Genetic variations in the low-affinity Fc Gamma Receptor (FcγR) region have been associated with various diseases and treatment efficacies. Using a high-throughput in vitro screen called Regulatory-Element Sequencing (Reel-Seq) we have identified over 400 candidate functional Single Nucleotide Polymorphisms (fSNPs) from the genomic region. In this study, we aim to identify regulatory function of these candidate non-coding fSNPs in THP-1 monocytes.
At each candidate FcγR fSNP position, we introduced random mutation in the THP-1 monocyte line using CRISPR-Cas9 based genome editing and generated over 30 knockout clones per fSNP site. We stimulated these THP-1 knockout clones with various treatments including Interferon and Lipopolysaccharide (LPS). Flow cytometry and qPCR were performed to measure the changes in cell-surface FcγR protein abundances and FcγR transcript abundances. Antibody-Dependent Cellular Phagocytosis (ADCP) assays are being conducted on selected clones to evaluate whether normal phagocytosis function has changed through the alteration of FcγR abundances.
We have observed significant changes in cell-surface FcγR abundance in these THP-1 knockout cells based on flow cytometry analysis. Detailed analysis of stimulated cells is ongoing, and we will share the full results at the meeting.
Noncoding genetic variants identified by our in vitro screen may play important roles in monocyte function, through the regulation of cell-surface FcγR abundance.
NIH NIAID #1R01AI187318-01, NCSU CVM Internal Grant 2022, CMI-EID Associate member Professional Development Scholarship 2023
Immune Response Regulation: Molecular Mechanisms (IRM)
Kaleigh Diveley, Nasif Mahmood, Xinxia Peng et al.· Journal of Immunology· 0 citations
Single-cell RNA-seq (scRNA-seq) analyses rely on accurate gene annotations, a challenge for many species with less completely curated genomes. Rhesus macaque (RM), a widely used model for human biomedical research, is one such case where missing gene annotations have hindered the study of immune responses. We aim to develop a computational framework to identify and reintegrate missing gene features, improving immune response characterization in RM.
In a preliminary analysis, we computationally searched in a RM peripheral blood mononuclear cell (PBMC) scRNA-seq dataset from a kidney allograft study for unannotated but transcriptionally active regions (uTARs). We then performed cell clustering twice, once on annotated-gene expression and again on uTAR expression, and assessed uTAR expression for cell-type specificity and association with immune-related pathways.
We identified >5,500 uTARs, indicating that numerous features–e.g., long non-coding RNAs or alternative transcripts of existing genes–are missing from current RM annotations. uTARs exhibit cell-type-specific expression and, when used to group cells, separate major cell types, paralleling cell clustering using annotated rhesus genes. These findings illustrate substantial gaps in the RM genome annotation and highlight the biological relevance of these missing genes or transcripts.
uTARs likely harbor many previously unannotated genes or transcripts that are involved in immune regulation in RM. Ongoing work will denoise the signals in scRNA-seq data and prioritize a subset of uTARs as candidate transcriptional regulators. We will also infer regulatory relationships between candidate regulators and downstream targets. Our immediate goal is to identify drivers of transplant rejection. However, this framework is broadly applicable to scRNA-seq datasets across species and experimental contexts.
NIAID U19 AI131471
Technological Innovations in Immunology (TECH)
Ethan Smith, Matthew Tunbridge, T. Tollison et al.· Journal of Immunology· 0 citations
Long non-coding RNAs (lncRNAs) can play a major role in modulating innate immune responses by acting as positive or negative regulators. LncRNAs can mediate protein interactions with chromatin and other proteins by serving as scaffolds, decoys, and guides. The expression pattern of most lncRNAs is cell and context specific, making them attractive targets for precise therapeutic intervention. However, only a small number of lncRNAs have been well characterized due to their low abundance, context dependent expression, and complex modes of action. We have previously identified lncRNA VILMIR as an interferon-stimulated gene that regulates host interferon response to viral infection and interferon treatment. In this study, we investigated molecular mechanisms through which VILMIR modulates host immune response.
Using in vitro RNA pulldown assays followed by in vivo RNA immunoprecipitation validation, we identified several VILMIR-interacting proteins, including FUBP1, an RNA-binding protein implicated in transcriptional regulation. Mapping experiments with truncated VILMIR fragments revealed specific regions involved in protein binding.
Our findings suggest that VILMIR modulates transcription of interferon-stimulated genes by forming an RNA-protein complex with FUBP1.
Future studies will focus on elucidating how the VILMIR-FUBP1 complex influences the transcriptional landscape during viral infection and exploring its potential as a therapeutic target to fine-tune antiviral immune responses.
National Institutes of Health Grant R21AI147187
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Nasif Mahmood, Kristen John, Alexandra Istishin et al.· Journal of Immunology· 0 citations
Two novel interferon-responsive human lncRNAs that may play important roles in the regulation of the host interferon response across different cell types are identified.
Alexandra Istishin, Kristen John, Ethan Smith et al.· Journal of Immunology· 0 citations
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