Cell Surface Free Thiols Are Elevated On Antigen-Specific Cells Undergoing Recent TCR Stimulation.

<p>Naïve mice were infected with either LCMV-Armstrong (Acute) or LCMV-Clone 13 (Chronic) and harvested 8 days postinfection. To determine if cell surface free thiol levels correlated with expression of granzyme B (A), splenocytes were stained with anti-CD8α, D<sup>b</sup>GP33-41, isotype control or anti-Granzyme B and maleimide-Alexa Fluor 488. The histogram is gated on CD8<sup>+</sup>D<sup>b</sup>GP33-41<sup>+</sup> T cells and the shaded area indicates isotype staining. For each population the Granzyme B expression was divided into negative (neg), intermediate (int) , and high (high) levels of staining. (B) The cell surface free thiol levels compared to those on CD8<sup>+</sup>CD44<sup>lo</sup>Granzyme B<sup>-</sup> T cells from uninfected mice were determined for each antigen-specific population and the average and standard deviation are plotted. To determine whether cell surface free thiols were elevated on antigen specific CD8<sup>+</sup> T cells that were actively proliferating (C) splenocytes were stained with anti-CD8α, D<sup>b</sup>GP33-41, isotype control or anti-Ki-67, and maleimide-Alexa Fluor 488 . Dot plots are gated on total CD8<sup>+</sup> T cells and the values in each corner indicate the percent of cells that are in that quadrant. (D) The cell surface free thiol levels compared to those on CD8<sup>+</sup>CD44<sup>lo</sup>Ki-67<sup>-</sup> T cells from uninfected mice were determined for each antigen-specific population and the average and standard deviation are plotted. (E) To determine recent TCR stimulation splenocytes were stained with anti-CD8α, anti-CD69, anti-PD-1, D<sup>b</sup>GP33-41 and maleimide-Pacific Blue. The dot plots are gated on either CD8<sup>+</sup>CD44<sup>lo</sup> (naïve) or CD8<sup>+</sup>D<sup>b</sup>GP33-41<sup>+</sup> (infected) T cells. The value in each corner indicates the percent of cells in that quadrant. (F) The cell surface free thiol levels compared to those on CD8<sup>+</sup>PD-1<sup>-</sup>CD69<sup>-</sup> T cells from uninfected mice were determined for each antigen-specific population and the average and standard deviation are plotted. Six mice were analyzed in two independent experiments. *, significant difference between acute and chronic infection, P<u><</u>0.05.</p>