Integrative Analysis of Spatial Transcriptome with Single-cell Transcriptome and Single-cell Epigenome in Mouse Lungs after Immunization

Immunological memory is key to productive adaptive immunity. An unbiased, high through-put gene expression profiling of tissue-resident memory T cells residing in various anatomical location within the lung is fundamental to understand lung immunity but still lacking. In this study, using a well-established model on Klebsiella pneumoniae, we performed an integrative analysis of spatial transcriptome with single-cell RNA-seq and single-cell ATAC-seq on lung cells from mice after Immunization using the 10x Genomics Chromium and Visium platform. We employed several deconvolution algorithms and established an optimized deconvolution pipeline to accurately decipher specific cell-type composition by location. We identified and located 12 major cell types by scRNA-seq and spatial transcriptomic analysis. Integrating scATAC-seq data from the same cells processed in parallel with scRNA-seq, we found epigenomic profiles provide more robust cell type identification, especially for lineage-specific T helper cells. When combining all three data modalities, we observed a dynamic change in the location of T helper cells as well as their corresponding chemokines for chemotaxis. Furthermore, cell-cell communication analysis of spatial transcriptome provided evidence of lineage-specific T helper cells receiving designated cytokine signaling. In summary, our first-in-class study demonstrated the power of multi-omics analysis to uncover intrinsic spatial- and cell-type-dependent molecular mechanisms of lung immunity. Our data provides a rich research resource of single cell multi-omics data as a reference for understanding spatial dynamics of lung immunization. Overall design: In this study, we inoculated mice with heat-killed K. pneumoniae and assigned them into two groups, the immunized and the re-challenged group. The immunized group was inoculated twice on day 0 and day 7, whereas the re-challenged group was additionally inoculated on day 13. On day 14, both groups of mice were sacrificed for tissue harvesting. Slices A1 and A2 were sectioned from the fresh frozen lung of the immunized mouse, whereas slices A3 and A4 were from the re-challenged mouse. In a separate cohort, lung tissue from the re-challenged mouse were harvested and single-cell suspension was obtained after enzymatic digestion. The four lung slices were used for spatial transcriptomics, and the single-cell suspension from the re-challenged mouse were subjected to scRNA-seq and scATAC-seq analysis.