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

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

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

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

Image_1_Granulocyte-Derived Extracellular Vesicles Activate Monocytes and Are Associated With Mortality in Intensive Care Unit Patients.pdf

To understand how extracellular vesicle (EV) subtypes differentially activate monocytes, a series of in vitro studies were performed. We found that plasma-EVs biased monocytes toward an M1 profile. Culturing monocytes with granulocyte-, monocyte-, and endothelial-EVs induced several pro-inflammatory cytokines. By contrast, platelet-EVs induced TGF-β and GM-CSF, and red blood cell (RBC)-EVs did not activate monocytes in vitro. The scavenger receptor CD36 was important for binding of RBC-EVs to monocytes, while blockade of CD36, CD163, CD206, TLR1, TLR2, and TLR4 did not affect binding of plasma-EVs to monocytes in vitro. To identify mortality risk factors, multiple soluble factors and EV subtypes were measured in patients’ plasma at intensive care unit admission. Of 43 coagulation factors and cytokines measured, two were significantly associated with mortality, tissue plasminogen activator and cystatin C. Of 14 cellular markers quantified on EVs, 4 were early predictors of mortality, including the granulocyte marker CD66b. In conclusion, granulocyte-EVs have potent pro-inflammatory effects on monocytes in vitro. Furthermore, correlation of early granulocyte-EV levels with mortality in critically ill patients provides a potential target for intervention in management of the pro-inflammatory cascade associated with critical illness.</p