Document resource
Background T cells rely on metabolic reprogramming to support activation, proliferation, and effector function, particularly in nutrient-limited environments such as solid tumors. CD28 costimulation is a critical component of T cell activation and promotes the expression of metabolic regulators that enable this transition. Using proteomic profiling, we identified the mitochondrial enzyme PCK2 as one of the most strongly CD28-induced proteins. PCK2 catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, redirecting TCA cycle intermediates into biosynthetic pathways. However, its function in T cell biology has not been previously defined.Methods We used proteomic profiling to identify proteins regulated by CD28 costimulation in murine CD8 + T cells, which led us to investigate the role of PCK2. We assessed its regulation and function in both murine and human CD8+ T cells. PCK2 knockout (KO) model was generated in OT-1 CD8+ T cells. Functional consequences were evaluated in vivo using tumor challenge and skin graft rejection models, and in vitro using serial co-culture assays to assess persistence and cytotoxic activity under nutrient-limited conditions.Results CD28 signaling induced strong upregulation of PCK2 protein without corresponding increases in transcript levels, consistent with post-transcriptional regulation. Proteomic analysis identified PCK2 as one of the most significantly CD28-induced proteins, with a fold change of 0.719 and a p-value of 1.3 × 10 – 8 (figure 1). This induction was conserved in both mouse and human CD8+ T cells. In vivo, PCK2KO T cells proliferated comparably to wild-type cells, undergoing eight rounds of division following antigenic stimulation. In an allogeneic skin graft model, both KO and WT T cells mediated full graft rejection by day 16, indicating intact activation and effector capacity in a non-stressed setting. However, in a B16 tumor model, PCK2KO T cells failed to control tumor growth; by day 15, average tumor volumes were 1100 mm³ in KO-treated mice versus 150 mm³ in WT-treated controls (figure 2). In serial co-culture assays, PCK2KO T cells exhibited diminished persistence and loss of cytotoxic activity after early rounds of stimulation.Conclusions These findings identify PCK2 as a CD28-regulated metabolic enzyme that supports T cell persistence in nutrient-stressed environments. By linking costimulatory signaling to biosynthetic flexibility, this work defines PCK2 as a critical determinant of T cell fitness in metabolically hostile settings.Abstract 762 Figure 1CD28 costimulation induces post-transcriptional upregulation of PCK2 in CD8+ T cells. (A) Schematic of activation conditions. (B) Proteomic volcano plot highlighting PCK2 induction. (C) Metabolic pathway diagram showing CD28-induced glycolytic remodelingAbstract 762 Figure 2PCK2 is required for T cell-mediated tumor control in vivo. (A) PCK2KO and WT OT-1 T cells proliferate similarly following activation. (B-C) PCK2KO T cells fail to control tumor growth in B16 and MC38 models compared to WT counterparts