T cell fate and clonality inference from single-cell transcriptomes

Abstract

We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4(+) T cells as well as mature effector and memory cells.

Figure 1

Distributions of lengths of reconstructed TCR sequences. Reconstructed sequences were trimmed to include the region derived from the V gene, junction and J gene. The lengths of these sequences are plotted as histograms and kernel density estimates for TCRa (upper) and TCRb (lower). Dotted lines represent the interquartile range of lengths of full-length sequences derived from the combinatorial recombinome files.

Figure 2

Assessment of clonal CD4⁺ T cell expansion during Salmonella typhimurium infection. (a) Schematic of timeline for Salmonella infection experiment. (b) Distribution of expanded clonotypes within splenic CD4⁺ T cell populations analysed by single-cell RNA-seq. The x-axis indicates the number of cells within the expanded clonotypes whilst the y-axis represents the number of clonotypes of each size. (c) Clonotype network graph from day 14, mouse 1. Each node in the graph represents an individual splenic CD4⁺ T lymphocyte. Coloured bars within the nodes indicate the presence of reconstructed TCR sequences that were detected for each cell. Dark coloured identifiers are productive, light coloured are non-productive. Red edges between the nodes indicate shared TCRα sequences whilst blue edges indicate shared TCRβ sequences. Edge thickness is proportional to the number of shared sequences.

Figure 3

Distribution of expanded clonotypes throughout the Th1 response to S. typhimurium infection. (a) Dimensionality reduction of single-cell gene expression data by independent component analysis (ICA). Each single CD4⁺ T cell is plotted in reduced two-dimensional space according to its gene expression profile. Points are colored according to the timepoint from which they were sampled or according to their expression of marker genes indicative of their phenotype. Where the expression of a set of genes (Th1 genes and proliferation markers) is plotted, this is the sum of TPM values for the genes within the set. (b) Clonotype distribution in gene-expression space. Three representative expanded clonotypes from day 14 mouse 1 are shown as purple points on top of all other cells within the gene expression space.