(35). characterized by fever, cough, fatigue, loss of taste, and smell and might range from moderate to severe acute respiratory distress syndrome (ARDS) leading to reduction in the number of proliferating lymphocytes (lymphopenia) in severe patients (4). In these patients, studies have also associated the disease with immune hyper-responsiveness called cytokine storm, characterized by increased interleukins (IL-2, IL-7, and IL-10), granulocyte-colony stimulating factor (GCSF), interferon-gamma inducible protein 10 (IP10), monocyte chemoattractant protein 1 (MCP1), and tumor necrosis factor-alpha (TNF-alpha) (4, 5). However, not all individuals exposed to SARS-CoV-2 show COVID-19 disease symptoms; few might be asymptomatic, suggesting that natural immunity can effectively combat this virus. Understanding humoral and adaptive immunity against SARS-CoV-2 is important for vaccine development, interpretation of the disease pathogenesis, and calibration of pandemic control measures (6). Delamanid (OPC-67683) Most of the studies have focused on the adaptive immune responses in COVID-19-positive patients, and as in the case of all viral infections, the Delamanid (OPC-67683) role of B and T cells have been widely explored in patients (7C11). Studies on humoral immune response have shown the presence of elevated levels of IgG and IgM antibody titers in Rabbit Polyclonal to NFE2L3 patients, the former being significantly elevated in severe patients (7). Several reports have appreciated the role of serum IgA for early neutralizing response against SARS-CoV-2 and their longevity for months after onset of symptoms (12C15). The role of T-cell immunity to SARS-CoV-2 has also been explored by researchers, and one such study elaborates on specific CD4+ and CD8+ memory T-cell responses in convalescent patients (16). Phenotyping based on and revealed that SARS-CoV-2-specific CD4+ T cells were biased towards T central memory (Tcm) phenotype, whereas SARS-CoV-2-specific CD8+ T cells were biased toward terminally differentiated effector (Temra) cells (17). CD8+ Temra cells are terminally differentiated effector memory cells with low expression of and high expression of (average expression 1) and T cells that expressed (average expression 1) were removed from the analysis, thus yielding a total of 7,848 cells. The feature barcoded samples were demultiplexed using Seurats multimodal analysis. Individual samples were normalized for cell counts using Sctransform (30) method in Seurat. Sample Integration To remove batch effects, the individual normalized samples were integrated using the integration method implemented in Seurat. Dimension Reduction and Clustering For the integrated object, principal component analysis (PCA) was performed (npcs = 30) to reduce the dimensionality of the dataset. Thirty principal components were then used to compute the k-nearest neighbor graph, which was further used to find clusters. We visualized the clusters by UMAP. We checked for marker genes in each cluster using the function FindAllMArkers (min.pct = 0.25 and logfc. threshold = 0.38). We used the default non-parametric Wilcoxon rank-sum test for the differential expression analysis. Clusters that had enriched ribosomal (RPS and RPL) genes were regarded as low-quality clusters and discarded from the analysis, further performing the above-mentioned dimension reduction and clustering analysis again. Cell-Type Annotation The function FindAllMarkers was used to find out the marker genes in each of the clusters. Average expression and DoHeatmap functions were Delamanid (OPC-67683) used to visualize the expression of the top marker genes in each cell cluster. These top markers and other known canonical markers were used to determine the cell type of each cluster. Differential analysis of selected clusters was performed using Find Markers function (min.pct = 0.25 and logfc. threshold = 0.38). TCR and BCR Data Integration TCR-seq and BCR-seq data were assembled using Cell Ranger pipeline (v5, 10 Genomics) with the cell ranger multi-command using the reference genome (refdata-cellranger-vdj-GRCh38-atlas-ensembl-3.10). For each of the samples, the output file, filtered_contig_annotations.csv, containing TCR- chain and TCR- chain CDR3 nucleotide sequences for single cells were generated. Using a custom code (https://www.biostars.org/p/383217/), the V(D)J information from filtered_contig_annotations.csv was added into the metadata of Seurat.
(35)
