Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria

Differentiation of naive CD4 + T cells into functionally distinct T helper (T H ) subsets is crucial for the orchestration of immune responses. Because of extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis with a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of T H 1 and T FH (T follicular helper) cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous T cell receptor sequences, we first demonstrated that T H 1/T FH bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with T H 1 or T FH fates and demonstrated a T cell–intrinsic role for Galectin-1 in supporting T H 1 differentiation. We also revealed the close molecular relationship between T H 1 and interleukin-10–producing Tr1 cells in this infection. T H 1 and T FH fates emerged from a highly proliferative precursor that up-regulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell interaction in driving T H 1/T FH fates. In particular, we found that precursor T H cells were coached toward a T H 1 but not a T FH fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources: a database, www.PlasmoTH.org, which facilitates discovery of novel factors controlling T H 1/T FH fate commitment, and, more generally, GPfates, a modeling framework for characterizing cell differentiation toward multiple fates.