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 (T_H1) and follicular helper T (T_(FH)) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T_H1 and T_(FH) trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T_(FH) and central memory were revealed, with antimalarials modulating these responses and boosting T_H1 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/)

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 (T_H1) and follicular helper T (T_(FH)) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T_H1 and T_(FH) trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T_(FH) and central memory were revealed, with antimalarials modulating these responses and boosting T_H1 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/)

Quantification of host-mediated parasite clearance during blood-stage Plasmodium infection and anti-malarial drug treatment in mice

A major mechanism of host-mediated control of blood-stage Plasmodium infection is thought to be removal of parasitized red blood cells (pRBCs) from circulation by the spleen or phagocytic system. The rate of parasite removal is thought to be further increased by anti-malarial drug treatment, contributing to the effectiveness of drug therapy. It is difficult to directly compare pRBC removal rates in the presence and absence of treatment, since in the absence of treatment the removal rate of parasites is obscured by the extent of ongoing parasite proliferation. Here, we transfused a single generation of fluorescently-labelled Plasmodium berghei pRBCs into mice, and monitored both their disappearance from circulation, and their replication to produce the next generation of pRBCs. In conjunction with a new mathematical model, we directly estimated host removal of pRBCs during ongoing infection, and after drug treatment. In untreated mice, pRBCs were removed from circulation with a half-life of 15.1 h. Treatment with various doses of mefloquine/artesunate did not alter the pRBC removal rate, despite blocking parasite replication effectively. An exception was high dose artesunate, which doubled the rate of pRBC removal (half-life of 9.1 h). Phagocyte depletion using clodronate liposomes approximately halved the pRBC removal rate during untreated infection, indicating a role for phagocytes in clearance. We next assessed the importance of pRBC clearance for the decrease in the parasite multiplication rate after high dose artesunate treatment. High dose artesunate decreased parasite replication ∼46-fold compared with saline controls, with inhibition of replication contributing 23-fold of this, and increased pRBC clearance contributing only a further 2.0-fold. Thus, in our in vivo systems, drugs acted primarily by inhibiting parasite replication, with drug-induced increases in pRBC clearance making only minor contributions to overall drug effect

Plasmodium-specific antibodies block in vivo parasite growth without clearing infected red blood cells.

Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC

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 (T1) and follicular helper T (T) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T1 and T trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T and central memory were revealed, with antimalarials modulating these responses and boosting T1 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/)