ProjecTILs murine reference atlas of virus-specific CD4+ T cells

GP66:I-A^b+ spleen T cells were sorted from LCMV infected animals either 7 or 21 days after Clone 13 infection, conditions referred to as Early and Late Chronic. In addition, similar populations were isolated 7, 21 and >60 days after LCMV Armstrong infection (samples referred to as Acute, Early and Late Memory, respectively). A total of 11 datasets were processed with droplet-based (10x Chromium) single-cell RNA sequencing resulting in over 35,000 high-quality virus-specific CD4⁺ T cell transcriptomes. In addition, selected samples were used to measure simultaneously TCR usage and transcriptome at the single-cell resolution.

For the construction of the CD4+ T cell reference atlas, datasets were downsampled to balance the contribution from different types of infection and time point. To this end, a maximum of 5,000 cells were randomly selected for each of the 5 subsets: acute day7, acute day21, acute day60, chronic day7 and chronic day21. To mitigate batch effects between samples, we integrated the 11 samples using STACAS (Andreatta & Carmona 2020) with the following parameters: number of variable genes=800, dist.thr=0.6, dims=20. On the integrated data, unsupervised clusters were calculated using the FindNeighbors and FindClusters functions from Seurat (Hao et al. 2021) with parameters: k.parameter=10, resolution=0.4. Finally, unsupervised clusters were manually annotated to obtain nine “functional clusters” that summarize the diversity of CD4⁺ T cells in acute and chronic infection.

The dataset is available as a Seurat object, with functional cluster, sample of origin and several other measurement for each cell stored in the object metadata. The object can be loaded into an R session using the readRDS() function, and directly used as a reference atlas for ProjecTILs (Andreatta et al. 2021). Sequencing data underlying this object can be obtained at NCBI GEO under accession number GSE182320.