IRF7-deficient cDCs show hyper-responsiveness to PAM₃CSK₄<i>in vivo</i> even in an IRF7-sufficient environment.

<p>B6.<i>Irf7</i>^−/− or C57BL/6 microchimeric mice were generated by bone marrow transfer into Busulfan-treated congenic B6J.CD45.1 hosts. <b>A</b>. 7 to 14 days post- engraftment, microchimeric mice bearing B6.<i>Irf7</i>^−/− or C57BL/6 chimeric cell populations received 5 µg/mouse PAM₃CSK₄ intravenously. Splenic CD11c^hiMHCII^hi cDC compartments in microchimeric animals were comprised of endogenous (CD45.1⁺) and chimeric (CD45.1^-) cell populations (<b>B</b>). After 24 hours, activation of splenic cDCs after PAM₃CSK₄ administration <i>in vivo</i> was assessed by quantifying changes in surface expression of CD80 (<b>C</b>) and CD86 (<b>D</b>) by flow cytometry. Flow plots in <b>B</b> are representative. Data in <b>C</b> and <b>D</b> show the mean fold change in indicated surface protein ± SEM on endogenous (open bars) and chimeric (black bars) cDCs after PAM₃CSK₄ injection, compared to the same populations of cDCs in mice receiving PBS. n = 4 per group, representative of two experiments. ** =  p<0.01.</p

IRF7 Regulates TLR2-Mediated Activation of Splenic CD11c^hi Dendritic Cells

<div><p>Members of the Interferon Regulatory Factor (IRF) family of transcription factors play an essential role in the development and function of the immune system. Here we investigated the role of IRF7 in the functional activation of conventional CD11c^hi splenic dendritic cells (cDCs) <em>in vitro</em> and <em>in vivo</em>. Using mice deficient in IRF7, we found that this transcription factor was dispensable for the <em>in vivo</em> development of cDC subsets in the spleen. However, IRF7-deficient cDCs showed enhanced activation in response to microbial stimuli, characterised by exaggerated expression of CD80, CD86 and MHCII upon TLR2 ligation <em>in vitro</em>. The hyper-responsiveness of <em>Irf7</em>^−/− cDC to TLR ligation could not be reversed with exogenous IFNα, nor by co-culture with wild-type cDCs, suggesting an intrinsic defect due to IRF7-deficiency. <em>Irf7</em>^−/− cDCs also had impaired capacity to produce IL-12p70 when stimulated <em>ex vivo</em>, instead producing elevated levels of IL-10 that impaired their capacity to drive Th1 responses. Finally, analysis of bone marrow microchimeric mice revealed that cDCs deficient in IRF7 were also hyper-responsive to TLR2-mediated activation <em>in vivo</em>. Our data suggest a previously unknown function for IRF7 as a component of the regulatory network associated with cDC activation and adds to the wide variety of situations in which these transcription factors play a role.</p> </div

IRF7-deficient splenic cDCs are hyper-responsive to a TLR2 agonist <i>in vitro</i>. A

<p>. CD11c^hi cDCs were sorted to <b>∼</b>99% purity from the spleens of C57BL/6 and B6.<i>Irf7</i>^−/− mice. Cells were cultured in triplicate at 1×10⁶ cells/ml in the presence of 10 µg/ml PAM₃CSK₄. At the indicated times post-stimulation, cells were removed and assessed by flow cytometry for expression of CD80, CD86 and MHCII. Representative flow plots showing progressive cDC activation in terms of CD86 and MHCII expression are shown in <b>B</b>, fold increases in surface expression of CD80, CD86 and MHCII on cDCs at the indicated time point over unstimulated cDCs are shown in <b>C</b>, <b>D</b> and <b>E</b>, respectively. <b>C</b>–<b>E</b> show mean fold increase ± SEM in surface expression of indicated proteins on cDCs from C57BL/6 (open bars) or B6.<i>Irf7</i>^−/−(closed bars) mice, compared to unstimulated cDCs from the same strain. Data are pooled from three individual experiments. * = p<0.05 ** =  p<0.01, *** = p<0.001.</p

Exaggerated CD86 expression after TLR2 stimulation occurs in the presence of IRF7-sufficient cDCs or exogenous IFNα.

<p>A. Representative purity of splenic CD11c^hi cDCs sorted from C57BL/6, B6.<i>Irf7</i>^−/− and congenic B6J.CD45.1 mice. B. Representative staining of cDCs from B6.<i>Irf7</i>^−/− and B6J.CD45.1 mice after co-culture at <b>∼</b>50∶50 ratio in the presence of 10 µg/ml PAM₃CSK₄. C. The fold increase in expression of CD86 after TLR2 stimulation for cDCs from either strain was determined by flow cytometry at the indicated times post-stimulation. D. C57BL/6 or B6.<i>Irf7</i>^−/− cDCs were cultured in the presence of 10 µg/ml PAM₃CSK₄ and 1000 U/ml IFNα. At the indicated times post- stimulation, cells were removed and assessed by flow cytometry expression of CD86. Data are presented as mean fold increase ± SEM in surface expression of CD86 relative to unstimulated cDCs from the same strain. Data are from two experiments. * = p<0.05 ** =  p<0.01, *** = p<0.001.</p

Cytokine production by IRF7-deficient cDCs <i>in vitro</i>.

<p>CD11c^hi cDCs were sorted from spleens of C57BL/6 and B6.<i>Irf7</i>^−/− mice and cultured for 24 hours in the presence of 10 µg/ml PAM₃CSK₄, 1 µg/ml LPS and 1000U/ml IFNα, or combinations thereof. Supernatants were assessed by ELISA for presence of IL-12p70 (<b>A</b>&<b>C</b>) or IL-10 (<b>B</b>&<b>D</b>). Data show mean concentration of cytokine from triplicate wells ± SEM and are representative of two experiments. ** = p<0.01 *** = p<0.001.</p

Splenic cDC subset development and TLR2 expression is independent of IRF7.

<p>The presence of splenic CD11c^hiMHCII^hi cDCs in C57BL/6 and B6.<i>Irf7</i>^−/− mice was determined by flow cytometry (<b>A</b> & <b>B</b>). CD4⁺, DN and CD8α⁺ cDC subset frequency was determined in the CD11c^hiMHCII^hi compartment of wildtype and IRF7-deficient animals (<b>C</b> & <b>D</b>). Expression of TLR2 on CD11c^hiMHCII^hi cells from C57BL/6 and B6.<i>Irf7</i>^−/− mice was determined by flow cytometry (<b>E</b> & <b>F</b>). Flow plots and histograms in <b>A</b>, <b>C</b> and <b>E</b> are representative, data in <b>B</b>, <b>D</b> and <b>F</b> show mean frequency ± SEM of indicated cell population in C57BL/6 (open bars) and B6.<i>Irf7</i>^−/− (closed bars) mice. Histograms in <b>E</b> are gated on CD11c^hiMHCII^hi cells gated as in <b>A</b> and show specific TLR2 staining (open line) compared to isotype control antibody (filled histogram). Data are from n = 4–5 mice per group and representative of three separate experiments.</p

The recruitment of B cells to <i>L. donovani</i>-induced hepatic granulomas.

<p>B^green/T^red mice (n = 6) were infected with <i>L. donovani</i> and at d14 (<b>A</b>), d21 (<b>B</b>) and d28 (<b>C</b>) p.i. liver explants were imaged using 2-photon microscopy to visualize T cells and B cells. <b>D.</b> Number of T cells and B cells at d14 (open bars) d21 (grey bars) and d28 (black bars) p.i. derived from 25–35 hepatic granulomas per time point from 2 mice per group. Data are shown as mean ± SEM along with the T cell: B cell ratio. <b>E.</b> B cells aggregate in hepatic granuloma. <b>F.</b> The number of B cell aggregates per granuloma was determined from 25–35 granulomas (mean ± SEM). <b>G.</b> C57BL/6 mice were infected with <i>L. donovani</i> (n = 3). Frozen sections were labeled with B220 (red), CD21/35 (green) and counterstained with DAPI (blue, except left panel). 60 hepatic granulomas were imaged. Control staining of spleen was performed (left panel).</p

Endogenous B cell behavior in hepatic granulomas.

<p><b>A.</b> B cell velocity in granulomas of d21-infected B^green/T^red mice. Each symbol represents an individual B cell. Bar shows median velocity calculated from 71 B cells in 12 granulomas imaged in two mice. Liver explants tissue from B^green/T^red mice was imaged using 2-photon microscopy and B cell-T cell contacts (<b>B</b>) identified by static imaging. The second harmonic signal is also visible (blue). Data represents the mean ± SEM % B cells interacting with T cells (<b>C</b>) and were derived from 25 and 35 granulomas from 2 mice per time point.</p

Naïve and immune B cells are recruited into hepatic granulomas.

<p><b>A.</b> B cells were CD19⁺ MACS-purified (>90% B220⁺) from naïve and d21-infected mice and labeled with Hoechst and CFSE, respectively. <b>B.</b> CCR6 expression on CD19⁺ B cells from naïve and d21-infected mice (n = 4). <b>C.</b> Data showing frequency of CCR6⁺CD19⁺ B cells in naïve (grey bars) and infected (black bars) mice. <b>D.</b> Protocol for mixed adoptive transfer of labeled B cells into T^red mice. <b>E and F.</b> Maximum projection image of transferred labeled B cells in granuloma of T^red mouse, shown with (E) and without (F) DsRed channel switched on. <b>G.</b> Number of labeled naïve (grey bars) and immune (black bars) B cells found per granuloma. Data are shown as mean ± SEM for 50 granulomas derived from imaging livers of two mice.</p

CD4⁺ Recent Thymic Emigrants Are Recruited into Granulomas during <i>Leishmania donovani</i> Infection but Have Limited Capacity for Cytokine Production

<div><p>Recent thymic emigrants (RTEs) represent a source of antigen-naïve T cells that enter the periphery throughout life. However, whether RTEs contribute to the control of chronic parasitic infection and how their potential might be harnessed by therapeutic intervention is currently unclear. Here, we show that CD4⁺ recent thymic emigrants emerging into the periphery of mice with ongoing <i>Leishmania donovani</i> infection undergo partial activation and are recruited to sites of granulomatous inflammation. However, CD4⁺ RTEs displayed severely restricted differentiation either into IFNγ⁺ or IFNγ⁺TNFα⁺ effectors, or into IL-10-producing regulatory T cells. Effector cell differentiation in the chronically infected host was not promoted by adoptive transfer of activated dendritic cells or by allowing extended periods of post-thymic differentiation in the periphery. Nevertheless, CD4⁺ RTEs from infected mice retained the capacity to transfer protection into lymphopenic RAG2^-/- mice. Taken together, our data indicate that RTEs emerging into a chronically inflamed environment are not recruited into the effector pool, but retain the capacity for subsequent differentiation into host protective T cells when placed in a disease-free environment.</p></div