GSE168859
Coupled Single Cell RNA Sequencing and TCR Profiling of T Cells in Large Granular Lymphocytosis to Infer Pathophysiology and Mechanism of Drug Action
Background and methods:  T-cell large granular lymphocytosis (T-LGL) is a lymphoproliferative disease usually presenting with cytopenia, and typically characterized by clonal expansion of terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs). Conventional methods including flow cytometry and sequencing of Vb chains provided incomplete signature of TCR repertoire. Efforts to characterize transcriptome of clonal CTLs using whole peripheral blood or after enrichment of specific clones by flow cytometry have not provided satisfying resolution and scale due to heterogeneity among individual patients. We applied single cell TCR-sequencing coupled with single cell RNA sequencing to CD3+ T cells from a relatively large cohort (N=13) of T-LGL patients in a clinical protocol to prospectively test therapy with alemtuzumab in refractory disease, to characterize TCR repertoire and to define pathophysiologic mechanism at the single cell level, and to determine mechanisms of response and non-response to therapy. Results: T cells have less clonal diversity in T-LGL patients, compared with that in healthy donors. TCR repertoires in T-LGLL follow power law distribution but at a larger scale. There are no significant evidence of common TCR clonotypes among T-LGLL patients. TCR usage and T cell activation states shape T cell phenotypes. Cell survival and apoptosis gene programs are deregulated in T-LGL. Immunosuppressive treatment modulates clonality and gene expression in T-LGL. Conclusion: Our work complements and adds to previous modeling of T cell clonal expansion. Single cell sequencing of  transcriptomes supports a theory of fluctuation in clone fitness, antigenic and environmental factors in concert with clone-specific and cell-specific germline and somatic genetic factors (HLA and STAT3). Overall design: We applied scTCR-seq coupled with scRNA-seq to CD3+ T cells obtained from a relatively large cohort of T-LGLL patients and 7 healthy donors