Schematic diagram of the effects of treatment with RTK inhibitors during <i>L.donovani</i> infection.

<p>Cartoon to depict processes inhibited by the broad spectrum RTKi (Sm) and the selective Nrtk2 inhibitor (ANA-12) during infection-associated neovascularization of white pulp.</p

Altered gene expression in F4/80^hiCD11b^lo cells compared to control macrophages.

<p>Altered gene expression in F4/80^hiCD11b^lo cells compared to control macrophages.</p

Infection-induced expression of Bdnf and Ntrk2 in spleen.

<p>Bdnf (black line) was expressed at significantly higher levels by F4/80^hiCD11b^lo cells compared to the other MP populations tested, as revealed by delta median fluorescence intensity levels (ΔMFI) using intracellular flow cytometry (A). Isotype control staining is highlighted by the solid grey histogram. Ntrk2 (red) expression in naïve mouse spleen is found in a subset of splenic red pulp macrophages (F4/80⁺, white) in naïve mouse spleen (B). Scale bar = 200microns. In <i>L. donovani</i> infected mice splenic white pulp vessels expressed Ntrk2 (C: red). F4/80⁺ (white) CD11c⁺ (green) MPs are found in close association with Nrtk2⁺ vessels (C: merge and inset). Endothelial cells (Meca-32, green) but not smooth muscle actin-positive cells (SMA, red) in white pulp express Nrtk2 (white, D and inset). All sections were stained with DAPI (blue). Scale bars = 100 microns and 50 microns in high magnification merge image.</p

Comparison of growth factor mRNA abundance in F4/80^hiCD11b^lo cells compared to other MP populations isolated from <i>L. donovani</i>-infected spleens.

<p>Comparison of growth factor mRNA abundance in F4/80^hiCD11b^lo cells compared to other MP populations isolated from <i>L. donovani</i>-infected spleens.</p

F4/80^hiCD11b^lo MPs are located in close proximity to white pulp vasculature and possess angiogenic properties.

<p>F4/80^hiCD11b^lo cells (FITC-dextran, green; yellow arrows) identified in fresh frozen sections as located either in or bordering the white pulp (A,B). Red pulp F4/80⁺ macrophages are also shown (white). F4/80^hiCD11b^lo cells (FITC-dextran, green) were found in close association with endothelial cells (C, E; Meca-32, magenta) but not follicular dendritic cells (D; FDCM1, red). High magnification image of area depicted by yellow circle in e (F). All sections were counterstained with DAPI (blue). Scale bars = 100 microns. F4/80^hiCD11b^lo cells, but not other splenic MPs tested, drive SVEC4–10 endothelial cell tube formation on a gelled basement membrane extract (G). Representative images are shown. An optimised cocktail of growth factors (EGM) was used as a positive control. Quantitative analysis of SVEC4–10 mean loop area (H) and difference in tube length (I), in the presence of each MP population or control growth factors. Mean loop area in the absence of growth factors or MPs is shown as a dotted line in h. *p = 0.05, **p = 0.02. Images were analysed using WimTube software and data are expressed as mean ± SEM from at least three independent experiments.</p

Phenotypic analysis of F4/80^hiCD11b^locells.

<p>Splenocytes isolated from <i>L.donovani</i> infected mice at 28 days post infection were stained with a panel of myeloid cell markers. CD11c⁺MHCII⁺F4/80^hiCD11b^lo MPs were positive for CD80, CD68 and a small proportion (15%) expressed CD115 (A: isotype control, filled grey histogram). Strong SIGNR1 (white) and FITC-dextran (green) labeling co-localise in the marginal zone of naïve mice (B), whereas in infected mice FITC-dextran⁺ (green) cells had low expression of SIGNR1 (white). FITC-dextran⁺ (green) cells were negative for GR1 (white) in both naïve and infected mice (C). Scale bars = 100 microns.</p

Sensitivity of mononuclear phagocytes in <i>L.donovani</i> infected mice to RTKi treatment.

<p>CD11c⁺MHCII⁺ MPs in d28 <i>L. donovani</i> infected mice were distinguished on the basis of F4/80 and CD11b expression, forward/side scatter profile and morphology into: (A) large CD11c⁺MHCII⁺F4/80^hiCD11b^lo (F4/80^hiCD11b^lo) cells with macrophage-like morphology, of which ∼80% harbored intracellular parasites; (B) slightly smaller CD11c⁺MHCII⁺F4/80^loCD11b^hi (F4/80^loCD11b^hi) cells with classic macrophage morphology, of which <5% harbored parasites; (C) much smaller CD11c⁺MHCII⁺F4/80^loCD11b^lo (F4/80^loCD11b^lo) cells with dendritic cell-like morphology and no observable parasites. Representative dot plots show pre-sorted populations with ellipsoid sort gates based on F4/80 and CD11b expression. Scale bar in micrographs = 10microns. The frequency and absolute numbers of each population is given in the right hand panels in naïve mice, infected mice and infected mice treated orally with sunitinib (Sm) for 7 days. P values = * <0.05, ** <0.008, ***<0.001, ns = not significant.</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

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

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