Differences in T cell number and phenotype for subjects with asymptomatic or neuroinvasive WNV infection.

<p>A) WNV-specific T cells from a total of 11 asymptomatic subjects (open symbols) and 9 subjects with neuroinvasive infection (filled symbols) as enumerated by ex vivo tetramer staining. Each data point represents tetramer staining for a single WNV epitope (2–3 epitopes were measured per subject) measured in a single subject. Subjects with neuroinvasive infection has significantly more WNV specific T cells (p<0.0001). B) Comparison of the relative proportion of naïve (CD45RA+CCR7+), TEM (CD45RA-CCR7-), TCM (CD45RA-CCR7+), and TEMRA (CD45RA+CCR7-) WNV specific T cells as determined by ex vivo tetramer analysis. Subjects with neuroinvasive infection (filled symbols) had a significantly lower proportion of naïve WNV specific T cells than asymptomatic subjects (open symbols) (p < 0.05) and a significantly higher proportion of TEM. C) Comparison of the relative proportion of WNV specific T cells within various defined functional subsets. Subjects with neuroinvasive infection had a significantly lower proportion of WNV specific T cells that were Th1-like (CXCR3+CCR4-CCR6-) (p < 0.0001) than asymptomatic subjects (p < 0.05) and a significantly higher proportion of Th1^ (CXCR3+CCR4+CCR6-) cells (p < 0.0001). D) Comparison of intracellular IFN-γ, IL-4, and IL-17 staining for WNV specific T cell lines from asymptomatic subjects (open symbols) or subjects with neuroinvasive infection (filled symbols). A significantly higher percentage of WNV specific T cells from subjects with neuroinvasive infections were positive for IL-4 (p<0.05). All other cytokines were not significantly different.</p

Comparing the absolute number of various subsets of WNV specific T cells in WNV subjects with neuroinvasive versus asymptomatic infection.

<p>A) Neuroinvasive subjects (9 subjects, designated by filled symbols) had higher numbers of WNV specific CD4+ T cells that were TEM or TCM (p < 0.0001 and p < 0.01 respectively) than asymptomatic subjects (11 subjects, designated by open symbols). B) Neuroinvasive subjects (filled symbols) had higher numbers of WNV specific CD4+ T cells that were Th1 or Th1^ (p < 0.0001 and p < 0.01 respectively) than asymptomatic subjects (open symbols).</p

Subjects with neuroinvasive WNV infections have an atypically polarized cytokine response.

<p>The large pie charts depict the proportion of WNV specific T cell lines obtained from 8 subjects with neuroinvasive infections (upper panel) or 8 subjects with asymptomatic infections (lower panel) that stained positive for IFN-γ only, IL-4 only, IL-10 only and IL-17 only, or any combination of these cytokines ('multiple'). Within the 10.7% and 4.1% of T cells respectively that produced multiple cytokines, the small pie charts indicate the proportion of cells in subjects with neuroinvasive infections (upper panel) or asymptomatic infections (lower panel) that stained positive for IFN-γ and IL-4; IFN-γ and IL-17; IL-4 and IL-17; or IFN-γ, IL-4, and IL-17. For all other possible combinations the percentage observed was negligible. A significantly higher percentage (p<0.0001) of WNV-specific T cell lines isolated from subjects with neuroinvasive infections were positive for more than one cytokine than from subjects with asymptomatic infections The elevated percentage of WNV-specific T cells that produced dual cytokines in subjects with neuroinvasive infections was almost exclusively due to T cells that co-produced IFN-γ and IL-4. These cells occurred at a significantly higher proportion (p<0.0001) in subjects with neuroinvasive infections than in subjects with asymptomatic infections whereas the proportion of T cells that co-produced other combinations of cytokines did not differ.</p

Altered Treg phenotype in subjects with neuroinvasive WNV.

<p>A) CD4+ Tregs were evaluated in PBMC samples from 9 subjects with neuroinvasive infection or 8 subjects with asymptomatic WNV by gating on CD25+CD4+ T cells and subsequently gating on CD127lowFoxP3++ T cells (designated by the upper left rectangle). B) Each symbol represents the overall percentage of Treg (CD4+CD25+CD127lowFoxP3++ T cells) within the PBMC of a single WNV infected subject. The percentage of CD4+ Tregs was not significantly different between subjects with neuroinvasive or asymptomatic WNV. C) Each symbol represents the percentage of Treg that were CTLA-4 positive for a single WNV infected subject. Subjects with neuroinvasive WNV had a significantly lower percentage of CTLA-4 positive Tregs (p = 0.0097). D) Each symbol represents the percentage of Treg that were CCR4 positive for a single WNV infected subject. The percentage of Treg that were CCR4+ was not significantly different between subjects with neuroinvasive or asymptomatic WNV.</p

Cytokine profiles of WNV-specific T cells A) WNV-specific T cell lines specific for single epitopes were isolated from WNV infected subjects and analyzed for IFN-γ, IL-4, IL-10, and IL-17 content by intracellular cytokine staining.

<p>Each symbol indicates the percentage tetramer positive T cells that were cytokine positive within a single cell line. B) Averaging across all subjects tested, the large pie charts depicts the proportion of WNV specific T cells in subjects with WNV infections that stained positive for IFN-γ only, IL-4 only, IL-10 only and IL-17 only, or any combination of these cytokines ('multiple'). Within the 7.8% of T cells that produced multiple cytokines, the small pie charts indicate the proportion of cells that stained positive for IFN-γ and IL-4; IFN-γ and IL-17; IL-4 and IL-17; or IFN-γ, IL-4, and IL-17. For all other possible combinations the percentage observed was negligible. These data were obtained from a total of 144 WNV specific lines isolated from 16 different subjects.</p

Enumeration and phenotypic characterization of WNV-specific T cells.

<p>A) Direct ex vivo tetramer staining of DRB1*01:01/NS1 205–220 specific T cells in 30 million peripheral blood mononuclear cells (PBMC) from a representative WNV infected subject. This tetramer labeled a clear population of CD4+ T cells (left panel) that were predominantly CD45RA- (right panel). B) Direct ex vivo tetramer staining of DRB1*01:01/NS1 205–220 specific T cells in 90 million PBMC from an HLA matched control subject. This tetramer labeled a diffuse population of CD4+ T cells (left panel) that were predominantly CD45RA+ (right panel). C) Summary of naïve and memory T cell phenotypes for WNV infected subjects based on ex vivo surface staining of CD45RA and CCR7. D) Functional heterogeneity of memory T cell phenotypes for WNV infected subjects based on ex vivo surface expression of various combinations of CXCR3, CCR4, and CCR6 on CD45RA- cells. For A-D, T cells specific for single WNV protein epitopes were co-stained with PE-labeled tetramers and CD45RA, CXCR3, CCR4, CCR6, CCR7, and CD38 antibodies. Each data point represents the percent expression of the appropriate combination of surface markers by T cells stained with a single tetramer from a single subject. Staining was performed for a total of 22 subjects using 3 tetramers per subject (66 total stains).</p