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/ ).

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.

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.

Characterising the effect of antimalarial drugs on the maturation and clearance of murine blood-stage Plasmodium parasites in vivo.

The artemisinins are the first-line therapy for severe and uncomplicated malaria, since they cause rapid declines in parasitemia after treatment. Despite this, in vivo mechanisms underlying this rapid decline remain poorly characterised. The overall decline in parasitemia is the net effect of drug inhibition of parasites and host clearance, which competes against any ongoing parasite proliferation. Separating these mechanisms in vivo was not possible through measurements of total parasitemia alone. Therefore, we employed an adoptive transfer approach in which C57BL/6J mice were transfused with Plasmodium berghei ANKA strain-infected, fluorescent red blood cells, and subsequently drug-treated. This approach allowed us to distinguish between the initial drug-treated generation of parasites (Gen0), and their progeny (Gen1). Artesunate efficiently impaired maturation of Gen0 parasites, such that a sufficiently high dose completely arrested maturation after 6h of in vivo exposure. In addition, artesunate-affected parasites were cleared from circulation with a half-life of 6.7h. In vivo cell depletion studies using clodronate liposomes revealed an important role for host phagocytes in the removal of artesunate-affected parasites, particularly ring and trophozoite stages. Finally, we found that a second antimalarial drug, mefloquine, was less effective than artesunate at suppressing parasite maturation and driving host-mediated parasite clearance. Thus, we propose that in vivo artesunate treatment causes rapid decline in parasitemia by arresting parasite maturation and encouraging phagocyte-mediated clearance of parasitised RBCs.

Host-mediated impairment of parasite maturation during blood-stage Plasmodium infection.

Severe malaria and associated high parasite burdens occur more frequently in humans lacking robust adaptive immunity to Plasmodium falciparum Nevertheless, the host may partly control blood-stage parasite numbers while adaptive immunity is gradually established. Parasite control has typically been attributed to enhanced removal of parasites by the host, although in vivo quantification of this phenomenon remains challenging. We used a unique in vivo approach to determine the fate of a single cohort of semisynchronous, Plasmodium berghei ANKA- or Plasmodium yoelii 17XNL-parasitized red blood cells (pRBCs) after transfusion into naive or acutely infected mice. As previously shown, acutely infected mice, with ongoing splenic and systemic inflammatory responses, controlled parasite population growth more effectively than naive controls. Surprisingly, however, this was not associated with accelerated removal of pRBCs from circulation. Instead, transfused pRBCs remained in circulation longer in acutely infected mice. Flow cytometric assessment and mathematical modeling of intraerythrocytic parasite development revealed an unexpected and substantial slowing of parasite maturation in acutely infected mice, extending the life cycle from 24 h to 40 h. Importantly, impaired parasite maturation was the major contributor to control of parasite growth in acutely infected mice. Moreover, by performing the same experiments in rag1-/- mice, which lack T and B cells and mount weak inflammatory responses, we revealed that impaired parasite maturation is largely dependent upon the host response to infection. Thus, impairment of parasite maturation represents a host-mediated, immune system-dependent mechanism for limiting parasite population growth during the early stages of an acute blood-stage Plasmodium infection.

IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection.

Parasite-specific antibodies protect against blood-stage Plasmodium infection. However, in malaria-endemic regions, it takes many months for naturally-exposed individuals to develop robust humoral immunity. Explanations for this have focused on antigenic variation by Plasmodium, but have considered less whether host production of parasite-specific antibody is sub-optimal. In particular, it is unclear whether host immune factors might limit antibody responses. Here, we explored the effect of Type I Interferon signalling via IFNAR1 on CD4+ T-cell and B-cell responses in two non-lethal murine models of malaria, P. chabaudi chabaudi AS (PcAS) and P. yoelii 17XNL (Py17XNL) infection. Firstly, we demonstrated that CD4+ T-cells and ICOS-signalling were crucial for generating germinal centre (GC) B-cells, plasmablasts and parasite-specific antibodies, and likewise that T follicular helper (Tfh) cell responses relied on B cells. Next, we found that IFNAR1-signalling impeded the resolution of non-lethal blood-stage infection, which was associated with impaired production of parasite-specific IgM and several IgG sub-classes. Consistent with this, GC B-cell formation, Ig-class switching, plasmablast and Tfh differentiation were all impaired by IFNAR1-signalling. IFNAR1-signalling proceeded via conventional dendritic cells, and acted early by limiting activation, proliferation and ICOS expression by CD4+ T-cells, by restricting the localization of activated CD4+ T-cells adjacent to and within B-cell areas of the spleen, and by simultaneously suppressing Th1 and Tfh responses. Finally, IFNAR1-deficiency accelerated humoral immune responses and parasite control by boosting ICOS-signalling. Thus, we provide evidence of a host innate cytokine response that impedes the onset of humoral immunity during experimental malaria.