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

Differentiation of naïve CD4+ T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to 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 using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR 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 a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards 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 modelling framework for characterizing cell differentiation towards multiple fates

Development of circulating CD4+ T‐cell memory

The ability of circulating CD4 T cells to retain memories of previous antigenic encounters is a cardinal feature of the adaptive immune system. Over the past two decades, since the first description of central and effector memory T cells, many studies have examined molecular mechanisms controlling CD8 T-cell memory, with comparatively less research into CD4 T-cell memory. Here, we review a number of seminal studies showing that circulating memory CD4 T cells develop directly from effector cells; and in so doing, preserve features of their effector precursors. We examine mechanisms controlling the development and phenotypes of memory CD4 T cells, and provide an updated model that accommodates both the central and effector memory paradigm and the diverse T helper cell classification system

Single-cell transcriptomics of alloreactive CD4+ T cells over time reveals divergent fates during gut graft-versus-host disease

Acute gastrointestinal (GI) graft-versus-host disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem cell transplantation (alloSCT). The condition is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN-γ, IL-17A, or GM-CSF and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between Th cell states during priming in mesenteric lymph nodes (mLNs) and effector function in the GI tract remain undefined at genome scale. We applied scRNA-Seq and computational modeling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLNs. Importantly, we inferred an unexpected second trajectory, categorized by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4β7 before gut migration and failed to express cytokines. These cells exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced T cell factor 1 (TCF1). Thus, scRNA-Seq suggested divergence of alloreactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogeneous priming in vivo. These findings, which are potentially the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT

Development of a novel CD4+ TCR transgenic line that reveals a dominant role for CD8+ dendritic cells and CD40 signaling in the generation of helper and CTL responses to blood-stage malaria

We describe an MHC class II (I-Ab)-restricted TCR transgenic mouse line that produces CD4 T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4 T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8 T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4 T cells and the previously described PbT-I CD8 T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8 DC (a subset of XCR1 DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4 T cell responses. Depletion of CD8 DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4 T cell immunity during malaria and provides evidence that CD4 T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8 DC