Profiling Of Peripheral Blood Mononuclear Cells Research Articles

Concordant B and T Cell Heterogeneity Inferred from the Multiomic Landscape of Peripheral Blood Mononuclear Cells in a Crohn's Disease Cohort.

Crohn's disease is characterized by inflammation in the gastrointestinal tract due to a combination of genetic, immune, and environmental factors. Transcriptomic and epigenomic profiling of intestinal tissue of Crohn's disease patients have revealed valuable insights into pathology, however have not been conducted jointly on less invasive peripheral blood mononuclear cells (PBMCs). Furthermore, the heterogeneous responses to treatments among individuals with Crohn's disease imply hidden diversity of pathological mechanisms. We employed single nucleus multiomic analysis, integrating both snRNA-seq and snATAC-seq of PBMCs with a variety of open source bioinformatics applications. Our findings reveal a diverse range of transcriptional signatures among individuals, highlighting the heterogeneity in PBMC profiles. Nevertheless, striking concordance between three heterogeneous groups was observed across B cells and T cells. Differential gene regulatory mechanisms partially explain these profiles, notably including a signature involving TGFß signaling in two individuals with Crohn's disease. A mutation mapped to a transcription factor binding site within a differentially accessible peak associated with the expression of this pathway, with implications for a personalized approach to understanding disease pathology. This study highlights how multiomic analysis can reveal common regulatory mechanisms that underlie heterogeneity of PBMC profiles, one of which may be specific to inflammatory disease.

Increased monocyte activity identified in Alzheimer’s disease by in depth single‐cell genomic and proteomic analysis

AbstractBackgroundAlzheimer’s disease (AD) patients show sustained levels of inflammation in the brain and the peripheral immune system. It is not known how various peripheral blood mononuclear cells (PBMCs) differ in AD patients and whether those differences can act as biomarkers of AD. Here we performed a multi‐omic profiling of PBMCs from AD patients and compared the composition of their cell type and cell state as well as their gene and protein expression to normal controls.MethodSingle‐cell proteogenomics analysis was performed on PBMCs from 20 AD patients and 15 controls using Singleron Biotechnologies’ ESCAPE platform. Seven of the AD and four control samples were additionally analyzed for bulk protein expression using Sciomics’ scioDiscover platform.ResultBulk proteomics identified 100 proteins with a significant differential abundance between AD patients and controls. Data point to a higher platelet activation and degranulation, as well as changes in the EGFR / MAPK3 and VEGF signaling in AD. As an individual marker CD163 was identified at a higher abundance in AD PBMCs pointing to an increase in monocyte / macrophage activity.From the single‐cell analysis we found that AD patients had significantly more CD14+ monocytes. We further found that the CD14+ monocytes could be split into seven clusters based on their gene expression with only two clusters having a significantly higher number of cells in AD patients. One of the overrepresented clusters showed high expression of Alarmin genes, suggesting an increased inflammatory environment, while the other cluster showed a higher level of HLA expression suggesting a state primed for activation.ConclusionWe found significant changes in both gene and protein expression in PBCMs from AD patients that indicate an increased inflammatory state . While there was a good overlap of findings between gene and protein expression, some of the changes are only seen at the protein level, while others are only observed at the level of gene expression. The combination of the two measurement techniques provides us with additional insights into inflammatory nature of the peripheral immune system in AD patients and provide hints at the mechanisms different cells use to generate those inflammatory signals.

Changes in peripheral blood immune cell population in thyroid cancer patients treated with lenvatinib

This study evaluated changes in the peripheral blood immune cell population in patients with advanced thyroid cancer receiving lenvatinib treatment to confirm the immune-modulatory effect of lenvatinib. After obtaining informed consent from patients, we prospectively collected 20 ml of whole blood at 2–3 months intervals 2–4 times from each patient; peripheral blood mononuclear cells (PBMCs) were separated, and the Maxpar Direct Immune Profiling Assay was performed. A total of 10 patients were enrolled, and 31 blood samples were obtained. The median age of patients was 65 years, and all patients showed durable responses to the lenvatinib treatment. When we compared the PBMC profiles between the pre-treatment, on-treatment, and off-treatment samples, the peripheral natural killer (NK) cell proportion differed significantly. The proportion of NK cells among total live cells significantly increased from 9.3 ± 4.5 (%) in the pre-treatment samples to 20.8 ± 7.9 (%) in the on-treatment samples (P = 0.009) and decreased to 13.3 ± 3.1 (%) in the off-treatment samples (P = 0.07). There was a significant increase in the peripheral NK cell population with lenvatinib treatment in advanced thyroid cancer patients. This finding confirms the immune-modulatory effect of lenvatinib.

Characterization of the circulating transcriptome expression profile and identification of novel miRNA biomarkers in hypertrophic cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM), one of the most common genetic cardiovascular diseases, but cannot be explained by single genetic factors. Circulating microRNAs (miRNAs) are stable and highly conserved. Inflammation and immune response participate in HCM pathophysiology, but whether the miRNA profile changes correspondingly in human peripheral blood mononuclear cells (PBMCs) with HCM is unclear. Herein, we aimed to investigate the circulating non-coding RNA (ncRNA) expression profile in PBMCs and identify potential miRNAs for HCM biomarkers.MethodsA Custom CeRNA Human Gene Expression Microarray was used to identify differentially expressed (DE) mRNAs, miRNAs, and ncRNAs (including circRNA and lncRNA) in HCM PBMCs. Weighted correlation network analysis (WGCNA) was used to identify HCM-related miRNA and mRNA modules. The mRNAs and miRNAs from the key modules were used to construct a co-expression network. Three separate machine learning algorithms (random forest, support vector machine, and logistic regression) were applied to identify potential biomarkers based on miRNAs from the HCM co-expression network. Gene Expression Omnibus (GEO) database (GSE188324) and experimental samples were used for further verification. Gene set enrichment analysis (GSEA) and competing endogenous RNA (ceRNA) network was used to determine the potential functions of the selected miRNAs in HCM.ResultsWe identified 1194 DE-mRNAs, 232 DE-miRNAs and 7696 DE-ncRNAs in HCM samples compared with normal controls from the microarray data sets. WGCNA identified key miRNA modules and mRNA modules evidently associated with HCM. We constructed a miRNA‒mRNA co-expression network based on these modules. A total of three hub miRNAs (miR-924, miR-98 and miR-1) were identified by random forest, and the areas under the receiver operator characteristic curves of miR-924, miR-98 and miR-1 were 0.829, 0.866, and 0.866, respectively.ConclusionsWe elucidated the transcriptome expression profile in PBMCs and identified three hub miRNAs (miR-924, miR-98 and miR-1) as potential biomarkers for HCM detection.

MOBIDIC trial: Use of peripheral blood mononuclear cells (PBMC) as biomarkers of diabetic cardiomyopathy

Type 2 diabetes (T2D) patients, especially when associated with metabolic syndrome (MS) are at high risk to develop heart failure with preserved ejection fraction (HFpEF) or heart failure with mildly reduced ejection fraction (HFmrEF), and the specific impact of T2D + MS on cardiac function impairment is usually known as “diabetic cardiomyopathy” (DCM). Cardiac remodeling (i.e. hypertrophy) and subtle myocardial dysfunction are highly prevalent in T2D + MS but not specific enough to predict further HFpEF or HFmrEF. Also, current biomarkers can identify but do not predict HFpEF or HFmrEF in T2D patients. Therefore, specific biomarkers are needed. Recently, peripheral blood mononuclear cells (PBMC) have emerged as complementary and non-invasive biomarkers to stratify the patients in several pathologies, but not yet in DCM. We hypothesize that the calcic and inflammatory profile of PBMC may reflect the degree of cardiac dysfunction encountered during DCM. To this end, we will perform a clinical study including 30 T2D + MS and 30 non-diabetic and non-MS patients without HF or with HFpEF/HFmrEF. A deep characterization of their cardiac function will be assessed by echocardiography. A biocollection of PBMC will be done to study by flow cytometry the calcium fluxes and the inflammatory cell subtypes. MOBIDIC trial is expected to start on February 2023. We expect that PBMC obtained from a peripheral blood sample would provide insights from calcic and inflammatory pathways, and may identify more specific molecular signatures shared between T2D + MS and HFpEF/HFmrEF.

Gene Expression Analysis of Immune Regulatory Genes in Circulating Tumour Cells and Peripheral Blood Mononuclear Cells in Patients with Colorectal Carcinoma.

Information regarding genetic alterations of driver cancer genes in circulating tumour cells (CTCs) and their surrounding immune microenvironment nowadays can be employed as a real-time monitoring platform for translational applications such as patient response to therapeutic targets, including immunotherapy. This study aimed to investigate the expression profiling of these genes along with immunotherapeutic target molecules in CTCs and peripheral blood mononuclear cells (PBMCs) in patients with colorectal carcinoma (CRC). Expression of p53, APC, KRAS, c-Myc, and immunotherapeutic target molecules PD-L1, CTLA-4, and CD47 in CTCs and PBMCs were analysed by qPCR. Their expression in high versus low CTC-positive patients with CRC was compared and clinicopathological correlations between these patient groups were analysed. CTCs were detected in 61% (38 of 62) of patients with CRC. The presence of higher numbers of CTCs was significantly correlated with advanced cancer stages (p = 0.045) and the subtypes of adenocarcinoma (conventional vs. mucinous, p = 0.019), while being weakly correlated with tumour size (p = 0.051). Patients with lower numbers of CTCs had higher expression of KRAS. Higher KRAS expression in CTCs was negatively correlated with tumour perforation (p = 0.029), lymph node status (p = 0.037), distant metastasis (p = 0.046) and overall staging (p = 0.004). CTLA-4 was highly expressed in both CTCs and PBMCs. In addition, CTLA-4 expression was positively correlated with KRAS (r = 0.6878, p = 0.002) in the enriched CTC fraction. Dysregulation of KRAS in CTCs might evade the immune system by altering the expression of CTLA-4, providing new insights into the selection of therapeutic targets at the onset of the disease. Monitoring CTCs counts, as well as gene expression profiling of PBMCs, can be helpful in predicting tumour progression, patient outcome and treatment.

Integrated whole-genome gene expression analysis reveals an atlas of dynamic immune landscapes after myocardial infarction.

Myocardial infarction (MI) is a deadly medical condition leading to irreversible damage to the inflicted cardiac tissue. Elevated inflammatory response marks the severity of MI and is associated with the development of heart failure (HF), a long-term adverse outcome of MI. However, the efficacy of anti-inflammatory therapies for MI remains controversial. Deciphering the dynamic transcriptional signatures in peripheral blood mononuclear cells (PBMCs) is a viable and translatable route to better understand post-MI inflammation, which may help guide post-MI anti-inflammatory treatments. In this work, integrated whole-genome gene expression analysis was performed to explore dynamic immune landscapes associated with MI. GSEA and GSVA showed that pathways involved in the inflammatory response and metabolic reprogramming were significantly enriched in PBMCs from MI patients. Based on leukocyte profiles generated by xCell algorithm, the relative abundance of monocytes and neutrophils was significantly increased in PBMCs from MI patients and had positive correlations with typical inflammation-associated transcripts. Mfuzz clustering revealed temporal gene expression profiles of PBMCs during the 6-month post-MI follow-up. Analysis of DEGs and gene sets indicated that PBMCs from HF group were characterized by elevated and lasting expression of genes implicated in inflammation and coagulation. Consensus clustering generated 4 metabolic subtypes of PBMCs with molecular heterogeneity in HF patients. In summary, integrated whole-genome gene expression analysis here outlines a transcriptomic framework that may improve the understanding of dynamic signatures present in PBMCs, as well as the heterogeneity of PBMCs in MI patients with or without long-term clinical outcome of HF. Moreover, the work here uncovers the diversity and heterogeneity of PBMCs from HF patients, providing novel bioinformatic evidence supporting the mechanistic implications of metabolic reprogramming and mitochondrial dysfunction in the post-MI inflammation and HF. Therefore, our work here supports the notion that individualized anti-inflammatory therapies are needed to improve the clinical management of post-MI patients.

Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLA2s.

Bothrops venom contains a high amount of secreted phospholipase A2 (sPLA2s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA2s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA2s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-β and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA2s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.

Abstract P2-26-07: Alteration of gut microbiota signatures and its association with diarrhea during abemaciclib treatment: A multicenter prospective cohort study (KBCRN-A002 study)

Abstract Background: Abemaciclib is a selective CDK4 and CDK6 inhibitor with demonstrated efficacy in hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. The most common adverse event across previous trials was early-onset diarrhea, affecting the patients’ quality of life and necessitating dose reductions. However, the exact mechanism for the lower rate of diarrhea in the other CDK4 and CDK6 inhibitors compared with abemaciclib is unknown. Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with the anti-tumor response; however, reported microbial signatures associated with adverse events by anti-cancer agent are inconsistent. To determine the underlying mechanism, we evaluated the correlation between diarrhea with abemaciclib and microbiota signatures in a metastatic breast cancer cohort. Methods: The KBCRN-A002 study is a multicenter, prospective cohort study, which aims to evaluate the association between gut microbiota signatures and abemaciclib-induced diarrhea in breast cancer patients. Patients with metastatic breast cancer who were receiving abemaciclib were eligible. The primary objective of this study is the correlation between diarrhea and the microbiota signatures and immune profile. Incidence and severity of diarrhea were evaluated by the Bristol stool scale at baseline, from day 1 to day 14, and at day 90 of treatment. Stool samples were collected at baseline and at day 90 after the start of abemaciclib treatment. The gut microbiota signature was evaluated by 16S rRNA analysis. Blood samples were collected at baseline and at days 14 and 90 after starting abemaciclib to evaluate the correlation between the gut microbiota signatures and the systemic immune profile in peripheral blood mononuclear cells (PBMCs). The immune profile was evaluated by mass cytometry, multi-plex cytokines assay, and RNA-sequencing of bulk PBMCs. We characterized the gut microbiota signatures, immune cell composition, immune cell signature, comprehensive cytokines, and severity of diarrhea in all patients. Results: We analyzed 39 patients, 77 stool samples, and 117 blood samples. In the preplanned interim analysis, among the 39 patients, 90% experienced diarrhea. Depleted gut microbiome α-diversity was positively associated with abemaciclib treatment and the severity of diarrhea. The relative abundances of 10 intestinal bacteria species increased and those of 18 intestinal bacteria decreased significantly after abemaciclib treatment, including bacteria known to be involved in diarrhea severity and anti-tumor immunity, such as Faecalibacterium (Table). The immune cell and cytokine profiles in PBMCs were also associated with the gut microbiota signatures. Conclusions: Gut microbiota signatures are associated with abemaciclib-induced diarrhea and the immune profile in metastatic breast cancer patients. These findings can help to elucidate the mechanism of diarrhea caused by abemaciclib and offer strategies for its management and prevention. Intestinal Microbiota Altered by Abemaciclib Citation Format: Kosuke Kawaguchi, Yurina Maeshima, Hiroshi Ishiguro, Kazuhiko Yamagami, Sachiko Takahara, Hirofumi Suwa, Masae Torii, Shigenori Nagai, Yasuaki Sagara, Wakako Tsuji, Hiroyasu Yamashiro, Takeshi Kotake, Shinji Fukuda, Kuniaki Saito, Yasuko Yamamoto, Masako Kataoka, Yuki Himoto, Atsushi Yonezawa, Yukiko Fukui, Yuki Nakamura, Wei Li, Sunao Tanaka, Satoshi Morita, Masakazu Toi. Alteration of gut microbiota signatures and its association with diarrhea during abemaciclib treatment: A multicenter prospective cohort study (KBCRN-A002 study) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-07.

Activated autophagy of innate immune cells during the early stages of major trauma.

Trauma-induced immune dysfunction has been a major barrier to achieving reduced mortality, which is poorly understood. Autophagy is a crucial catabolic mechanism of immune cells during times of stress. Few studies have investigated the immune regulatory effects induced by autophagy after trauma. Here, we use single-cell transcriptomics analysis in a major trauma cohort to demonstrate the dominant role of autophagy in innate immune cells during the early stages of major trauma. Single-cell transcriptional profiling of peripheral blood mononuclear cells (PBMCs) was performed, which were sampled from three control participants and five major trauma patients within 6 hours of injury. In detail, after single-cell RNA-sequence data processing, cell type annotation and cluster marker identification were performed. A genetic toolbox with 604 autophagy-related genes was used to monitor the autophagy levels in immune cells. In addition, all transcriptome RNA sequencing data obtained from PBMCs in a cohort of 167 major trauma patients were downloaded from gene expression omnibus (GEO) datasets (GSE36809). Key deregulated biological processes and important autophagic hub genes involved in immune cells were identified by weighted gene co-expression network analysis and gene ontology enrichment analysis. A total of 20,445 differentially expressed genes were identified and five co-expression modules were constructed. Enrichment analysis indicated that activated autophagy is the most important biological process during the early stages of major trauma, and JMY (autophagy-related genes) were identified as hub genes. The single-cell transcriptional profiling of PBMCs demonstrated that all components of adaptive immune cells were significantly decreased, whereas components of innate immune cells (monocytes and neutrophils) were significantly increased in major trauma patients compared with control participants. Activated autophagy was detected in monocytes and neutrophils by monitoring the dynamic transcriptional signature of the autophagy-related genetic toolbox. Biological process analysis shows that antigen uptake, processing presentation, and major histocompatibility complex (MHC) class II protein complex assembly pathways were up-regulated in autophagy-positive monocytes, whereas antigen processing and presentation of endogenous antigen and type I interferon signaling pathways were up-regulated in autophagy-positive neutrophils during the early stages of major trauma. Our study demonstrated that autophagy is a biological process crucial to the development of immune disorders in the early stages of major trauma. Furthermore, the results of our study generated a comprehensive single-cell immune landscape for major trauma patients, in which we determined that autophagy profoundly affects the main functions of innate immune cells and provides insight into the cellular basis of immune dysregulation after major trauma.

Standard immunosuppressive treatment reduces regulatory B cells in children with autoimmune liver disease.

Autoimmune hepatitis (AIH) is a chronic liver disease caused by a perturbed immune system. The scarcity of short- and long-term immune monitoring of AIH hampered us to comprehend the interaction between immunosuppressive medication and immune homeostasis. We recruited children with AIH at the time of diagnosis and at the 1st, 3rd, 6th, 12th, 18th, and 24th months of immunosuppression (IS). We also enrolled children with AIH being on IS for >2 years. Children with drug-induced liver injury (DILI), and those receiving tacrolimus after liver transplantation (LT), were enrolled as disease/IS control subjects. Healthy children (HC) were also recruited. Peripheral blood mononuclear cells (PBMCs) were isolated from all participants. Healthy liver tissue from adult donors and from livers without inflammation were obtained from children with hepatoblastoma. By using flow cytometry, we performed multi-parametric immune profiling of PBMCs and intrahepatic lymphocytes. Additionally, after IS with prednisolone, tacrolimus, rapamycin, or 6-mercaptopurine, we carried out an in vitro cytokine stimulation assay. Finally, a Lifecodes SSO typing kit was used to type HLA-DRB1 and Luminex was used to analyze the results. Untreated AIH patients had lower total CD8 T-cell frequencies than HC, but these cells were more naïve. While the percentage of naïve regulatory T cells (Tregs) (CD4+FOXP3lowCD45RA+) and regulatory B cells (Bregs, CD20+CD24+CD38+) was similar, AIH patients had fewer activated Tregs (CD4+FOXP3highCD45RA - ) compared to HC. Mucosal-associated-invariant-T-cells (MAIT) were also lower in these patients. Following the initiation of IS, the immune profiles demonstrated fluctuations. Bregs frequency decreased substantially at 1 month and did not recover anymore. Additionally, the frequency of intrahepatic Bregs in treated AIH patients was lower, compared to control livers, DILI, and LT patients. Following in vitro IS drugs incubation, only the frequency of IL-10-producing total B-cells increased with tacrolimus and 6MP. Lastly, 70% of AIH patients possessed HLA-DR11, whereas HLA-DR03/DR07/DR13 was present in only some patients. HLA-DR11 was prominent in our AIH cohort. Activated Tregs and MAIT cell frequencies were lower before IS. Importantly, we discovered a previously unrecognized and long-lasting Bregs scarcity in AIH patients after IS. Tacrolimus and 6MP increased IL-10+ B-cells in vitro.

The circular RNA landscape in multiple sclerosis: Disease-specific associated variants and exon methylation shape circular RNA expression profile.

Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background. We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out. Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool. We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as "circ-QTLs", and proposing a role for exon-based DNA methylation in regulating circRNA expression.

Pharmacokinetics of oral and subcutaneous methotrexate in red and white blood cells in patients with early rheumatoid arthritis: the methotrexate monitoring trial

ObjectiveTo investigate the pharmacokinetics of methotrexate polyglutamate (MTX-PG) accumulation in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) in patients with early rheumatoid arthritis (RA) after oral and...

31P The IOpener study: Tyrosine kinase activity profiling to predict response to immune checkpoint inhibitors in patients with advanced stage non-small cell lung cancer

Treatment with immune checkpoint inhibitors (ICIs) is successful, but only in a subset of patients with advanced stage non-small cell lung cancer (NSCLC). Blood-based kinase profiling of peripheral blood mononuclear cells (PBMCs) has previously shown its predictive value for response to ICI treatment in a discovery cohort (Hurkmans et al., JITC 2020). The aim of the current prospective study was to perform a blind validation of the predictive value of kinase activity profiles in PBMCs for ICI response in patients with NSCLC in a standardized setting.

Influence of SARS-COV-2 Infection on Cytokine Production by Mitogen-Stimulated Peripheral Blood Mononuclear Cells and Neutrophils in COVID-19 Intensive Care Unit Patients.

We sought to investigate the influence of SARS-CoV-2 infection on the cytokine profiles of peripheral blood mononuclear cells (PBMCs) and neutrophils from coronavirus disease 2019 (COVID-19) intensive care unit (ICU) patients. Neutrophils and PBMCs were separated and stimulated with the mitogen phytohemagglutinin. Culture supernatants of mitogen-stimulated PBMCs and neutrophils from 88 COVID-19 ICU patients and 88 healthy controls were evaluated for levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-2, -4, -5, -6, -9, -10, -12, -17A, and tumor necrosis factor (TNF)-α using anti-cytokine antibody MACSPlex capture beads. Cytokine profiles of PBMCs showed significantly lower levels of GM-CSF, IFN-γ, IL-6, IL-9, IL-10, IL-17A, and TNF-α (p < 0.0001) in COVID-19 ICU patients. In contrast, COVID-19 ICU patients showed higher median levels of IL-2 (p < 0.001) and IL-5 (p < 0.01) by PBMCs. As for neutrophils, COVID-19 ICU patients showed significantly lower levels of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-17A, IL-12, TNF-α (p < 0.0001), and IFN-α (p < 0.01). T-helper (Th)1:Th2 cytokine ratios revealed lower inflammatory cytokine for PBMCs and neutrophils in COVID-19 ICU patients. Cytokine production profiles and Th1:Th2 cytokine ratios suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has an immunomodulatory effect on PBMCs and neutrophils. This study also suggests that the increased levels of several cytokines in the serum are not sourced from PBMCs and neutrophils.

The Specific microRNA Profile and Functional Networks for Children with Allergic Asthma.

BackgroundAllergic asthma is the most common type of asthma and often occurs in early life with increasing comorbidities, including atopic dermatitis and allergic rhinitis. MicroRNAs (miRNAs) are involved in the pathogenesis of numerous immune and inflammatory disorders, particularly allergic inflammation. The specific miRNA profiles of children with allergic asthma have not been fully delineated and still require in-depth study.ObjectiveThis study aimed to identify the expression profile of miRNAs and constructed a network of the interactions between differentially expressed miRNAs and target mRNAs to provide novel insights into understanding the pathogenesis of allergic asthma.Materials and MethodsIn this study, we performed high-throughput sequencing of peripheral blood mononuclear cells (PBMCs) from children in the acute phase of asthma. Bioinformatics approaches, including miRanda, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, were employed to predict novel therapeutic and diagnostic targets for allergic asthma. Real-time quantitative PCR was conducted to detect the expression of aberrantly expressed miRNAs.ResultsOne hundred and sixty-one differentially expressed miRNAs were identified in children with allergic asthma, including 140 conserved miRNAs and 21 novel miRNAs. A total of 8929 targeted mRNAs (44,186 transcripts) associated with differentially expressed miRNAs were predicted and significantly enriched in the cGMP-PKG signalling pathway, cholinergic synapse, and salivary secretion. We also found that miRNA-370-3p targeted PKG and MLCP molecules in the cGMP-PKG signalling pathway and was involved in the pathogenesis of allergic asthma.ConclusionWe identified the miRNA profile of PBMCs in children with allergic asthma and also found that miRNA-370-3p targeted PKG and MLCP molecules in the cGMP-PKG signalling pathway, which provides a novel insight into understanding the pathogenesis of allergic asthma and investigating new targets for the treatment of allergic asthma in children.

Abstract 250: Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy

Abstract Canonical p53-activated pathways can influence a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages (TAMs). We examined whether p53 activity in the tumor microenvironment (TME) influences antitumor immunity and show that p53 signaling induced pharmacologically with APR-246 (eprenetapopt) can augment the efficacy of immune checkpoint blockade (ICB) in preclinical models, a strategy that is also being tested in patients (NCT04383938). We first investigated the effects of combining APR-246 with ICB in wildtype C57BL6 (B6) mice bearing syngeneic p53 wildtype MC38 colon cancer and B16 melanoma tumors. The combination of an anti-PD-1 antibody (RMP1-14) with APR-246 in mice significantly delayed tumor growth (p < 0.001) and improved survival of tumor-bearing mice, compared to monotherapies (p < 0.01). To further dissect the effects of APR-246 on myeloid and T cells in the TME, we used a conditional knockout of p53 in CSF1R+myeloid cells (CSF1Rcre/p53fl mice), or T cells (CD8cre/p53fl mice). CSF1Rcre/p53fl had loss of tumor control and worse survival with APR-246+anti-PD-1. CD8cre/p53fl had intact tumor control. To study enhanced p53 activity in the TME, we performed flow cytometry, cytokine multiplex and global transcriptional profiling by RNA seq. We found enhanced p53-activity led to increased infiltration of T cells, increased MHC-II expression in TAMs and downregulation of M2-associated cytokines. This was associated with cellular senescence in TAMs and induction of canonical p53-induced senescence-associated secretory phenotype (SASP). Our preclinical findings informed the development of a phase I/II clinical trial using APR-246 with pembrolizumab for patients with advanced solid tumors (NCT04383938). We studied peripheral blood samples from two of the patients with tumor regression and two patients in whom tumors progressed on therapy. We analyzed peripheral blood mononuclear cells (PBMCs) and serum prior to therapy, and at the beginning of cycle 2 and 5 for the patients with tumor control, and at the end of therapy for patients who had progression. Single cell RNA-seq of PBMCs demonstrated a signature consistent with T cell activation and proliferation, and SASP-associated changes in the myeloid compartment as seen in mice. T cell profiling of PBMCs by flow cytometry demonstrated strong proliferation of T cells in patients with tumor control. Serum cytokine analysis demonstrated robust in IL-12, IFN-gamma and Eotaxin-1 in the two responders, which was not seen in the patients whose tumors progressed. Our study illustrates p53-induced SASP in TAMs as a mechanism to reprogram the TME and augment responses to ICB. Ongoing studies will help determine biomarkers that are predictive of response to APR-246+ICB therapy. Citation Format: Arnab Ghosh, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, Nathan Suek, Aliya Holland, Yu-Jui Ho, Mohsen Abu-Akeel, Luis Felipe Campesato, Levi Mark Bala Mangarin, Cailian Liu, Hong Zhong, Sadna Budhu, Andrew Chow, Roberta Zappasodi, Marcus Ruscetti, Scott W. Lowe, Taha Merghoub, Jedd D. Wolchok. Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 250.

Strategy for Microscale Extraction and Proteome Profiling of Peripheral Blood Mononuclear Cells.

Peripheral blood mononuclear cells (PBMCs) play vital roles in physiological and pathological processes and represent a rich source for disease monitoring. Typical molecular profiling on PBMCs involves the sorting of cell subsets and thus requires a large volume of peripheral blood (PB), which impedes the clinical practicability of omics tools in PBMC measurements. It would be clinically invaluable to develop a convenient approach for preparing PBMCs from small volumes of PB and for deep proteome profiling of PBMCs. To this end, here, we designed an apparatus (PBMC-mCap) for microscale enrichment and proteome analysis of PBMCs, which pushed the needed PB volume from the normal 2 mL or higher to 100 μL or lower, comparable to the volume of a drop of finger blood. A PBMC-specific mass spectra library containing 8869 proteins and 121,956 peptides was further built, which, in combination with the optimized data-independent acquisition strategy, helped to identify 6000 and 6500 proteins from PBMCs with 100 μL and 1 mL of PB as initial materials, respectively. Further application of the strategy for PBMC proteomes revealed a steady difference between gender (male vs female) and upon stimulus (COVID-19 vaccination). For the latter, we observed differentially expressed genes and pathways involving the activation of immune cells, including the NF-κB pathway, inflammation response, and antiviral response. Our strategy for the proteome analysis of microscale PBMCs may provide a convenient clinical toolkit for disease diagnosis and healthy state monitoring.

Altered non-coding RNA profiles and potential disease marker identification in peripheral blood mononuclear cells of patients with NMOSD

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelination disorder, and dysregulation of RNAs contributes to its pathogenesis. We aimed to reveal the expression profiles of RNAs, including messenger RNA (mRNA), circular RNA (circRNA) and long non-coding RNA (lncRNA), in the peripheral blood mononuclear cells (PBMCs) of patients with NMOSD. Seven NMOSD patients and seven healthy controls (HCs) were enrolled in the competitive endogenous RNA (ceRNA) microarray analysis. Bioinformatics analysis was then performed on the microarray data. Selected RNAs were validated by RT-qPCR. Differentially expressed (DE) RNA profiles of patients and HCs were related to NK cell mediated cytotoxicity, the IL-17 signaling pathway, and the B cell receptor signaling pathway. Moreover, DE non-coding RNAs (DE ncRNAs) including DE circRNAs and DE lncRNAs, may participate in the transforming growth factor beta (TGF-β) signaling pathway, leukocyte migration and neutrophil chemotaxis. Immune cell infiltration analysis showed that the abundance of M1 macrophages and plasma cells significantly increased, while that of M2 macrophages significantly decreased in the NMOSD group. Finally, through RT-qPCR validation, lnc-HELZ-7:1 (95% confidential interval of area under curve [95%CI of AUC] = 0.6633–1.0000), ring finger protein-LIM domain interacting (RLIM; 95%CI of AUC = 0.6980–1.0000), and hsa_circ_0026993 (95%CI of AUC = 0.7550–1.0000) could discriminate NMOSD from HCs by receiver operating characteristic curve analysis. To our knowledge, this is the first study to preliminarily investigate the RNA profiles, especially circRNA profiles in PBMCs of NMOSD patients from North China. We identified lnc-HELZ-7:1, RLIM, and hsa_circ_0026993 as the potential disease markers for NMOSD.

Impact of Diminished Expression of circRNA on Multiple Sclerosis Pathomechanisms.

Circular RNA (circRNA) molecules represent a novel and unique class of endogenous non-coding RNAs controlling the expression and function of microRNA (miRNA) and post-transcriptional regulation. Recent studies implicated circRNA in the pathomechanism of multiple sclerosis (MS). Hybridization microarray was used to define the circRNA profile in the peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with relapsing–remitting MS (RRMS: 10 in relapse, 10 in remission) and 10 healthy controls (HCs). We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with an independent cohort of 45 RRMS patients (18 in relapse, 27 in remission) and 27 HCs. Microarray analysis revealed 246 circRNAs differentially downregulated (P < 0.05) in RRMS patients versus HCs. We validated two circRNAs of the three showing the lowest levels of differential expression in the RRMS remission group versus the HC group: hsa_circRNA_101145 and hsa_circRNA_001896. Their expression was significantly decreased during remission in RRMS (P = 0.0000332, FC = 0.385 and P = 0.0455, FC = 0.591, respectively) and in patients showing a lower level of disability (hsa_circRNA_101145, P = 0.0695; hsa_circRNA_001896, P = 0.0008). Bioinformatic analysis revealed 10 miRNAs interacting with these circRNAs in a complementary manner and led to the discovery of three protein-coding mRNAs downregulated in patients with RRMS during remission. These transcripts have been previously implicated in oxidative stress, blood–brain barrier permeability, microglia function, and extracellular matrix molecules altering the microenvironment and inhibiting oligodendrocyte progenitor cells. circRNAs displayed a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNAs with low expression in important mechanistic pathways of RRMS.