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Monocytic-Myeloid Derived Suppressor Cells Suppress T-Cell Responses in Recovered SARS CoV2-Infected Individuals

ABSTRACT: The goal of this study was to determine changes in the expression of genes in monoctic myleoid derived suppressor cells (M-MDSC) as a result of SARS CoV2 infection. The study aimed to investigate if M-MDSC are functionally active and inhibit T cell function in response to SARS CoV2 antigens 5 months after first detection of the virus. Methods: Peripheral blood mononuclear cells (PBMC) were collected from CoV2 (-) and CoV2 (+) donors (N=5 each group). M-MDSC were isolated by flow cytometry, and RNA extracted for RNA-seq studies. Filtering low quality reads and removal of the 3’ adapter sequences were performed using the Trim Galore tool. Reads were mapped to the latest version of the human genome (build hg38) using HISAT2. Mapped reads were counted against the human GENCODE annotation (v37) using HT-Seq. The EdgeR library in the R computing environment was used for quality control of the RNA-Seq data, and ComBat-seq method for correction of batch effects. Differential gene expression analysis was conducted using EdgeR. Pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Results: An average of 34 million reads per sample were acquired and mapped to the human genome (build hg38). After applying filtering criteria, 9,217 human genes were identified with the HISAT2 and HTSeq workflow. Differential expression analysis was performed between CoV2 (+) and CoV2 (-) samples using EdgeR. A total of 188 differentially expressed genes (DEGs) were identified with nominal p-value <0.05; of which 63 were up- and 125 downregulated in CoV2 (+) samples. A total of 12 DEGs were identified with false discovery rate corrected p-value <0.05, of which 2 were up- and 10 downregulated. Pathway enrichment analysis identified pathways involved in immune response and innate immune signaling. Conclusion: The study demonstrated that CoV2 infection modulated the expression of genes involved in immune response and innate immune signaling. Most of the genes remained downregulated even after 5 months of first detection of SARS CoV2.

ORGANISM(S): Homo sapiens

PROVIDER: GSE199286 | GEO | 2022/06/03

REPOSITORIES: GEO

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Project description:The goal of this study was to investigate transcriptome remodeling induced by treatment with a camptothetin analog, Topotecan (TPT), of a primary T cell model of HIV latency. The study aimed to determine whether TPT has a global inhibitory effect on gene expression in primary CD4+ T cells, and identify mechanisms of action of this drug as an HIV “block and lock” agent. Methods: CD4+ T cells were isolated from blood of HIV seronegative study participants (N=3) and utilized to generate an in vitro model of latent HIV infection (model developed in the Spina laboratory and previously described by Spina et al., 2013 and Soto et al., 2022). Following generation of the model, cells were treated with 10 uM Topotecan or its solvent dimethyl sulfoxide (DMSO) for 24 hours. Cells were lyzed with RLT buffer containing beta-mercaptoethanol from a RNeasy micro kit (Qiagen, Inc. cat # 74004). ERCC spikes (Thermo Fisher Scientific, Inc.) were added to cell lysates based on cell number in each sample (10 ul of 1:800 dilution per million cells). RNA was extracted and subjected to deep sequencing at the Institute for Genomics Medicine (IGM) Genomics Center at the University of California San Diego. Filtering low quality reads and removal of the 3’ adapter sequences were performed using the Trim Galore tool. Reads were mapped to the human genome (build hg38) using HISAT2, and to HIV and ERCC references using bowtie. Mapped human reads were counted against the human GENCODE annotation (v37) using HT-Seq. ERCC counts were used to determine whether TPT had global inhibitory effect on transcription in primary CD4+ T cells. Because no such effect was observed, differential gene expression analysis was performed using library EdgeR in Bioconductor R without correction of expression based on ERCC spikes. Genes with false discovery rate (FDR)-corrected p-value less than 0.05 and an absolute fold change in TPT-treated samples compared to DMSO controls greater than 2, were considered significantly modulated by TPT. Pathway and gene ontology (GO) term enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v2022q3. Pathways and GO terms with FDR-corrected p-value less than 0.05 were considered significantly enriched for differentially expressed genes. Results: An average of 19.7 million reads per sample were acquired and mapped to the human genome (build hg38). After applying filtering criteria, 9,678 human genes were identified with the HISAT2 and HTSeq workflow. Differential expression analysis was performed between TPT- and DMSO-treated samples using EdgeR. A total of 1,749 differentially expressed genes (DEGs) were identified with FDR p-value <0.05 and an absolute fold change greater than 2; of which 522 were up- and 1227 downregulated by TPT. Pathway and GO term enrichment analysis revealed that TPT interferes with gene transcription and cell signaling pathways (e.g. T cell receptor signaling, positive regulation of JNK cascade, regulation of actin cytoskeleton and positive regulation of GTPase activity were affected by TPT treatment). Conclusion: The study demonstrated that TPT induces multiple effects on transcriptome in primary CD4+ T cells. Some of these changes may represent direct and indirect mechanisms of action of TPT as an HIV “block and lock” agent.

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Monocytic-Myeloid Derived Suppressor Cells Suppress T-Cell Responses in Recovered SARS CoV2-Infected Individuals

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