Figure 3. POWV-specific T cells are present during infection.

(A) Schematic representation of the POWV-LB polyprotein (not to scale). Structural proteins capsid (red) (C) pre-membrane (pink, prM), and envelope (purple) (E) are located near the N terminus. Also depicted is the coverage of the POWV peptide library, comprised of a 15-mer peptide library designed with a 10-amino-acid (aa) overlap.

(B) Potential CD8⁺ T cell epitope hits as determined by interferon-gamma (IFN-γ) production by CD8⁺ T cells (CD19⁻ CD8⁺) in response to the peptide library illustrated in (A). Candidate epitopes were determined in two independent experiments by stimulating splenocytes ex vivo from POWV-LB infected B6 mice s.c. infected with 10³ FFU (n = 2) 7 dpi in the presence of brefeldin A for 6 h. Positive hits were considered those that elicited a T cell response 2-fold above background (dashed line).

(C) Potential CD4⁺ T cell epitope hits determined in the same manner as (B), at 11 dpi (n = 2).

(D) Representative flow plots depicting CD8⁺ T cell responses to POWV-LB infection described in (B). The cytokines tumor necrosis factor alpha (TNF-α) and IFN-γ are shown on the x and y axes, respectively. Reponses to no stimulation (top left), α-CD3 (top right), POWV56/57 peptide (bottom left) and POWV70/71 peptide (bottom right) are shown.

(E) Representative flow plots depicting CD4⁺ T cell responses to POWV-LB infection described in (B). Reponses to no stimulation (left), α-CD3 (middle), POWV104/105 peptide (top right), and POWV125/126 peptide (bottom right) are shown.

(F) Representative image of tetramer staining for POWV-E₂₈₂-specific CD8⁺ T cells using H2-b restricted D^b tetramer from naive B6 mice (left) or mice challenged s.c. with 10³ FFU POWV-LB and harvested 7 dpi. Cells are gated on CD19⁻ CD8⁺.

(G) Frequency of POWV-E₂₈₂-specific CD8⁺ T cells by tetramer staining relative to total CD8⁺ T cells from B6 mice challenged s.c. with 10³ FFU POWV-LB and bled longitudinally over a period of 7 days. All data are reported as mean ± SEM.