Background Single-cell transcriptomics is a transformative technology to explore heterogeneous cell populations such as T cells, one of our most potent weapons against cancer and viral infections. Recent advances in this technology and the computational tools developed in their wake provide unique opportunities to build reference cell atlases that can be used to interpret new single-cell RNA-sequencing (scRNA-seq) data and systematically compare data sets derived from different models or therapeutic conditions.

Materials and Methods We have developed ProjecTILs (https://github.com/carmonalab/ProjecTILs), a novel computational method to project new data sets into a reference map of T cells, enabling their direct comparison in a stable, annotated system of coordinates. ProjecTILs enables the classification of query cells into curated, discrete states, but also over a continuous space of intermediate states. We illustrate the projection of several data sets from recent publications over two cross-study murine T cell reference atlases: the first describing tumor-infiltrating T lymphocytes (TILs), the second characterizing acute and chronic viral infection.

Results ProjecTILs accurately predicted the effects of multiple perturbations, including the ablation of genes controlling T cell differentiation, such as Tox, Ptpn2, miR-155 and Regnase-1, and identified novel gene programs that were altered in these cells (such as a Lag3-Klrc1 inhibitory module), revealing mechanisms of action behind these immunotherapeutic targets and opening new opportunities for the identification of novel targets. By comparing multiple samples over the same reference map, and across alternative embeddings, our method allows exploring the effect of cellular perturbations (e.g. as the result of therapy or genetic engineering) in terms of transcriptional states and altered genetic programs.

Conclusions The proposed computational method will likely contribute to reveal the mechanisms of action of experimental immunotherapies and guide novel therapeutic interventions in cancer and beyond.

Disclosure Information M. Andreatta: None. S.J. Carmona: None.