IL-27 secreted from <i>T gondii</i> infected-DC promotes T-bet⁺ Th1-Treg cell differentiation through stimulating IL-27R on Treg cells.

<p><b>(A, B)</b> CD4⁺CXCR3^-GFP⁺ Treg cells isolated from naive Foxp3^GFP mice were cultured with DCs from <i>CD11c</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/fl</i> or WT control mice day 6 post <i>T</i>. <i>gondii</i> infection. Isotype control or IL-27 neutralizing antibodies were added at the beginning of culture. <b>(C, D)</b> CD4⁺CXCR3^-CD25^hi Treg cells isolated from naive IL-27Rα-deficient or WT control mice were cultured with DCs from WT mice day 6 post <i>T</i>. <i>gondii</i> infection in the presence of antibodies as indicated. FACS plots and histograms represent two or three independent experiments (*p<0.05; **p<0.01; ***p<0.001). </p

Minimal role of Treg cell-intrinsic IFNγ signaling in promoting T-bet⁺CXCR3⁺ Treg cells.

<p>FACS analysis and frequencies of T-bet⁺ or CXCR3⁺ cells in Foxp3⁺CD4⁺ Treg cells in <b>(A)</b> the spleen (Spl) and <b>(B)</b> the lamina propria (LP) of small intestine. FACS analysis and frequencies of T-bet⁺ or IFNγ⁺ Foxp3^-CD4⁺ Teff cells in <b>(C)</b> Spl and <b>(D)</b> LP isolated from <i>Foxp3</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/fl</i> or <i>Foxp3</i>^<i>cre</i><i>IFNγR2</i>^<i>+/+</i> mice. FACS plots shown are representative of three independent experiments.</p

Dispensable role of IFNγR in DC maturation and function at steady state.

<p>FACS analysis of MHC class II and CD86 in <b>(A)</b> CD11c⁺ DCs with or without LPS stimulation for 24hr from <i>CD11c</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/f</i> and WT control mice. <b>(B)</b> Proliferation of OTII T cells co-cultured with DCs isolated from indicated mice pulsed with different does of OVA protein was shown by CFSE dilution. <b>(C)</b> Gene expression volcano plot, with—log 10 of the p value on the y axis and log 2 fold change on the x axis, such that genes with higher expression in WT DCs are on the right and genes with higher expression in KO DCs are on the left. <b>(D)</b> Signals (log2 intensity) of individual probe sets of <i>Ifngr2</i> gene and their locations on corresponding exons. All data are representative of three independent experiments. (*p<0.05).</p

IFNγR2-deficient DCs failed to produce IL-27 as well as other molecules potentially important for Th1-Treg cell differentiation during <i>T</i>. <i>gondii</i> infection.

<p><b>(A)</b> GSEA of T cell activation pathway genes (GO:0050863) between IFNγR2-deficient and—sufficient DCs [normalized enrichment score (NES) = 1.7312976, nominal P = 0.0, false discovery rate (FDR) < 25%] 8 days post <i>T</i>. <i>gondii</i> infection. <b>(B)</b> Comparison of genes with significant difference (p < 0.05) is shown of normalized expression values of candidate Th1-associated genes. Data are row normalized and presented as a heat map. <b>(C)</b> Expressions of IL-27(p28) and CXCL9 are further confirmed and quantified by qRT-PCR. <b>(D)</b> FACS analysis and <b>(E)</b> frequencies of CXCL9⁺ or IL-27⁺ CD11b⁺CD11c^int or CD11b^-CD11c⁺ cells in LP isolated from indicated mice 8 days post <i>T gondii</i> infection. Data are representative of three independent experiments (*p<0.05; **p<0.01; ***p<0.001).</p

DC-derived IL-27 is critical for maintaining normal T-bet⁺ Th1-Treg cell population in both physiological and <i>T</i>. <i>gondii</i> infection settings.

<p>FACS analysis and frequencies of T-bet⁺ cells within Foxp3⁺CD4⁺ T cell population from spleen and LP in <i>CD11c</i>^<i>cre</i><i>IL27p28</i>^<i>fl/fl</i> mice and WT littermate controls <b>(A)</b> at steady state or <b>(B)</b> 8 days after <i>T</i>. <i>gondii</i> infection. FACS plots are representative of three independent experiments (*p<0.05; **p<0.01; ***p<0.001).</p

Reduced T-bet⁺ Th1-Treg cells in <i>CD11c</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/fl</i> mice resulted in unrestrained IFNγ-mediated Th1 inflammation during <i>T</i>. <i>gondii</i> infection.

<p><b>(A,B)</b> Histological assessment of ileum from infected <i>CD11c</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/fl</i> and WT control mice (n = 12). <b>(C)</b> ELISA analysis of serum IFNγ levels and <b>(D)</b> PCR analysis of parasite burden in LP at days 8 after infection. <b>(E)</b> Frequencies of total Foxp3⁺ Treg cells from LP in <i>CD11c</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/fl</i> and WT control mice at day 8 post <i>T</i>. <i>gondii</i> infection. FACS analysis and frequencies of T-bet⁺ cells in Foxp3⁺CD4⁺ Treg cells and IFNγ⁺ cells in Foxp3^-CD4⁺ Teff cells from LP in <i>CD11c</i>^<i>cre</i><i>IFNγR2</i>^<i>fl/fl</i><b>(F)</b> without or <b>(G)</b> with Treg cell collapse and their corresponding WT control mice at day 8 post <i>T</i>. <i>gondii</i> infection. FACS data are representative of three to four independent experiments. (*p<0.05; **p<0.01; ***p<0.001).</p

Blimp-1 inhibits the control of parasite growth during experimental malaria.

<p><b>(A)</b><i>Prdm1</i>^fl/fl and <i>Prdm1</i>^ΔT C57BL/6 mice were infected with PcAS and peripheral parasitemia was measured beginning at day 4 post infection (p.i.). (<b>B)</b> Splenic CD4⁺ T cell responses were assessed by flow cytometry, as indicated, at day 15 p.i.. The frequency and number of activated CD4⁺ T cells (CD11a⁺CD49d⁺) (<b>C)</b>, Th1 cells (IFNγ⁺Tbet⁺) (<b>D)</b> and Tr1 cells (IFNγ⁺IL-10⁺) (<b>E)</b> were measured. Representative of 3 independent experiments, mean ±SEM, n = 5 in each group in each experiment, **p<0.01, *p<0.05, Mann-Whitney U test.</p

IFNγ signalling drives TNF production and contributes to splenic pathology.

<p>C57BL/6 IFNγR^-/- and WT mice were infected with <i>L</i>.<i>donovani</i>. (<b>A</b>) Representative images of MZMs at day 28 p.i. (objective 20x, scale bars 500 μm; staining as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005398#ppat.1005398.g004" target="_blank">Fig 4D</a>). Spleen weights were measured at times indicated (<b>B</b>) and the number of MZMs per mm² of spleen tissue was determined at day 28 p.i. (<b>C</b>), as described in the Materials and Methods. Spleen and liver parasite burdens were measured (<b>D</b>) at day 28 p.i. as was serum TNF and IFNγ levels (<b>E</b>) Representative of 3 similar experiments, mean ±SEM, n = 6 in each group in each experiment, **p<0.01, *p<0.05, Mann-Whitney U test.</p

Blimp-1 controls CD4⁺ T cell derived IL-10 production.

<p><b>(A)</b><i>Prdm1</i>^<i>GFP/+</i> (black-filled histograms) and WT (grey-filled histograms) mice were infected with PcAS. Blimp-1/GFP expression measured on CD4⁺ T cells producing IL-10, IFNγ and TNF at day 15 p.i.. (<b>B)</b> Kinetics of Blimp-1/GFP expression measured in various cytokine producing CD4⁺ T cells, as indicated, beginning at day 5 p.i.. <b>(C)</b><i>Prdm1</i>^<i>GFP/+</i> and WT mice were infected with <i>L</i>. <i>donovani</i> and the kinetics of Blimp-1 expression measured in the spleen and liver. (<b>D)</b> FACS plots showing gated CD4⁺ T cell IL-10 and IFNγ production from <i>Prdm1</i>^<i>GFP/+</i> mice treated with 500 μg of polyclonal rat IgG (black bars in (<b>E</b>)) or anti-IL-12 (C17.8; white bars in (<b>E</b>)), as indicated, prior to infection and every 3 days following infection until day 14 p.i.. (<b>E</b>) The frequencies of IL-10 and IFNγ-producing CD4⁺ and CD8⁺ T cells, as well as Blimp-1 expressing CD4⁺ and CD8⁺ T cells, were determined by flow cytometry at day 14 p.i.. Representative of 3 similar experiments, mean ±SEM, n = 5 in each group in each experiment, **p<0.01, Mann-Whitney U test.</p

Blimp-1 inhibits the control of parasite growth during experimental visceral leishmaniasis.

<p><i>Prdm1</i>^fl/fl and <i>Prdm1</i>^ΔT C57BL/6 mice were infected with <i>L</i>. <i>donovani</i> and spleen (<b>A</b>) and liver (<b>D</b>) parasite burdens were measured at times indicated. The frequency of Th1 and Tr1 cells in the spleen (<b>B</b>) and liver (<b>E)</b> were measured by flow cytometry. Organ weights and frequencies of TNF producing CD4⁺ T cells in the spleen (<b>C</b>) and liver (<b>F)</b> were also assessed, as were serum TNF and IFNγ levels (<b>G</b>). Antigen-specific Th1 cell frequency (<b>H</b>), IFNγ, TNF and IL-10 production (<b>I</b>) were measured in splenocytes after 24 hours of culture in the presence of parasite antigen at times indicated. In all panels, closed shapes represent <i>Prdm1</i>^fl/fl mice, while open shapes indicate <i>Prdm1</i>^ΔT littermates. Representative of 5 similar experiments, mean ±SEM, n = 5–6 in each group in each experiment, **p<0.01, *p<0.05, Mann-Whitney U test.</p