Macrophage Colony Stimulating Factor derived from CD4+ T cells aids in control of an intracellular infection [RNA-seq]
ABSTRACT: Background: In malaria, parasites of the genus Plasmodium elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. In a microarray analysis of pooled, activated CD4+ T cells from mice infected with P. chabaudi, we detected inducible expression of Csf1, which promotes macrophage proliferation. To better characterize Csf1-producing T cells, single-cell RNA-Seq was performed. Results: Robust Csf1 expression was detected in a subset of sampled CD4+ T cells (n = 14/35), whereas the remainder of cells had no detectable Csf1. Further, we identified ~ 400 genes that were differentially expressed between Csf1+ and Csf1- T cells. Conclusions: This work defines the transcriptional landscape of a subset of activated CD4+ T cells that produce the cytokine Csf1. These cells are expected to be important in infections with intracellular pathogens such as Plasmodium. Overall design: Antigen-experienced (CD11a+ CD49d+) CD4+ T cells were isolated by double-sorting from the blood of C57BL/6 adult female mice 6 days post-infection with Plasmodium chabaudi. Single cells were isolated and processed for RNA sequencing using a Fluidigm C1 integrated fluidic circuit chip. 35 biological replicates were analyzed.
Project description:Expression data from antigen-experienced Nfat1+/+ and Nfat1-/- CD4+ T cells following 21 days of Plasmodium yoelii 17XNL infection. Overall design: Total RNA was isolated from CD4+ CD49d+ CD11a-high cells sorted from spleens following 21 days Plasmodium yoelii 17XNL infection and subjected to microarray analysis.
Project description:BACKGROUND:Determining the efficacy of human vaccines that induce antigen-specific protective CD4 T cell responses against pathogens can be particularly challenging to evaluate. Surface expression of CD11a and CD49d has been shown to identify antigen-specific CD4 T cells against viral pathogens in mice. We hypothesized that CD11a and CD49d would also serve as markers of human antigen-specific T cells responding to vaccination. METHODS:A phase I vaccine trial enabled us to evaluate a novel gating strategy based on surface expression of CD11a and CD49d as a means of detecting antigen-specific, cytokine producing CD4 and CD8 T cells induced after vaccination of naïve individuals against leishmaniasis. Three study groups received LEISH-F3 recombinant protein combined with either squalene oil-in-water emulsion (SE) alone, SE with the synthetic TLR-4 ligand glucopyranosyl lipid adjuvant (GLA-SE), or SE with Salmonella minnesota-derived monophosphoryl lipid A (MPL-SE). Individuals were given 3 vaccine doses, on days 0, 28 and 168. RESULTS:Starting after the first vaccine dose, the frequency of both CD11ahiCD49d+ CD4 and CD11ahiCD49d+ CD8 T cells significantly increased over time throughout the 24-week trial. To confirm the role of CD11ahiCD49d+ expression in the identification of the antigen-specific T cells, cytokine production was measured following LEISH-F3 stimulation. All of the IFN-γ, TNF-α, and IL-2 producing cells were found within the CD11ahiCD49d+ population. CONCLUSIONS:Our results suggest that the change in the frequency of CD11ahiCD49d+ T cells can be used to track antigen-specific CD4 and CD8 T cell responses following T cell-targeted vaccination.
Project description:Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.
Project description: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.
Project description: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.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 7, and 28 to undergo processing and generate scRNA-seq dataset. At Day 28, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). scRNA-seq dataset was analysed to investigate transcriptome dynamics of CD4+ T cells from effector to memory states.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 7, 10, 14, 17, 21, 28 to undergo processing and generate scRNA-seq dataset. From Day 10 onwards, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). scRNA-seq dataset was analysed to investigate transcriptome dynamics of CD4+ T cells from effector to memory states.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 4, 7, and 32 to undergo processing and to generate scATAC-seq dataset. At Day 7, CXCR5+ and CXCR6+ cells were recovered separately. At Day 32, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). scATAC-seq dataset was analysed to investigate epigenomic landscapes of CD4+ T cells from effector to memory states.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 7, and 28 to undergo processing and to generate ATAC-seq dataset (2 independent biological repeats per time point). At Day 28, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). Bulk ATAC-seq dataset was analysed to investigate epigenomic landscapes of CD4+ T cells from effector to memory states.
Project description:CD4+ T cells are critical for defense against the Plasmodium parasites that cause malaria. To better understand CD4+ T cell effector mechanisms during malaria, we performed microarray analysis of CD4+ T cells from naïve and infected mice. Comparison of activated (CD44 hi CD62L lo) CD4+ T cells from infected mice to bulk CD4+ T cells from naïve mice revealed a subset of genes that were upregulated by infection with Plasmodium chabaudi. These results help generate a more complete picture of CD4+ T cell function in malaria. Overall design: Total CD4+ T cells were double-sorted from the blood of adult naïve female mice; activated (CD44 hi CD62L lo) CD4+ T cells were double-sorted from the blood of adult female mice 6 d post-infection with P. chabaudi. Four independent biological replicates were performed per condition.