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

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posted on 13.11.2013 by Samuel Troy Pellom, Ryan D. Michalek, Katie E. Crump, P. Kent Langston, Daniel G. Juneau, Jason M. Grayson

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, DbGP33-41, isotype control or anti-Granzyme B and maleimide-Alexa Fluor 488. The histogram is gated on CD8+DbGP33-41+ 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+CD44loGranzyme B- 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+ T cells that were actively proliferating (C) splenocytes were stained with anti-CD8α, DbGP33-41, isotype control or anti-Ki-67, and maleimide-Alexa Fluor 488 . Dot plots are gated on total CD8+ 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+CD44loKi-67- 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, DbGP33-41 and maleimide-Pacific Blue. The dot plots are gated on either CD8+CD44lo (naïve) or CD8+DbGP33-41+ (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+PD-1-CD69- 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<0.05.

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