Spatial Profiling

"We next employed spatial transcriptomics analysis on the four treatment groups, with duplicates for each group, to further dissect whether immune cell types, particularly NK cells, were enriched in the +/+ treated group. After data aggregation and batch-effect removal, dimensionality reduction using Seurat generated a UMAP plot consisting of 17,974 spatial spots. The spatial spots of the uninfected samples (−/− and +/−) clustered closely together, whereas those of the −/+ and +/+ samples were separated into different clusters. Graph-based unsupervised clustering identified 20 Visium clusters across all combined treatment samples. The −/− and +/− samples shared the majority of the clusters, whereas the clusters of −/+ and +/+ samples were distinct from each other. Spatial clustering patterns overlaid with the tissue map revealed distinct clusters in the conducting parts and respiratory parts of the lung. Secondary annotation categorized the Visium clusters into 12 major lung cell-type groups. Mesenchymal cells were detected almost exclusively in the −/+ samples, implying tissue remodeling after substantial damage caused by SARS-CoV-2 infection. In contrast, the +/+ treatment group was characterized by robust recruitment of immune cells. NK cell marker genes exhibited higher average expression in the +/+ treatment group, specifically in Visium clusters 4, 9, and 18. These clusters also showed higher expression of additional NK cell marker genes and ADCC-associated genes, with cluster 18 showing high expression of Fcer1g and Fcgr3. A two-tailed Wilcoxon rank-sum test confirmed that the NK cell module score was significantly higher in the infected +/+ group than in the −/+ group, suggesting an increase in NK cell abundance in mAb-pretreated samples. Functional enrichment analysis of the NK cell-enriched clusters identified the “cell killing” biological process in cluster 18, with Ptprc, Tyrobp, and Coro1a enriched. The high expression of Fcer1g and Fcgr3 further supports the involvement of NK cells in ADCC activity. In cluster 4, the GO term “negative regulation of viral genome replication,” including Ccl5, Oas2, and Oas2l, was enriched, highlighting the antiviral effect of the ADCC-inducing antibody."