are contacted monthly for tolerance and adherence. Blood samples and FNA adipose are used for metabolic measurements and processed for biomarkers of inflammation and immune response. Peripheral blood mononuclear cells (PBMC) will be used for RNA single-cell analysis to assess for immune cell profiles and expression with an interest in evaluating MDSC. Red blood cells will be assessed for fatty acid profiles. Eligibility: Postmenopausal, obese (BMI ≥30, Asian ≥25) women with high risk for breast cancer who can swallow pills are eligible. Patients are high risk if their relative risk of breast cancer is ≥2 times that of the general population; this includes women with a strong family history, a genetic mutation associated with hereditary breast cancer, a high-risk breast histology, Gail model 5-year risk &amp;gt;1.67, or a previous history of stage I-II breast cancer. Participants are excluded for an active malignancy within 5 years, ongoing active or preventative cancer treatment, or lab abnormalities of serum creatinine and total bilirubin &amp;gt;1.5 the upper limit of normal, hemoglobin of &amp;lt; 9.0 g/dL, platelets of &amp;lt; 100,000/mm3, or a total white blood cell count of &amp;lt; 3,500/mm3. Participants cannot be taking mushroom supplements, immunosuppressants, or non-steroidal anti-inflammatories. Specific Aims: This study aims to evaluate the effects of WBM on regulating immune response and inflammation in obese women at high risk for breast cancer. Our primary objective is the assessment of PBMC, specifically MDSC immune profiles before and after treatment. Our secondary objective is to evaluate changes in biomarkers of inflammation, immune cell profiles in breast adipose tissue, BMI, waist circumference, and metabolic biomarkers. Additionally, the study aims to assess tolerability of the WBM dose and effects on diet quality. Statistical Methods: 26 patients will provide 80% power to detect a change in %MDSC of -0.67, assuming a standard deviation of 1.15, and a 2-sided type I error of 5%. We will have similar power to evaluate changes in other immune subsets, inflammatory markers, metabolic markers, anthropometric measurements, and diet quality. FNA adipose tissue analysis will be used as a safety signal to evaluate the potential for unwanted inflammation with taking WBM. 13 patients with FNA will provide 80% power to detect a change of 85% of the standard deviation of the difference in the measure of biomarkers such as Interleukin-6 with a 2-sided type I error of 5%. As a safety signal, the signal will be reported without adjustment for multiple comparisons. Any conclusions based on such secondary endpoints will make note of the multiple comparison issue. Accrual: The accrual target is 26 evaluable patients with an estimated need to enroll 31. The study has enrolled 27 patients, 14 completed the study and 5 are currently on treatment. 8 participants were removed; 3 never started treatment, 1 was diagnosed with breast cancer, and 4 had other medical issues preventing continuation. Contact: Jessica Dzubnar, MD: jdzubnar@coh.org Citation Format: Jessica Dzubnar, Lisa Yee, Shiuan Chen. White Button Mushroom and Biomarkers of Immune Cell and Inflammatory Responses in Obese Postmenopausal Women at High Risk of Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-19-02.

Abstract PO2-25-05: Spatial multiplexing of protein biomarkers for immune cell profiling of the tumor microenvironment with ChipCytometry

Abstract Understanding the spatial distribution of key immune cell populations is critical in advancing our understanding of cancer and the development of novel therapeutics. Historically, the spatial analysis of the tumor microenvironment (TME) has been limited to relatively low-plex immunohistochemical (IHC) or immunofluorescent (IF) assays, which were inadequate for deep immune cell profiling of the TME. Here, we present the analysis of fresh-frozen human breast tissue using spatial multiplexing technology called ChipCytometry, which combines iterative immuno-fluorescent staining using open-source antibodies for quantitative measure of virtually unlimited protein biomarkers on the same sample and high dynamic range imaging to facilitate quantitative phenotyping with single-cell resolution. In addition to traditional pathology review of the high-plex dataset, subsequent image analysis can be done using open-source or commercial software for advanced spatial analyses, including cluster analysis, neighborhood analysis and advanced data visualization. The results show precise expression levels for each of the protein biomarkers in the assay for each individual cell within the sample while maintaining spatial information about each cell. Dozens of immune cell subtypes were identified and quantified based on protein expression profiles. Spatial analysis of the samples revealed quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for in-depth immune profiling in clinical samples. The ChipCytometry platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Combined with the single-cell spatial information, such datasets provide an opportunity for the discovery of new complex multiplexed biomarker signatures to inform therapeutic development and personalized medicine. Citation Format: Benton Berigan. Spatial multiplexing of protein biomarkers for immune cell profiling of the tumor microenvironment with ChipCytometry [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-05.

Abstract PO5-16-07: Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is known for its aggressive behavior with poor survival outcomes. Influenced by different expression levels of Programmed Death-Ligand 1 (PD-L1) and several subtypes of tumor-infiltrating lymphocytes (TILs), tumor microenvironment (TME) plays a crucial role in TNBC response to treatment and progression. Objective: To assess the prevalence and prognostic role of PD-L1 expression in advanced TNBC, defined as unresectable stage III or stage IV disease, in patients treated with a standard cytotoxic chemotherapy regimen. TILs composition was also evaluated as an exploratory objective. Methods: The internal database of the Brazilian National Cancer Institute was queried out for women diagnosed and treated with advanced TNBC from January 2018 to December 2022. Formalin-fixed paraffin-embedded samples of a maximum of four years old were analyzed. PD-L1 expression using 22C3 pharmDX assay was estimated through combined positive score (CPS) dichotomization (CPS &amp;lt; 10 vs. CPS≥10). Tissue microarrays comprising the samples of biopsies were subjected to immunohistochemistry, targeting specific markers including CD3, CD4, CD8, CD56, CD68, CD117, FOXP3, PD-1, the immune cell profiles were carefully examined and their correlation with the PD-L1 CPS was determined. Results: A total of 150 patients were included. The expression of PD-L1 was undetermined in 2 cases. The median age was 51.5 years (IQR 41.8-60.2) and 20.9% of the cases had CPS≥10. Most patients were &amp;lt; 65 years (73.0%), were postmenopausal (56.8%) and belonged to non-white ethnicity (70.3%). Postmenopausal women predominated in the CPS≥10 subgroup (74.2%) (Table 1). No significant differences in demographic characteristics and clinicopathological variables were observed based on PD-L1 subgroups, 117 patients (79.1%) had CPS &amp;lt; 10, with 91 (77.8%) being de novo stage IV metastatic disease. CD3+ (p=0.037), CD4+ (p=0.005) and CD8+ (p=0.001) TILs had higher expression in tumors with PD-L1 CPS≥10 (Table 2). According to the treatment received, almost half of the patients only received first-line chemotherapy (47%), as the following second line was applied to 28.8% of the total group. The third and fourth lines were administered in 12.9% and 9.1% of patients, respectively. Only 2 patients went through five palliative chemotherapy lines, and only one received up to 6 treatment lines. Between the subgroups CPS≥10 versus CPS &amp;lt; 10 no statistically significant differences were observed in the median progression-free survival (PFS) (5.1 vs. 5.0 months, p=0.89) and overall survival (OS) (8.7 vs. 8.8 months, p=0.6). Conclusion: This study offers insights into the expression patterns of PD-L1 and TILs subtypes in advanced TNBC cases in Brazil. PD-L1 CPS did not influence survival outcomes for this cohort of patients treated with cytotoxic chemotherapy only. The composition of TILs subtypes within the TME appears to vary based on PD-L1 CPS. Table 1. Advanced TNBC population characteristic by CPS (Nf148). Table 2. Tumor Infiltrating Lymphocytes characterization (CD3, CD4, CD8, CD56, CD68, CD 117, FOXP3 and PD-1) and its correlation with CPS status. Citation Format: Alexssandra Lima Siqueira dos Santos, Jesse Lopes da Silva, Lucas Zanetti de Albuquerque, Antônio Lucas Araújo Neto, Cecília Ferreira da Silva, Luana Aguiar Mesquita Cerva, Isabele Ávila Small, Cicera Pimenta Marcelino, Paula de Mendonça Batista, Maria Aparecida do Carmo Rego, Maria Amélia Carlos Souto Maior Borba, Andreia Cristina de Melo. Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-07.