Varodom Charoensawan

Blood provides an accessible window into human health, yet the absence of a peripheral blood mononuclear cells (PBMCs) reference with standardized immune cell annotations has constrained comparisons between single-cell RNA sequencing (scRNAseq) studies. Here, we present HARP ( H uman Cell A tlas R eference for P BMCs), an integrated atlas of ∼9 million PBMCs from >2,600 donors across 15 studies spanning four continents, encompassing diverse populations, neonates to 97 years, across health and diverse immune-related diseases. We developed optimized integration workflows, including novel label-free metrics to assess integration quality, and generated community-driven consensus annotations for 192 immune cell subsets, identifying rare populations representing as few as 0.004% of PBMCs. HARP reveals coordinated cellular modules associated with age, sex, and disease, identifies female-biased interferon and inflammatory gene programs and their perturbation by COVID-19, and uncovers a novel sexual dimorphism in prostaglandin signaling. Finally, we introduce scTiger, a hierarchical label-transfer framework that accurately projects these 192 cell annotations onto ∼18 million additional PBMCs, providing a robust and transferable reference for harmonizing future PBMC studies. HARP provides a high-resolution community-driven reference atlas for PBMC annotation and analysis, as a basis for emerging clinical applications of single-cell genomics.

Mainland Southeast Asia (MSEA) remains under-represented in global immunogenomic references despite its extensive genetic heterogeneity. We present the first single-cell immune atlas of an MSEA population, utilizing Thai individuals from the Asian Immune Diversity Atlas (AIDA) as a representative cohort. We demonstrate that the Thai population is highly genetically diverse, reflecting its history as a geographic nexus for Asian admixture. By integrating single-cell transcriptomics with high-resolution genotyping, we show that genetic ancestry significantly shapes innate immune profiles, specifically CD14⁺ monocytes, highlighting potential evolutionary adaptations to regional pathogens. We identify specific sex-by-ancestry interactions that may drive the baseline activation of pro-inflammatory pathways in females, providing a long-sought cellular rationale for the high prevalence of autoimmune disorders observed in Southeast Asian populations. Ultimately, our study reveals that population-specific genetic architecture dictates immune heterogeneity often missed by self-reported ethnicity or country of origin, providing a critical immunogenomic reference for precision medicine in MSEA regions.

Dengue virus (DENV) is a major global health threat, with secondary heterotypic infections potentially inducing detrimental memory immune responses. Antigen-specific CD8 + T cells contribute to both protection and pathogenicity, yet how their phenotypic heterogeneity relates to disease severity remains unclear. Here, we performed plate-based single-cell RNA sequencing of circulating DENV-specific CD8 + T cells identified by HLA tetramers loaded with DENV NS3-derived epitopes. Using tetramer binding to peptides corresponding to the currently and serologically inferred dominant previously infecting serotypes, we identify distinct CD8 + T cell subsets associated with disease severity. Asymptomatic dengue is enriched for lower tetramer binding cells with moderate cytotoxic programs, whereas dengue hemorrhagic fever is associated with high tetramer binding CX3CR1 + CD8 + T cells exhibiting enhanced expression of genes related to T cell receptor signaling and cytotoxicity. T cell receptor repertoires are similar among symptomatic cases but displayed temporal dynamics. Overall, DENV NS3-specific CD8 + T cells across disease severity and time are associated with distinct transcriptomic states and T cell receptor features.

Genome-scale gene expression analysis has become a standard approach for discovering biomarkers and understanding molecular mechanisms. Recent advances in omics technologies now enable investigations beyond conventional case–control comparison and standard gene-centric differential expression (DE) analyses. In this review, we highlight conceptual and methodological advances in using transcriptomic and multimodal omic data to elucidate diverse mechanisms of gene expression. We first provide a comprehensive overview of different types of gene expression study designs, along with suitable statistical testing, as well as key considerations. We then describe strategies for inferring gene co-expression and regulatory networks, with particular emphasis on context-specific network models and machine learning methods that capture the multifactorial nature of gene expression regulation. Finally, we present perspectives on emerging modalities such as single-cell and spatial transcriptomics, which enable unprecedented resolution in mapping regulatory complexity. We envisage that the concepts and examples described here will raise awareness and encourage the application of advanced network-based analyses.

Intravitreal injection anti-vascular endothelial growth factor (VEGF) drugs offer significant benefits for individuals with retinal diseases, yet high cost and limited availability impede widespread use. Compounding and repackaging present a potential strategy for better drug utilization; however, earlier studies addressing the efficacy and sterility of repackaged anti-VEGF drugs focused on individual or few drugs, limiting their ability to provide comparative insights across different drugs and experimental settings. In this study, we survey the stability, efficacy, and sterility of five intravitreal anti-VEGF drugs, namely aflibercept, bevacizumab, brolucizumab, faricimab, and ranibizumab, after repackaging and storage for 14, 30, and 60 days. The drugs were aseptically repackaged into a minimum of three syringes per time points, which were all examined using one consistent protocol comprising six different experimental techniques: spectrophotometry, sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), enzyme-linked immunosorbent assay (ELISA), and microbial plating. All five intravitreal anti-VEGF drugs can be repackaged and stored in Tuberculin-type syringes up to 60 days, while maintaining sterility up to 60 days with minimal changes in protein concentrations, degradation products, aggregation, and functional binding to VEGF-A, if appropriate aseptic protocols are followed. Specific differences observed in biophysical properties between drugs were noted and discussed.

A comprehensive understanding of human systemic immune responses to mosquito-borne dengue virus (DENV) infection is vital for addressing challenges posed by viral heterologous serotypes and potential adverse memory immune responses. Asymptomatic DENV infection offers an opportunity to explore protective immunity because infected individuals effectively clear the virus without symptomatic manifestations. However, data on asymptomatic dengue are scarce because of limited sample availability during silent viremia. Here, we conducted single-cell RNA and immune receptor sequencing of peripheral blood mononuclear cells (PBMCs) from donors with varying disease severities including asymptomatic dengue and performed longitudinal analysis in a symptomatic dengue cohort, enabling identification of distinct immune responses. In asymptomatic dengue, we observed potential indications of enhanced viral antigen processing via MHC-I, correlating with increased CD8 effector T cell activities, distinct NK cell profiles, and enriched IGHA1 ⁺ plasmablasts. In contrast, symptomatic dengue cases exhibited indications toward antibody-mediated viral entry, elevated type I interferon responses, and IL-10–associated expansion of IGHG1 ⁺ plasmablasts with biased V(D)J gene usage and a shared B cell receptor clonotype network. Our study reports a gene expression and immune receptor repertoire resource for systemic immune responses to DENV infection and suggests distinct mechanisms for potential protection and pathogenicity in individuals with asymptomatic compared with symptomatic dengue.

Vibrio parahaemolyticus (VP) is a major bacterial species that causes early mortality syndrome (EMS) in shrimps. EMS can be classified into two groups based on histological signs of hepatopancreatic tissues, i.e., acute hepatopancreatic necrosis disease (AHPND) and non‐AHPND. To investigate how toxin‐producing AHPND and toxin‐lacking non‐AHPND VP could lead to EMS, growth characteristics and transcriptomic analyses of the representative strains, 5HP and 2HP, were compared. Non‐pathogenic VP represented by strain S02 was also used. Two types of growth media included rich medium represented by tryptic soy broth plus 1.5% NaCl (TSB) and depleted media represented by artificial seawater (SW) and spent seawater (SSW). SSW refers to a sterile used‐SW medium from healthy shrimp rearing or shrimp‐conditioned SW. Growth characteristics under these media indicated that TSB and SSW supported better growth than SW, suggesting shrimp‐conditioned SW is sufficient to support normal VP growth. Transcriptomic analyses revealed that both EMS isolates shared overall expression patterns. Metabolic stress adaptation systems of non‐AHPND strain 2HP under SW and SSW were more upregulated than in AHPND strain 5HP. Specific virulence genes (i.e., zot1 [zonula occludens toxin] and vopS [type III secretion effector]) and a general stress response gene (i.e., rpoS [stress response sigma factor]) were upregulated in strain 2HP under both SW and SSW. These expression profiles of strain 2HP suggest higher persistence, which might be useful for cell survival and non‐AHPND pathogenesis even without toxin production. We proposed that these genes encode virulence factor ‘candidates’ in non‐AHPND VP.

Droplet-based single-cell RNA sequencing (scRNA-seq) frequently encounters significant challenges from contamination of cell-free mRNAs, known as “ambient mRNAs”, which can substantially distort single-cell transcriptome data interpretation to a large extent. In this study, we investigate the impact of ambient mRNA contamination on differential gene expression and biological pathway enrichment analyses, using two independent scRNA-seq datasets: ten peripheral blood mononuclear cells (PBMCs) samples from dengue-infected patients and forty-two scRNA-seq samples of human fetal liver tissues. We apply two independent ambient mRNA correction approaches – CellBender (automate correction) and SoupX (using a predefined set of potential ambient mRNA genes). We demonstrate that ambient mRNA transcripts appear among differentially expressed genes (DEGs), subsequently leading to the identification of significant ambient-related biological pathways in unexpected cell subpopulations before ambient mRNA contamination correction. In contrast, after suitable correction, we observe a reduction in ambient mRNA expression levels, resulting in improved DEG identification and leading to the highlight of biologically relevant pathways specific to cell subpopulations. Our study underscores the importance of understanding and applying appropriate corrections for ambient mRNA contamination to enhance the reliability and accuracy of scRNA-seq data analyses, thereby improving the robustness of data interpretation in droplet-based scRNA-seq datasets.

While prostaglandin E₂ (PGE₂) is produced in human tumor microenvironment (TME), its role therein remains poorly understood. Here, we examine this issue by comparative single-cell RNA sequencing of immune cells infiltrating human cancers and syngeneic tumors in female mice. PGE receptors EP4 and EP2 are expressed in lymphocytes and myeloid cells, and their expression is associated with the downregulation of oxidative phosphorylation (OXPHOS) and MYC targets, glycolysis and ribosomal proteins (RPs). Mechanistically, CD8⁺ T cells express EP4 and EP2 upon TCR activation, and PGE₂ blocks IL-2-STAT5 signaling by downregulating Il2ra, which downregulates c-Myc and PGC-1 to decrease OXPHOS, glycolysis, and RPs, impairing migration, expansion, survival, and antitumor activity. Similarly, EP4 and EP2 are induced upon macrophage activation, and PGE₂ downregulates c-Myc and OXPHOS in M1-like macrophages. These results suggest that PGE₂-EP4/EP2 signaling impairs both adaptive and innate immunity in TME by hampering bioenergetics and ribosome biogenesis of tumor-infiltrating immune cells.