8 research outputs found
Blockade of IL-12 and IFNγ during co-infection preserves Th2 responses.
<p>A-C). C57BL/6 mice were orally infected with 200 <i>H</i>. <i>polygyrus</i> larvae. 6 days post-infection, mice were infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i>. At day 8-post infection with <i>P</i>. <i>chabaudi</i> (d14 <i>H</i>. <i>polygyrus</i>), mice were harvested. Mice were treated with 0.5 mg of anti-IL-12 and anti-IFNγ i.p. at days 0, 5, and 11. B). Total numbers of CD4<sup>+</sup>CD44<sup>hi</sup><i>Il4</i><sup>gfp+</sup> cells in the mesenteric lymph nodes. C). IgE measured in the serum by ELISA. Data are representative of 2 independent experiments with 6 mice per group. * denotes P<0.05.</p
<i>H</i>. <i>polygyrus/ P</i>. <i>chabaudi</i> co-infection leads to impaired Th2 responses.
<p>A-C). Triple reporter mice were orally infected with 200 <i>H</i>. <i>polygyrus</i> larvae. 6 days post-infection, mice were infected i.p. with 10<sup>5</sup><i>P</i>. <i>chabaudi</i>. At day 8 of <i>P</i>. <i>chabaudi</i> infection (d14 <i>H</i>. <i>polygyrus</i>), mice were harvested. B). Total numbers of CD4<sup>+</sup>CD44<sup>hi</sup><i>Il4</i><sup>gfp+</sup> cells in the mesenteric lymph nodes. Data are representative of 5 independent experiments with 2–4 mice per group. C). IgE measured in the serum by ELISA from 3 pooled experiments. D and E). C57BL/6 mice were infected with 200 <i>H</i>. <i>polygyrus</i> larvae, treated on 2 consecutive days (days 14–15) with pyrantel pamoate (5 mg), infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i>, and re-infected with <i>H</i>. <i>polygyrus</i>. Adult worms in intestine were counted on day 51. Data are representative of 4 independent experiments with 6–7 mice per group. * denotes P<0.05.</p
<i>In vitro</i> Th2 cells produce IFNγ and protect <i>Rag1</i><sup><i>–/–</i></sup>mice during <i>Plasmodium</i> infection.
<p>A). Experimental set-up: 2-week <i>in vitro</i> polarized Th2 cells were FACS sorted as CD4<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup><i>Ifng</i><sup><i>yfp–</i></sup><i>Il17a</i><sup><i>FP635–</i></sup> and transferred i.v. to <i>Rag1</i><sup><i>–/–</i></sup>mice. As a control, a group of <i>Rag1</i><sup><i>–/–</i></sup>mice received naïve CD4<sup>+</sup> T cells. A second control group received no T cells. Recipient mice were infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i> i.p. on day 14 post-transfer and harvested at day 8 post-infection. B). Percent and total number of CD4<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup> and <i>Ifng</i><sup><i>yfp+</i></sup> cells in the spleen, as determined by FACS. C). Serum IFNγ levels determined by ELISA. D). Percent parasitemia was determined by blinded counting of Giemsa-stained blood smears. E and F). Hemoglobin and eHred blood cell counts were measured in peripheral blood by Vetscan. Data is representative of at least 3 independent experiments, with 3–5 mice per group. G). Converted CD4<sup>+</sup>TCRβ<sup>+</sup><i>Ifng</i><sup><i>yfp+</i></sup><i>Il4</i><sup><i>gfp–</i></sup><i>Il17a</i><sup><i>FP635–</i></sup> cells were sorted from pooled spleens of 3 recipient <i>Rag1</i><sup><i>–/–</i></sup>mice at day 8 post-<i>P</i>. <i>chabaudi</i> infection. H). Sorted <i>Ifng</i><sup><i>yfp+</i></sup> cells were cultured <i>in vitro</i> in Th2 conditions for 5 days. ELISAs for IFNγ (H), IL-5 and IL-13 (I) were run on cell supernatants. Error bars represent technical replicates. Data are representative of 3 independent experiments. * denotes P<0.05.</p
TCR stimulation is critical for IFNγ production by Th2 cells.
<p>A–D). OTII <i>Rag1</i><sup><i>–/–</i></sup><i>Il4</i><sup><i>gfp</i></sup> or <i>Il4</i><sup><i>gfp</i></sup> Th2 cells (CD4<sup>+</sup>TCRβ<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup>) were transferred to <i>Rag1</i><sup><i>–/–</i></sup>recipient mice. 14 days later, mice were infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i>, and mice were harvested at d8 post-infection. Representative of 2 independent experiments with 5 mice per group. B). Cytokine expression in transferred cells in the spleen (IFNγ by ICS, <i>Il4</i><sup><i>gfp</i></sup> reporter expression). C). IFNγ protein in serum, measured by ELISA. D). Percent parasitemia, determined by blinded counting of Giemsa-stained blood smears. E–H). Th2 cells (CD4<sup>+</sup>TCRβ<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup><i>Ifng</i><sup><i>yfp-</i></sup><i>Il17a</i><sup><i>FP635-</i></sup>) were transferred to MHC Class II sufficient or deficient <i>Rag1</i><sup><i>–/–</i></sup>recipients. Mice were infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i> at day 14, and mice were harvested at day 8 post-infection. Representative of 2 independent experiments with 5 mice per group. F). Cytokine reporter expression in transferred cells in the spleen. G). IFNγ protein in serum, measured by ELISA. H). Percent parasitemia, determined by blinded counting of Giemsa-stained blood smears. * denotes P<0.05.</p
Th2 cells become IL-12 responsive following adoptive transfer.
<p>A and B). CD4<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup><i>in vitro</i> Th2 cells or naïve CD4 cells were transferred to <i>Rag1</i><sup><i>–/–</i></sup>mice for 2 weeks. CD4<sup>+</sup>TCRβ<sup>+</sup> cells were then sorted from spleens of recipient mice and treated with 10ng/mL IL-12 for 15 minutes (blue) (or untreated, red) and then stained for pSTAT4 by FACS. Representative of 2 independent experiments. C—G). Naïve CD4<sup>+</sup> T cells or <i>in vitro</i> Th2 cells (CD4<sup>+</sup>TCRβ<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup><i>Ifng</i><sup><i>yfp-</i></sup><i>Il17a</i><sup><i>FP635-</i></sup>) were transferred to <i>Rag1</i><sup><i>–/–</i></sup>recipient mice for 14 days. Mice were infected with <i>P</i>. <i>chabaudi</i> and harvested at day 8 post-infection. Mice were treated i.p. with 0.5mg of anti-IL12 at days -1, 6, 13, and 19. D–F). Cytokine reporter expression in transferred cells in the spleen, with or without anti-IL-12 treatment. G). Percent parasitemia, determined by blinded counting of Giemsa-stained blood smears. Data are representative of 2 independent experiments with 3–6 mice per group. * denotes P<0.05.</p
Blockade of IL-12 and IFNγ prevents optimal IFNγ production by Th2 cells.
<p>A–C). <i>In vitro</i> Th2 (CD4<sup>+</sup>TCRβ<sup>+</sup><i>Il4</i><sup>gfp+</sup>) cells were transferred to <i>Rag1</i><sup><i>–/–</i></sup>recipient mice for 14 days. Mice were infected with <i>P</i>. <i>chabaudi</i> and harvested at d8 post-infection. Mice were treated i.p. with 0.5mg anti-IL12 and anti-IFNγ at days -1, 6, 13, and 19. B). IFNγ production by transferred Th2 cells in the spleen, as determined by ICS. C). <i>Il4</i><sup><i>gfp</i></sup> expression in transferred cells in the spleen. Data representative of 2 independent experiments with 5 mice per group. * denotes P<0.05.</p
Converted Th2 cells are transcriptionally similar to Th1 cells.
<p>Purified <i>in vitro</i> Th2 cells (CD4<sup>+</sup>TCRβ<sup>+</sup><i>Il4</i><sup><i>gfp+</i></sup><i>Ifng</i><sup><i>yfp-</i></sup><i>Il17a</i><sup><i>FP635-</i></sup>) or naive CD4<sup>+</sup> T cells were sorted for RNA or transferred to <i>Rag1</i><sup><i>–/–</i></sup>mice. Recipients were then infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i>, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004994#ppat.1004994.g002" target="_blank">Fig 2</a>. CD4<sup>+</sup>TCRβ<sup>+</sup><i>Ifng</i><sup><i>yfp+</i></sup><i>Il4</i><sup><i>gfp-</i></sup><i>Il17a</i><sup><i>FP635–</i></sup> cells were then sorted from spleens of recipient mice at day 8 post-infection for RNA. RNA sequencing and IPA analysis was performed on the four cell populations. Data were expressed relative to naïve in A and B. A and B). Venn diagram and heatmap generated from differentially regulated genes (P<0.05, 2-fold relative to naïve T cells) of Th2 cells and Th1 cells, highlighting 1899 genes commonly expressed, which were not changed in Th2 cells. Th2 cells and converted Th2 cells shared 117 differentially regulated genes, which were not expressed in Th1 cells. C, D, and F). Normalized RNA-Seq reads of indicated genes. E). Upstream pathways analysis in Ingenuity Pathways Analysis (IPA) identified IL-12, type 1 IFN, and IFNγ as potential upstream regulators of converted Th2 cells. Samples were generated from 3 biological replicates (each sample representing cells from a single donor mouse).</p
IFNγ production by Th2 cells does not depend on lymphopenia.
<p>A). Th2 cells generated from <i>Il4</i><sup><i>gfp</i></sup> mice were polarized <i>in vitro</i> for 2 weeks, sorted as CD4<sup>+</sup>TCRβ<sup>+</sup><i>Il4</i><sup>gfp+</sup>, and 2.5x10<sup>6</sup> were transferred to OTII <i>Rag1</i><sup><i>–/–</i></sup>recipient mice. Control groups received no T cells or sorted naïve T cells. 2 days post-transfer, mice were infected with 10<sup>5</sup><i>P</i>. <i>chabaudi</i>. B and C). Cytokine production in donor CD45.1<sup>+</sup> or host cells in spleens, day 8 post-infection with <i>P</i>. <i>Chabaudi</i>, as determined by intracellular cytokine staining. D). IFNγ protein in serum, measured by ELISA. Data are representative of 2 independent experiments with 3–4 mice per group.</p