Understanding Mechanisms and Consequences of T Cell Co-Receptor Regulated RNA Maturation

In vitro: CD8+ T cells will be isolated from spleens of mice or human peripheral blood using Stem Cell Technologies CD8 magnetic kits. Four experiments are proposed: 1. WT C57BL/6J, CD28KO, CD28Y170F knock-in, CD28AYAA knock-in, and CD28Y170F,AYAA knock-in (5 groups) T cells will be isolated and activated in vitro using áCD3/CD28 beads + IL-2 (IL-2 added because CD28KO and knock-ins have known defects in IL-2 production). 2. WT C57BL/6J, ARS2iKO, and PKM2 iKO mice will be treated with tamoxifen prior to isolation of T cells (3 groups) and isolated cells activated as in 1. 3. *note iKO mice express two floxed alleles and express tamoxifen-inducible Cre recombinase CreERT2.CD28AYAA T cells transduced with control vector, ARS2 or PKM2 (3 groups) 4. Human T cells activated in vitro áCD3/CD28 beads in the presence of a control or PKM2 morpholino to disrupt splicing to PKM2 (2 groups).