Single-cell RNA-seq and computational analysis using temporal mixture modeling resolves TH1/TFH fate bifurcation in malaria

Differentiation of naïve CD4+ T cells into functionally distinct T helper (TH) 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 TH1 and TFH (T follicular helper) cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous T cell receptor sequences, we first demonstrated that TH1/TFH bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with TH1 or TFH fates and demonstrated a T cell–intrinsic role for Galectin-1 in supporting TH1 differentiation. We also revealed the close molecular relationship between TH1 and interleukin-10–producing Tr1 cells in this infection. TH1 and TFH 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 TH1/TFH fates. In particular, we found that precursor TH cells were coached toward a TH1 but not a TFH 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 TH1/TFH fate commitment, and, more generally, GPfates, a modeling framework for characterizing cell differentiation toward multiple fates.