Transcriptome dynamics of CD4+ T cells during malaria maps gradual transit from effector to memory.

The dynamics of CD4+ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4+ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (TH1) and follicular helper T (TFH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated TH1 and TFH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between TFH and central memory were revealed, with antimalarials modulating these responses and boosting TH1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4+ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).

CD4+ T cells display a spectrum of recall dynamics during re-infection with malaria parasites.

Children in malaria-endemic regions can experience repeated Plasmodium infections over short periods of time. Effects of re-infection on multiple co-existing CD4+ T cell subsets remain unresolved. Here, we examine antigen-experienced CD4+ T cells during re-infection in mice, using scRNA-seq/TCR-seq and spatial transcriptomics. TCR transgenic TEM cells initiate rapid Th1/Tr1 recall responses prior to proliferating, while GC Tfh counterparts are refractory, with TCM/Tfh-like cells exhibiting modest non-proliferative responses. Th1-recall is a partial facsimile of primary Th1-responses, with no upregulated effector-associated genes being unique to recall. Polyclonal, TCR-diverse, CD4+ T cells exhibit similar recall dynamics, with individual clones giving rise to multiple effectors including highly proliferative Th1/Tr1 cells, as well as GC Tfh and Tfh-like cells lacking proliferative capacity. Thus, we show substantial diversity in recall responses mounted by multiple co-existing CD4+ T cell subsets in the spleen, and present graphical user interfaces for studying gene expression dynamics and clonal relationships during re-infection.

Plasmodium infection induces phenotypic, clonal, and spatial diversity among differentiating CD4+ T cells.

Naive CD4+ T cells must differentiate in order to orchestrate immunity to Plasmodium, yet understanding of their emerging phenotypes, clonality, spatial distributions, and cellular interactions remains incomplete. Here, we observe that splenic polyclonal CD4+ T cells differentiate toward T helper 1 (Th1) and T follicular helper (Tfh)-like states and exhibit rarer phenotypes not elicited among T cell receptor (TCR) transgenic counterparts. TCR clones present at higher frequencies exhibit Th1 skewing, suggesting that variation in major histocompatibility complex class II (MHC-II) interaction influences proliferation and Th1 differentiation. To characterize CD4+ T cell interactions, we map splenic microarchitecture, cellular locations, and molecular interactions using spatial transcriptomics at near single-cell resolution. Tfh-like cells co-locate with stromal cells in B cell follicles, while Th1 cells in red pulp co-locate with activated monocytes expressing multiple chemokines and MHC-II. Spatial mapping of individual transcriptomes suggests that proximity to chemokine-expressing monocytes correlates with stronger effector phenotypes in Th1 cells. Finally, CRISPR-Cas9 gene disruption reveals a role for CCR5 in promoting clonal expansion and Th1 differentiation. A database of cellular locations and interactions is presented: https://haquelab.mdhs.unimelb.edu.au/spatial_gui/.

Systemic host inflammation induces stage-specific transcriptomic modification and slower maturation in malaria parasites.

Maturation rates of malaria parasites within red blood cells (RBCs) can be influenced by host nutrient status and circadian rhythm; whether host inflammatory responses can also influence maturation remains less clear. Here, we observed that systemic host inflammation induced in mice by an innate immune stimulus, lipopolysaccharide (LPS), or by ongoing acute Plasmodium infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. Importantly, plasma from LPS-conditioned or acutely infected mice directly inhibited parasite maturation during in vitro culture, which was not rescued by supplementation, suggesting the emergence of inhibitory factors in plasma. Metabolomic assessments confirmed substantial alterations to the plasma of LPS-conditioned and acutely infected mice, and identified a small number of candidate inhibitory metabolites. Finally, we confirmed rapid parasite responses to systemic host inflammation in vivo using parasite scRNA-seq, noting broad impairment in transcriptional activity and translational capacity specifically in trophozoites but not rings or schizonts. Thus, we provide evidence that systemic host inflammation rapidly triggered transcriptional alterations in circulating blood-stage Plasmodium trophozoites and predict candidate inhibitory metabolites in the plasma that may impair parasite maturation in vivo. IMPORTANCE Malaria parasites cyclically invade, multiply, and burst out of red blood cells. We found that a strong inflammatory response can cause changes to the composition of host plasma, which directly slows down parasite maturation. Thus, our work highlights a new mechanism that limits malaria parasite growth in the bloodstream.

An introduction to spatial transcriptomics for biomedical research.

Single-cell transcriptomics (scRNA-seq) has become essential for biomedical research over the past decade, particularly in developmental biology, cancer, immunology, and neuroscience. Most commercially available scRNA-seq protocols require cells to be recovered intact and viable from tissue. This has precluded many cell types from study and largely destroys the spatial context that could otherwise inform analyses of cell identity and function. An increasing number of commercially available platforms now facilitate spatially resolved, high-dimensional assessment of gene transcription, known as 'spatial transcriptomics'. Here, we introduce different classes of method, which either record the locations of hybridized mRNA molecules in tissue, image the positions of cells themselves prior to assessment, or employ spatial arrays of mRNA probes of pre-determined location. We review sizes of tissue area that can be assessed, their spatial resolution, and the number and types of genes that can be profiled. We discuss if tissue preservation influences choice of platform, and provide guidance on whether specific platforms may be better suited to discovery screens or hypothesis testing. Finally, we introduce bioinformatic methods for analysing spatial transcriptomic data, including pre-processing, integration with existing scRNA-seq data, and inference of cell-cell interactions. Spatial -omics methods are already improving our understanding of human tissues in research, diagnostic, and therapeutic settings. To build upon these recent advancements, we provide entry-level guidance for those seeking to employ spatial transcriptomics in their own biomedical research.

A diverse fibroblastic stromal cell landscape in the spleen directs tissue homeostasis and immunity.

The spleen is a compartmentalized organ that serves as a blood filter and safeguard of systemic immune surveillance. Labyrinthine networks of fibroblastic stromal cells construct complex niches within the white pulp and red pulp that are important for tissue homeostasis and immune activation. However, the identity and roles of the global splenic fibroblastic stromal cells in homeostasis and immune responses are poorly defined. Here, we performed a cellular and molecular dissection of the splenic reticular stromal cell landscape. We found that white pulp fibroblastic reticular cells (FRCs) responded robustly during acute viral infection, but this program of gene regulation was suppressed during persistent viral infection. Single-cell transcriptomic analyses in mice revealed diverse fibroblast cell niches and unexpected heterogeneity among podoplanin-expressing cells that include glial, mesothelial, and adventitial cells in addition to FRCs. We found analogous fibroblastic stromal cell diversity in the human spleen. In addition, we identify the transcription factor SpiB as a critical regulator required to support white pulp FRC differentiation, homeostatic chemokine expression, and antiviral T cell responses. Together, our study provides a comprehensive map of fibroblastic stromal cell types in the spleen and defines roles for red and white pulp fibroblasts for splenic function and orchestration of immune responses.

Francisella tularensis induces Th1 like MAIT cells conferring protection against systemic and local infection.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.

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 α4ß7 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.