LCMV Infection Induces Focal Destructive Lesions within the Developing Brains of Humans and Rats

<div><p>(A) Head CT scan from a 4-mo-old child with congenital LCMV infection. The scan reveals bilateral asymmetric regions of encephalomalacia (asterisks), strongly suggestive of a focal destructive process. Note also the periventricular calcifications (arrow), characterisitic of a prenatal viral infection.</p><p>(B) Section (50-μm-thick) through the cerebellar cortex of a neonatal rat infected with LCMV. The section has been immunohistochemically stained for LCMV antigens. The virus infects both Purkinje cells (arrows) and granule cells (arrowheads).</p><p>(C) Nissl-stained section (2-μm-thick) through the cerebellar vermis of an uninfected (control) 30-d-old rat. The ten lobules of the cerebellar vermis (I–X) are labelled according to the system of Larsell [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030149#ppat-0030149-b084" target="_blank">84</a>].</p><p>(D) Section (2-μm-thick) through the cerebellar vermis of a 30-d-old rat infected 3 wk earlier with LCMV. The dorsal cerebellum has undergone a destructive process (arrows). Most of lobules V, VI, VII, and VIII have been obliterated, while lobules I, II, III, and X have been relatively spared.</p><p>(E) Section (50-μm-thick) through the cerebellar cortex of a 14-d-old rat infected 10 d earlier with LCMV. The animal was sacrificed at the time that acute destruction of the cerebellum was occurring. The section has been immunohistochemically stained for CD8+ antigen, which labels a subset of lymphocytes. Note the dense infiltration of CD8+ lymphocytes (arrows).</p><p>Magnification bars represent 1 cm in (A), 100 um in (B), 500 um in (C), 500 um in (D), and 100 um in (E).</p></div

LCMV Infection Disrupts Neuronal Migration in the Developing Brain of Humans and Rats

<div><p>(A) MRI scan of a 3-y-old child with congenital LCMV infection. The MRI scan demonstrates microencephaly and a deficit of white matter (arrowheads) with a compensatory enlargement of the lateral ventricles (asterisks). There is also a diminished number of cortical sulci and an abnormally smooth cortical surface (white arrow). This is strongly suggestive of pachygyria, a developmental defect due to abnormal neuronal migration.</p><p>(B and C) are 2-μm-thick sections through the cerebellar cortex of uninfected control (B) and LCMV-infected (C) rats.</p><p>(B) Normal cerebellar cortex from a control (uninfected) adult rat demonstrating the trilaminar cytoarchitecture of the cortex, which consists of the molecular layer (M), Purkinje cell layer (P), and granule cell layer (G). Within the molecular layer, a few stellate cells and basket cells (arrowheads) are normally present. In contrast, granule cells (arrows) have migrated through the molecular layer to the granule cell layer. Granule cells no longer reside in the molecular layer in the normal cerebellum.</p><p>(C) Cerebellar cortex from an adult rat infected during early postnatal life with LCMV. Many granule cells (arrows) remain abnormally placed within the molecular layer. As a result of LCMV infection, these neurons have failed to migrate properly to their normal location within the granule cell layer and remain permanently ectopic within the molecular layer.</p><p>Magnification bars represent 100 um in (B and C).</p></div

The Cellular Targets of LCMV within the Cerebral Cortex Depend on Host Age

<div><p>Shown are 50-μm-thick sections through the cerebral cortex of rats infected on PD 1 (A), PD 4 (C), or PD 21 (E). In each case, the animals were sacrificed 25 d post-inoculation, and the brain sections were immunohistochemically stained for LCMV antigens.</p><p>Inoculation on PD 1 leads to infection of cerebral cortical neurons (arrows) and astrocytes (arrowheads). Note that a greater proportion of neurons are infected in superficial cortical layers than in deep cortical layers.</p><p>Inoculation on PD 4 leads to infection of astrocytes alone (arrowheads). Whereas countless neurons were infectable 3 d earlier, virtually no neurons are infectable by PD4.</p><p>Inoculation on PD 21 leads to infection of neither neurons nor astrocytes.</p></div

Congenital LCMV Infection of Humans Induces Periventricular Calcifications

<div><p>The neonatal rat model demonstrates that periventricular neurons are selectively vulnerable to infection with LCMV.</p><p>(A) Head CT scan from an infant with congenital LCMV infection. The scan reveals microencephaly and prominent periventricular calcifications (arrows). In addition, this scan reveals an abnormal cortical gyral pattern (arrowheads), suggestive of disturbed cortical neuronal migration.</p><p>(B) Horizontal section (50-μm-thick) through a 49-d-old rat brain immunohistochemically stained for LCMV. The rat was inoculated as a neonate with LCMV. Infection is localized to the periventricular region (arrows). L = lateral ventricle, S = septum, BG = basal ganglia.</p><p>(C) Higher magnification of the boxed area in (B) shows that the infected cells are neuronal in morphology. Viral antigen is present in neuronal cell bodies (arrows) and neurites (arrowheads).</p><p>Magnification bars represent 500 um in (B) and 100 um in (C).</p></div

M133 Tconv and Treg proliferation occurs in DCLN and CLN after FTY720 treatment.

<p>(<b>A</b>) Experimental design. M133 Tregs and M133 Tconv were mixed at a 1∶1 ratio and labeled with Violet. 1.5×10⁵ cells were transferred to Thy1 congenic mice one day prior to infection with rJ2.2. Mice were treated with FTY720 or saline 0.5 hour prior to infection and on days 1, 2, 3 p.i. (<b>B</b>) Representative plots showing Violet dilution at day 4 p.i. Numbers are percentage of divided Tregs (upper) or Tconv (lower). (C) Summary of data from three independent experiments with three mice in each.</p

IL-12 inhibits IL-2 expression by T cells.

<p>Lymphocytes were prepared from naïve <i>Foxp3^gfp</i> mice and treated with anti-CD3 mAb and IL-12 or IFN-γ. (A). The fraction of CD4 and CD8 T cells expressing IL-2 was diminished after 48 and 66 hr. Data are from 3–5 independent experiments. (B) Levels of IL-2, measured by ELISA, were reduced in IL-12 but not IFN-γ-treated cultures at 48 hr. Data are representative of 2 independent experiments performed in triplicate for stimulation, and each sample plated in duplicate for ELISA. *<i>P</i><0.05, ***<i>P</i><0.001, Student’s two-tailed un-paired <i>t</i> tests.</p

IL-12 does not induce apoptosis in Tconvs or Tregs.

<p>Lymphocytes were prepared from <i>Foxp3^gfp</i> mice and cultured in the presence of anti-CD3 mAb with or without IL-12 for 72 hr. (A) Gating strategy to identify live CD4 T cells is shown. (B) Representative FACS plots are shown. (C) Summary of data from 3 independent experiments, each performed in triplicate, is shown. ***<i>P</i><0.01, Student’s two-tailed un-paired <i>t</i> tests.</p

Initial proliferation of M133 Tconv occurs in the DCLN.

<p>(<b>A</b>) Experimental design. 1×10⁵ CFSE labeled M133 Tconv (Treg-depleted CD4 T cells) were transferred to Thy1 congenic mice one day prior to rJ2.2 infection. (<b>B</b>) Representative plots showing proliferation of transferred cells and CXCR3 expression. (<b>C</b>) CFSE levels on transferred cells at several times p.i. MFI, mean fluorescence intensity. (<b>D, E</b>) Frequency (<b>D</b>) and numbers (<b>E</b>) of M133 Tconv at the indicated times after infection. The data are representative of four independent experiments with 3 mice per time point in each.</p

IL-27 inhibits IL-2 production, but only modestly affects IL-2R expression by Tconv and CD8 T cells.

<p>Lymphocytes were prepared from naïve B6 mice and treated with anti-CD3 mAb and IL-27 (100 ng/ml) for 66 hr. (A) The fraction of CD4 and CD8 T cells expressing IL-2 was diminished. Data are from 3 independent experiments. (B) IL-27 diminished Foxp3 GMFI in Tregs. Samples were analyzed in triplicate. Data are from one experiment representative of 3 independent experiments. (C, D, E) Lymphocytes were labeled with CFSE prior to incubation. IL-27 reduced proliferation of Tregs and Tconvs but not CD8 T cells (C, D), resulting in a modest decrease in the percentage of Foxp3⁺ Tregs (E). Data are from 3 independent experiments. (F) IL-27 treatment resulted in small changes in CD25 expression on Tregs and CD8 T cells but not Tconvs and in a small decrease in CD122 expression on all three cell types. Data are representative of 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Student’s two-tailed un-paired <i>t</i> tests (A, B, E) or paired <i>t</i> tests (D).</p

Transferred M133 Tregs enhance survival and diminish the M133 Tconv immune response in the brain.

<p>10⁵ M133 Tregs, bulk Tregs or B6-derived Foxp3⁻CD4 T cells (control group) were transferred to Thy1 congenic mice one day prior to rJ2.2 infection. (<b>A</b>) M133 Treg numbers in spleen, DCLN and brain of recipient mice from days 0 to 41 p.i. (<b>B–D</b>) Survival (<b>B</b>), weight loss (<b>C</b>) and viral titers in the brains (<b>D</b>) of recipient mice were monitored. 18–22 mice in 4 independent experiments were analyzed for survival and weight loss. In (<b>C</b>), *<i>P</i><0.05, **<i>P</i><0.01 weights of M133 Treg recipients compared to mice that received bulk Tregs or control cells; (<b>E–G</b>) Lymphocytes were prepared from brains of recipient mice at day 7 p.i. and stimulated with the indicated peptides. (<b>E</b>) Frequencies of S510 and S598-specific cells within the CD8 T cell population and M133- and S358-specific cells within the CD4 T cell population are shown. 6–10 mice in 3–5 independent experiments were analyzed. (<b>F</b>) Brain-derived lymphocytes were harvested from mice that received control cells or M133 Tregs. Representative dot plots show IFN-γ and IL-10 expression by endogenous CD4 T cells after M133 peptide stimulation. Summary data show expression levels of IFN-γ by M133-specific CD4 T cells in the absence (<b>black</b>) or presence (<b>red</b>) of M133 Treg. (<b>G</b>) Representative dot plots (from the same sample as in <b>F</b> (right hand dot plot)) show IFN-γ and IL-10 expression by exogenous M133 Tregs. Note that lower levels of IFN-γ were expressed by M133 Tregs when compared to Tconv. Summary data show expression levels of IL-10 by IFN-γ^- (<b>black</b>) and IFN-γ⁺ M133 Tregs (<b>red</b>). Data in (<b>F</b>) and (<b>G</b>) are representative of three independent experiments with at least 3 mice/group. (<b>H</b>) Expression levels of MHC II on brain microglia at day 7 p.i. Data are representative of three independent experiments with 3–5 mice/group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p