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

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

Funding: This work was funded by a Career Development Fellowship (1028634) and a project grant (GRNT1028641) awarded to AHa by the Australian National Health & Medical Research Council (NHMRC). IS was supported by The University of Queensland Centennial and IPRS Scholarships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Visual system diversity in coral reef fishes

Coral reefs are one of the most species rich and colourful habitats on earth and for many coral reef teleosts, vision is central to their survival and reproduction. The diversity of reef fish visual systems arises from variations in ocular and retinal anatomy, neural processing and, perhaps most easily revealed by, the peak spectral absorbance of visual pigments. This review examines the interplay between retinal morphology and light environment across a number of reef fish species, but mainly focusses on visual adaptations at the molecular level (i.e. visual pigment structure). Generally, visual pigments tend to match the overall light environment or micro-habitat, with fish inhabiting greener, inshore waters possessing longer wavelength-shifted visual pigments than open water blue-shifted species. In marine fishes, particularly those that live on the reef, most species have between two (likely dichromatic) to four (possible tetrachromatic) cone spectral sensitivities and a single rod for crepuscular vision; however, most are trichromatic with three spectral sensitivities. In addition to variation in spectral sensitivity number, spectral placement of the absorbance maximum (λ) also has a surprising degree of variability. Variation in ocular and retinal anatomy is also observed at several levels in reef fishes but is best represented by differences in arrangement, density and distribution of neural cell types across the retina (i.e. retinal topography). Here, we focus on the seven reef fish families most comprehensively studied to date to examine and compare how behaviour, environment, activity period, ontogeny and phylogeny might interact to generate the exceptional diversity in visual system design that we observe

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

IFN regulatory factor 3 balances Th1 and T follicular helper immunity during nonlethal blood-stage Plasmodium infection

Differentiation of CD4Th cells is critical for immunity to malaria. Several innate immune signaling pathways have been implicated in the detection of blood-stageparasites, yet their influence over Th cell immunity remains unclear. In this study, we usedreactive TCR transgenic CD4T cells, termed PbTII cells, during nonlethalAS and17XNL infection in mice, to examine Th cell development in vivo. We found no role for caspase1/11, stimulator of IFN genes, or mitochondrial antiviral-signaling protein, and only modest roles for MyD88 and TRIF-dependent signaling in controlling PbTII cell expansion. In contrast, IFN regulatory factor 3 (IRF3) was important for supporting PbTII expansion, promoting Th1 over T follicular helper (Tfh) differentiation, and controlling parasites during the first week of infection. IRF3 was not required for early priming by conventional dendritic cells, but was essential for promoting CXCL9 and MHC class II expression by inflammatory monocytes that supported PbTII responses in the spleen. Thereafter, IRF3-deficiency boosted Tfh responses, germinal center B cell and memory B cell development, parasite-specific Ab production, and resolution of infection. We also noted a B cell-intrinsic role for IRF3 in regulating humoral immune responses. Thus, we revealed roles for IRF3 in balancing Th1- and Tfh-dependent immunity during nonlethal infection with blood-stageparasites

IFNAR1-signalling simultaneously limits splenic Th1 and Tfh cell responses.

<p>(A & B) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and absolute numbers of splenic (A) Th1 (IFNγ⁺ Tbet⁺) and (B) emerging Tfh (PD1⁺ CXCR5⁺) cells in WT <i>and Ifnar1</i>^<i>-/-</i> mice (n = 6/group), 6 days <i>p</i>.<i>i</i> with <i>Pc</i>AS. Data representative of two independent experiments. (C) Numbers of splenic ICOS⁺ Th1 cells (Tbet⁺ IFNγ⁺ CD4⁺ T cells) and Tfh cells (PD1⁺CXCR5⁺ CD4⁺ T cells) 6 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (D) Numbers of ICOS⁺ Tfh cells (PD1⁺CXCR5⁺ CD4⁺ T cells), 16 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL infection. (E) Proportions and absolute numbers of splenic CD4⁺ T-cells expressing Ki-67 in naïve, WT and <i>Ifnar1</i>^<i>-/-</i> mice 6 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (F) Proportions and absolute numbers of splenic CD4⁺ T-cells expressing Ki-67 in naïve mice, and WT mice, 6 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL and treatment with α-IFNAR1 or Control IgG. (G) Absolute numbers of splenocytes, CD4⁺ T-cells and B-cells, in WT naïve, infected WT and <i>Ifnar1</i>^<i>-/-</i> mice 6 days (n = 17–18, pooled from three independent experiments (n = 5–6 per expt)) and 8 days (n = 29, pooled from five experiments (n = 5–6 per expt)) <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (H) Absolute numbers of splenocytes, CD4⁺ T-cells and B-cells in WT naïve, infected WT and <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL (n = 17–18, pooled from three experiments (n = 5–6 per expt)) Mann-Whitney U-test **P<0.01, *P<0.05.</p

Antibody-mediated IFNAR1 blockade boosts humoral immune responses during blood-stage infection.

<p>WT mice (n = 5/group) were treated with anti-IFNAR1 blocking antibody (α-Ifnar1) or control IgG prior to and during infection with <i>Pc</i>AS. (A) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and absolute numbers of splenic ICOS⁺ CD4⁺ T cells in naïve and infected mice on days 6 and 7 <i>p</i>.<i>i</i>. (B) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and numbers of splenic Tfh cells (as PD1⁺CXCR5⁺ CD4⁺ T cells) in naïve and infected and antibody-treated mice, 7 days <i>p</i>.<i>i</i>. Bcl-6 expression is also shown in histograms for Tfh (PD1⁺CXCR5⁺; red gate) and non-Tfh cells (PD1^-CXCR5^-; blue gate), alongside Geometric Mean Bcl-6 expression by these populations in individual mice. (C and D) Numbers of splenic (C) plasmablasts and (D) GC B cells in naïve, and infected and treated mice, 7 days <i>p</i>.<i>i</i>. Data representative of 2 independent experiments. Mann-Whitney U test *P<0.05; **P<0.01.</p

IFNAR1-signalling limits splenic Tfh cell responses.

<p>(A) WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 5/group) were infected with <i>Py</i>17XNL, and splenic Tfh (PD1⁺ CXCR5⁺) cell proportions and absolute numbers were assessed at day 16 <i>p</i>.<i>i</i>. Representative FACS plots gated on CD4⁺ TCRβ⁺ live singlets. Data representative of three independent experiments; Mann-Whitney U test *P<0.05; **P<0.01. (B) WT <i>and Ifnar1</i>^<i>-/-</i> mice (n = 5-6/group) were infected with <i>Pc</i>AS, and splenic Tfh (PD1⁺ CXCR5⁺ or Bcl-6⁺ CXCR5⁺) cell proportions and absolute numbers were assessed at day 8 <i>p</i>. <i>i</i>. Representative FACS plots gated on CD4⁺ TCRβ⁺ live singlets. Data representative of three independent experiments; Mann-Whitney U test *P<0.05; **P<0.01.</p