Cellular requirements for splenic T1IFN transcriptional induction.

<p>(A) RPMs, but not other macrophage or dendritic cell subsets, induce <i>Ifna</i> and <i>Ifnb</i> at 24 h post-infection with <i>P. chabaudi</i> as detected by qRT-PCR in C57BL/6 mice. Fold mRNA induction represents fold induction of transcript in infected versus mock-infected normalized to beta-actin. Grey dots represent independent experiments conducted on different days; black bars denote the geometric means of the fold inductions. (B) pDCs are not required for splenic <i>Ifna</i> or <i>Ifnb</i> transcriptional induction in response to <i>P. chabaudi</i> in C57BL/6 mice. (C) Genetic deletion of RPMs in 129Sv mice results in diminished T1IFN transcriptional induction. (D) Genetic deletion of <i>Myd88</i> from dendritic cells does not impact transcriptional induction of T1IFNs in spleens of C57BL/6 mice. (E) Genetic deletion of <i>Myd88</i> from macrophages/neutrophils decreases transcriptional induction of T1IFNs by an order of magnitude in C57Bl/6 mice. Grey bars represent geometric means with 95% confidence intervals. Asterisks represent <i>p</i><0.05 in a Student’s <i>t</i>-test against control samples.</p

T1IFNs contribute to control of <i>P. chabaudi</i> infection.

<p>Infected mice were monitored for parasitemia by thin blood smear and survival. Wild type C57BL/6 and congenic knockout mice were infected and monitored for percent parasitemia (<i>n</i> = 5 per strain), which are represented as geometric means with standard deviations and Mann-Whitney <i>p</i>-value. A representative experiment of two is shown. Crosses indicate deaths due to parasitemia.</p

Mice lacking RPMs exhibit wild type infection kinetics.

<p>(A) Parasitemia courses in 129Sv <i>SpiC^+/−</i> (<i>n</i> = 4) and <i>SpiC^−/−</i> (<i>n</i> = 5) mice are represented as geometric means with standard deviations and Mann-Whitney <i>p</i>-value. (B) Ly6c^lo monocyte (CD11b⁺ F4/80⁺ Ly6g⁻ SSC^lo Ly6c^lo) frequencies in blood and spleen of 129Sv <i>SpiC^+/−</i> (white bars) and <i>SpiC^−/−</i> mice (black bars) during the course of infection. Days depicted in blue and orange represent a 1.5-fold decrease or increase, respectively, in Ly6c^lo monocyte frequencies in blood and spleen of mice infected with <i>P. chabaudi</i>. (C) Ly6c^lo monocyte frequencies on day 12 post-infection. Means are presented with standard errors; <i>p</i>-values represent a two-tailed <i>t</i>-test assuming unequal variances. Data represent three independent experiments (<i>n</i> = 6–7 mice per group total).</p

T1IFNs are produced during <i>P. chabaudi</i> infection.

<p>(A) Kinetics of early <i>Ifna, Ifnb</i>, and <i>Ifng</i> transcription using whole spleen qRT-PCR. Fold mRNA induction represents the ratio of transcript in infected over mock-infected C57BL/6 mice. (B) Reproducibility of T1IFN transcript induction as detected by whole spleen qRT-PCR. Each point represents an independent experiment with 4–6 animals, with horizontal bars displaying the geometric mean. (C) Plasma IFNA and IFNB at 24 h post-infection. Data are combined from two independent experiments with each point representing one animal. N.D. = not detected (<i>n</i> = 8).</p

<i>P. chabaudi</i> infection induces IFNB production in pDCs and RPMs.

<p>(A) No splenocytes are YFP⁺ in mock-infected samples, but some splenocytes become YFP⁺24 h after <i>P. chabaudi</i> infection of C57BL/6 <i>Ifnb</i> reporter mice. 2.5×10⁶ events are depicted in each dot plot. (B) pDCs and RPMs constitute over 90% of YFP⁺ events in C57BL/6 mice. (C) Both pDCs and RPMs contribute to splenic IFNB production in 129Sv mice. pDCs were depleted with a single 500 µg dose of anti-mPDCA1 antibody 18 h before infection with <i>P. chabaudi</i>. Grey dots represent individual mice, with horizontal bars representing the mean (B) or geometric mean (C).</p

T1IFN and IFNG signaling redundantly regulate early gene expression responses to <i>P. chabaudi</i> infection.

<p>A representative set of ISG is shown for the gene expression response in whole blood from animals infected for 24 h with <i>P. chabaudi</i> in C57BL/6 knockout mice. Each column represents an individual mouse.</p

Molecular requirements for splenic T1IFN transcriptional induction.

<p>(A) <i>Tlr9</i> and <i>Myd88</i> are required for full transcriptional induction of T1IFNs in spleens of <i>P. chabaudi</i>-infected C57Bl/6 mice. Grey dots represent the means of independent experiments using 4–6 total mice, with T1IFN fold mRNA induction in knockout mice represented as a percentage of induction in wild type animals. Black bars represent means; asterisks represent <i>p</i><0.05 as compared to wild type induction in a two-tailed Student’s <i>t</i>-test assuming unequal variances. (B) <i>Irf7</i> is required for full T1IFN induction, and <i>Irf3</i> is required for full <i>Ifna</i> but not <i>Ifnb</i> induction. (C) <i>Ifnar1</i> is required for full <i>Ifna</i> induction but dispensable for <i>Ifnb</i> induction, whereas <i>Ifngr1</i> is dispensable for all T1IFN induction.</p

Macrophage Colony Stimulating Factor Derived from CD4⁺ T Cells Contributes to Control of a Blood-Borne Infection

<div><p>Dynamic regulation of leukocyte population size and activation state is crucial for an effective immune response. In malaria, <i>Plasmodium</i> parasites elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. Here we show that myeloid expansion during <i>P</i>. <i>chabaudi</i> infection is dependent upon both CD4⁺ T cells and the cytokine Macrophage Colony Stimulating Factor (MCSF). Single-cell RNA-Seq analysis on antigen-experienced T cells revealed robust expression of <i>Csf1</i>, the gene encoding MCSF, in a sub-population of CD4⁺ T cells with distinct transcriptional and surface phenotypes. Selective deletion of <i>Csf1</i> in CD4⁺ cells during <i>P</i>. <i>chabaudi</i> infection diminished proliferation and activation of certain myeloid subsets, most notably lymph node-resident CD169⁺ macrophages, and resulted in increased parasite burden and impaired recovery of infected mice. Depletion of CD169⁺ macrophages during infection also led to increased parasitemia and significant host mortality, confirming a previously unappreciated role for these cells in control of <i>P</i>. <i>chabaudi</i>. This work establishes the CD4⁺ T cell as a physiologically relevant source of MCSF <i>in vivo;</i> probes the complexity of the CD4⁺ T cell response during type 1 infection; and delineates a novel mechanism by which T helper cells regulate myeloid cells to limit growth of a blood-borne intracellular pathogen.</p></div

A role for CD169⁺ macrophages in restriction of <i>Plasmodium</i>.

<p>(A) Immunofluorescent labeling for the macrophage marker CD169 on mesenteric lymph nodes excised from the indicated mice 14 d.p.i. Representative sections are shown. (B) The fraction of each lymph node section perimeter lined with CD169⁺ cells, relative to the total section circumference, was quantified. (C) Lymph node sections from mice of the indicated genotypes were obtained 14 d.p.i. and co-labeled with antibodies to CD169 and Ki67. The percentage of CD169⁺ cells that were also Ki67⁺ was quantified. In B and C, graphs depict mean + SEM (n = 3 mice per group with 4 technical replicates for each mouse). **, p < 0.01; ***, p < 0.001 by <i>t</i>-test. (D-G) CD169^+/DTR mice were infected with <i>P</i>. <i>chabaudi</i> and treated with diphtheria toxin (DT; n = 7) or a catalytically inactive mutant (DT*Glu; n = 6) to deplete CD169⁺ cells 12 d.p.i. (arrows). Representative results from one of two independent experiments are shown. Parasitemia (D), weight (E), and survival (F) were monitored. (G) The indicated myeloid frequencies were assessed in infected CD169^+/DTR mice 24 h after administration of DT. (H, I) <i>Lyz2</i>^{<i>Cre/Cre</i>}; Rosa-DTR mice were infected and treated with DT (n = 5) or DT*Glu (n = 6) 13 and 15 d.p.i. (arrows) to deplete Lyz2⁺ cells. Results are representative of three independent experiments. Depletion of bone marrow macrophages was confirmed by flow cytometry (H), and parasitemia was monitored (I). **, p < 0.01; ***, p < 0.001 by Mann-Whitney test. n.s., not significant.</p

Transcriptional and phenotypic analysis of <i>Csf1</i>^<i>+</i> CD4⁺ T cells.

<p>Antigen-experienced CD4⁺ T cells were sorted from blood 6 d.p.i. and subjected to single-cell RNA-Seq (n = 35 cells). (A-C) RNA-Seq measurements of the indicated transcripts in individual <i>Csf1</i>^<i>+</i> and <i>Csf1</i>^<i>-</i> T cells. TPM, transcripts per kilobase million. Dashed line indicates limit of detection. (D) Scatter plot depicting expression of all detected transcripts in <i>Csf1</i>^<i>+</i> versus <i>Csf1</i>^<i>-</i> cells. Red symbols indicate differentially expressed transcripts (FDR < 5%). (E) Expression of the indicated transcripts, as described for A-C. (F) <i>Csf1</i> expression was assessed by qRT-PCR in antigen-experienced CD4⁺ T cells sorted into CCR2 and CD39 high and low populations. Antigen-naive (CD11a^- CD49d^-) cells from infected mice were included for comparison. (G) Naive splenocytes were stimulated <i>in vitro</i> with plate-bound α-CD3 and α-CD28, either alone (Th0) or in conjunction with Th1- or Th2-polarizing cocktails. Expression of the indicated genes was assessed after 5 d by qRT-PCR. For <i>Csf1</i>, antigen-experienced CD4^<i>+</i> T cells isolated from an infected mouse 6 d.p.i. are included as a positive control (Pc); note log scale. In (F-G), representative results from one of two independent biological replicates are shown (n = 4 mice or wells per condition per replicate). *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Mann-Whitney (A) or <i>t</i>-test (others).</p