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

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

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

rGal-9 is a potent mediator of HIV transcription <i>ex vivo and synergizes with JQ1 in reactivating latent HIV</i>.

(A) Treatment of CD4+ T cells isolated from ART-suppressed HIV-infected individuals with DMSO 0.5% (negative control), PMA/ionomycin (2 nM / 500 nM), vorinostat (1μM), or varying concentrations of rGal-9 (500 nM and 1000 nM) for 24 hours. Fold increase in cell-associated HIV RNA was determined relative to the corresponding DMSO-treated control for each individual time point. Mean ± SEM is displayed, and statistical comparisons between rGal-9 and other treatments were performed using two-tailed paired Wilcoxon signed-rank tests. (B-C) CD4+ T cells were isolated from PBMCs of three HIV-infected ART-suppressed individuals using negative selection. Resting CD4+ T cells were further enriched through depletion of cells expressing CD69, CD25, or HLA-DR surface markers from half of the isolated CD4+ T cells. The remaining half was processed through the exact enrichment procedure, except PBS was added instead of the depleting antibodies. Both cell populations were treated with 0.5% DMSO (negative control), 500 nM rGal-9, 1000 nM rGal-9 or αCD3/αCD28-conjugated beads. Induction of cell-associated HIV RNA was measured 24 hours post-treatment using RT-qPCR. Each individual is represented with a different symbol. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed paired t tests. Percentages reported reflect average values measured in the CD69- / CD25- / HLA-DR- CD4+ T cells with respect to values observed in total CD4+ T cells. (D) CD4+ T cells from HIV-infected ART-suppressed individuals were treated with 500 nM of rGal-9, 1 μM vorinostat, 40 nM romidepsin, 10 nM bryostatin, 300 nM prostratin, 1 μM JQ1, or 30 nM panobinostat alone or in combination with 500 nM of rGal-9 for 24 hours, and fold induction of cell-associated HIV RNA was determined using quantitative real-time PCR. * = p(E) The Bliss independence model was utilized for calculation of synergy for drug combinations. Δfaxy = 0 signifies a pure additive effect. Δfaxy>0 signifies synergy, while Δfaxy<0 signifies antagonism. Statistical significance was calculated using a two-tailed paired t-test comparing predicted and observed drug combination effects. * = p < 0.05.</p

rGal-9 modulates the expression of genes involved in several signaling pathways associated with HIV latency.

<p>(<b>A</b>) Venn diagram showing the number of genes modulated by >2 fold with FDR<0.05 in sorted GFP-positive and GFP-negative cells containing reactivated (transcriptionally active) HIV proviruses, and latent (transcriptionally inactive) proviruses, respectively, after either rGal-9 treatment or αCD3/αCD28 stimulation. (<b>B</b>) Heat maps describing effects of rGal-9 treatment on host gene expression, organized by signaling pathways. All statistical comparisons were performed using t tests, and p values were adjusted for multiple comparisons using false discovery rate. Asterisks indicate >2-fold, statistically significant differences in gene expression between r-Gal9-treated, GFP+ cells and unstimulated control, as follows: * = FDR<0.05; ** = FDR<0.01, and *** = FDR<0.001.</p

rGal-9 induces the expression of several anti-HIV host restriction factors including APOBEC3G <i>ex vivo</i>.

<p>(<b>A</b>) Heat map representing expression levels of host restriction factors in CD4+ T cells isolated from ART-suppressed individuals, after treatment with either 0.5% DMSO as negative control, 500 nM rGal-9, 1000 nM rGal-9, 1μM vorinostat, or a combination of PMA (2 nM) and Ionomycin (0.5 μM). Heat colors indicate fold modulation compared to the DMSO control. Red indicates induction of expression, and blue indicates reduction of expression. Statistical comparisons were performed using t tests, and p values were adjusted for multiple comparisons using false discovery rate. Asterisks indicate >3-fold, statistically significant modulation of gene expression as compared to DMSO control, as follows: * = FDR<0.05; ** = FDR<0.01, and *** = FDR<0.001. (<b>B</b>) APOBEC3G expression in isolated CD4+ T cells from HIV-infected ART-suppressed individuals, treated as described in panel <b>A</b>. Statistical comparisons were performed using two-tailed Wilcoxon signed-rank tests compared to the DMSO-treated control. <b>(C-D)</b> APOBEC3G protein expression in CD4+ T cells treated with either 500 nM rGal-9 or interferon-α (5000 units/ml), as determined by western blot. Immunoblotting bands were quantified with ImageJ software. The quantified APOBEC3G protein expression levels were normalized to corresponding Tubulin protein levels to account for variation in loading.</p