Gating strategy for phenotyping IFN-γ secreting T cells in response to stimulation.

<p>The frequencies of Tim-3⁺ and Tim-3⁻ IFN-γ secreting CD4⁺ and CD8⁺ T cells were measured in PBMC collected at day 30 post-index from 6 HLA-A02 WNV-infected donors and incubated with or without anti-CD3/anti-CD28 mAbs, WNV peptide pool, and SVG9 tetramer. Tim-3⁻ and Tim-3⁺ CD4⁺ and CD8⁺ T cells were analyzed for IFN-γ secretion. The gates were set using fluorescence minus one controls. Dot-plots for CD8⁺ T cells from all 6 WNV+ subjects in different stimulation conditions are shown.</p

WNV+ subject characteristics.

<p>N/A, not available.</p>a<p>Highest number of symptoms reported on either questionnaire.</p>b<p>Female (F) and male (M).</p>c<p>Antibody interpretation at index: positive (+), negative (−), or equivocal (E).</p>d<p>AS is for asymptomatic when peak symptom number ≤3 and S is for symptomatic when peak symptom number ≥4.</p

Frequencies of Tim-3 and PD-1 expressing T cells compared over the course of WNV infection in groups with different disease outcome.

<p>The graphs below demonstrate the change of frequencies of (A) Tim-3⁺, (B) PD-1⁺, (C) Tim-3⁺PD-1⁻, and (D) Tim-3⁺PD-1⁺ CD4⁺ (left panel) and CD8⁺ (right panel) T cell subsets in asymptomatic (AS, circles/solid lines, n = 24) and symptomatic (S, squares/dash lines, n = 8) WNV-infected subjects 14, 30, 90, and 365 days post-index donation. The frequencies of the same cells in uninfected controls are displayed (WNV−, triangles, n = 26). The symbols indicate the means and the error bars represent the SEM. The <i>p</i>-values of the pairwise comparisons between asymptomatic and symptomatic groups are indicated by *<i>p</i> <0.05, ** <i>p</i> <0.01, and *** <i>p</i> <0.001 above time-points when groups were compared at a given time-point by Mann-Whitney test. The <i>p</i>-values of the comparison between asymptomatic and symptomatic groups are indicated by ** adjacent to the cell subset title for <i>p</i> <0.01 over the time of post-index by generalized estimated equation (GEE).</p

IL-1β synergizes with type I IFN to mediate direct antiviral actions and control of WNV infection in cortical neurons.

<p>Cortical neurons were prepared from d15 embryos of WT (closed circles) or <i>Il-1r^−/−</i> (open squares) animals and were assessed for viral load by plaque assay after infection with MOI (1) WNV-TX at 12, 24 and 48 hrs p.i. (<b>A</b>). Quantification of IL-1β (<b>B</b>) or IFN-β (<b>C</b>) from supernatants of mock or WNV MOI 1 infected WT and <i>Il-1r^−/−</i> neurons at 12, 24 and 48 hrs p.i. was assessed by Luminex array (<b>B</b>) or ELISA (<b>C</b>). IL-1β treatment reduces WNV load in cortical neurons. WT cortical neurons were either infected with MOI1 WNV alone (WT no treatment; closed circles) or with WNV MOI1+24 hr pre-treatment of 10 ng/ml recombinant IL-1β (WT 10 ng/ml IL-1β; open squares), (<b>D,E</b>) or with IFN-β (100 IU/ml) or both cytokines together (<b>E</b>) and viral load was assessed by plaque assay (<b>D,E</b>). Western blot analysis for WNV and interferon stimulated genes with IL-1β and IFN-β treatment (<b>F</b>). Data are shown as Mean+/−S.E.M. (<b>A–E</b>) for n = 3 per time-point and are representative of three independent experiments. * p<0.05, ** p<0.005, *** p<0.0005. Dashed line represents the lower limit of detection for each assay.</p

Phenotyping IFN-γ secreting T cells in response to stimulation.

<p>The frequency of IFN-γ-producing Tim-3⁻ and Tim-3⁺ CD4⁺ T cells (A) and CD8⁺ T cells (B) collected 30 days post-index from 6 HLA-A02 WNV-infected donors are shown after stimulation in the presence or absence of anti-CD3/anti-CD28 monoclonal antibodies, WNV peptide pool, and SVG9 peptide; Ratios of IFN-γ⁺/IFN-γ⁻ cells within Tim-3⁻ and Tim-3⁺ are shown for CD4⁺ T cells (C) and CD8⁺ T cells (D). The histograms indicate the means and the error bars represent the SEM. **<i>p</i> <0.01, *<i>p</i> <0.05, and *** <i>p</i> <0.001 by <i>t</i>-test.</p

Gating strategy for measuring CD28 differentiation and CD57 senescence makers on T cells.

<p>The plots show (A) the gating strategy for live CD3⁺ lymphocytes, (B) for CD4⁺ (left) and CD8⁺ (right) T cells. Gates were set on FMO no CD28 (C) and FMO no CD57 (D). Plots are shown for representative (E) West Nile virus (WNV) uninfected controls (WNV−) and (F) WNV infected subjects (WNV+) day 14 post-index donation.</p

Differentiation status and functional capacity of Tim-3⁺ T cells in acute West Nile virus infection.

<p>The graphs show, through the course of WNV infection, the frequencies of Tim-3⁺ CD28⁺CD57⁻, CD28⁻CD57⁻ and CD28⁻CD57⁺ (A) CD4⁺ and (B) CD8⁺ T cell subsets in asymptomatic (AS, circles/solid lines, n = 24) and symptomatic (S, squares/dash lines, n = 8) WNV+ subjects 14, 30, 90, and 365 days post-index donation. The frequencies of the same cells in uninfected controls are displayed (WNV-, triangles, n = 26). The symbols indicate the means and the error bars represent the SEM. **<i>p</i> <0.01, *<i>p</i> <0.05, and *** <i>p</i> <0.001 by Mann-Whitney. The <i>p</i>-values of the comparison between asymptomatic and symptomatic groups are indicated by ** adjacent to the cell subset title for <i>p</i> <0.01 over the time post-index by GEE.</p

Gating strategy for measuring Tim-3 and PD-1 inhibitory receptors on T cells.

<p>The plots show (A) the gating strategy for live CD3⁺ lymphocytes, (B) for CD4⁺ (left) and CD8⁺ (right) T cells. Gates were set on FMO no Tim-3 (C) and FMO no PD-1 (D). The gating of cells expressing Tim-3 and PD-1 is shown for representative (E) WNV− and (F) WNV+ subjects day 14 post-index donation.</p

IL-1β signaling is important for limiting neuropathology within the CNS.

<p>Histological analysis from day 10 p.i. sagittally cut brain sections from paraffin embedded tissue. Representative formalin-fixed hematoxylin and eosin-stained sections of day 10 p.i. brains from WT (top panels) and <i>Il-1r−/−</i> (bottom panels) sections (<b>A</b>). Brain areas as indicated in figure. Original magnification 600×. In all regions of the WT brain, the sections are histologically unremarkable whereas lesions were readily apparent in the <i>Il1r</i>^−/− tissues. Brainstem: black arrows indicate perivascular regions; note the mildly increased cellularity in the <i>Il1r</i>^−/− vessel wall and immediate perivascular space. Forebrain: there is a mild focus of acute perivascular hemorrhage (arrow). Midbrain: There is a focus of mild inflammation associated with shrunken and eosinophilic neuron suggestive of neuronal degeneration (white arrow) which was in association with inflammatory infiltrates. Meninges: Black arrow indicates expansion of the meninges with moderate inflammatory infiltrates; compare to WT which is histologically unremarkable. Data are representative of two animals per genotype. (<b>B</b>) Representative day 10 p.i. Mac-2 immunohistochemical stained sections (brain regions are as indicated in the figure) in WT (top panel) or <i>Il-1r^−/−</i> (bottom panel) sections. Positive signal is as indicated by brown staining; original magnification for all panels, 200×. Mac-2 ⁺ cells are present perivascularly (black arrows) and in the adjacent parenchyma (Non-specific staining of the choroid is present (WT top panel hippocampus-thalamus) and <i>Il-1r^−/−</i> (data not shown). (<b>C</b>) Quantification of MAC-2 signal to tissue ratio.</p

IL-1β signaling is critical for regulating CD8⁺ T cell effector activity within the CNS.

<p>Examination of T lymphocyte effector activity in the CNS by flow cytometry. Cell were isolated from the brains of mock or day 6–10 p.i. WT (closed circles) or <i>Il-1r^−/−</i>(open squares) and staining for CD3, CD8 and WNV-NS4b tetramer was performed. Quantitation of CD3⁺/CD8+ T lymphocytes (<b>A</b>), CD3⁺/CD8⁺/NS4b⁺ (<b>B</b>) as derived from total cell numbers or frequency of CD3⁺/CD8⁺/NS4b⁺ cells (<b>C</b>). Brains were harvested from day 8 or 10 p.i. and stimulated with 1 µM WNV-NS4b for 4 hrs (<b>D,E, H,I</b>) and assessed for cytokine expression by intracellular staining. Representative dot-plot analysis for IFN-γ and TNF-α expression at day 10 p.i. in the brain (<b>D</b>) or perforin and granzyme B (<b>H</b>). Data are shown as percent cytokine positive for WT (closed box) or <i>Il-1r^−/−</i> (open box) (<b>E,I</b>). Quantification of IFN-γ secretion by ELISA. Total brain cells (2.5e5) were stimulated with NS4b for 24 hrs and IFN-γ was quantified by ELISA for WT (closed circles) and <i>Il-1r^−/−</i> (open squares) cells (<b>F</b>). Data are shown as mean +/− S.E.M. for n = 3–4 mice per time-point (<b>A–E, H,I</b>) or n = 3–7 (<b>F</b>) and are representative of 2–3 independent experiments. *p<0.05, ** p<0.005.</p