Work

718 CD28 co-stimulation induces PCK2 expression which sustains T cell function in solid tumors

Year: 2026

Type: conference-abstract

Abstract: Background Within nutrient-limited solid-tumor microenvironments, T cells require metabolic reprogramming to support activation, proliferation, persistence, and effector function. This reprogramming is supported by metabolic programs promoted by CD28 co-stimulation, a central regulator of T-cell activation. Using unbiased proteomic profiling, we identified mitochondrial phosphoenolpyruvate carboxykinase, PCK2, as a key CD28-upregulated protein in T cells. PCK2 catalyzes conversion of oxaloacetate to phosphoenolpyruvate, linking mitochondrial and cytoplasmic pathways and potentially benefitting T cells in metabolic stress.Methods Naïve murine CD8 + T cells were activated with anti-CD3 alone or anti-CD3 plus CD28 in the presence of IL-2; cells were harvested on day 4 and analyzed by proteomics. PCK2 regulation was assessed at the transcript and protein levels by qPCR and immunoblotting, and CD28-signaling requirements were evaluated with CRISPR/Cas9-mediated disruption of CD28 intracellular signaling motifs and with pharmacologic inhibition. To define PCK2 function, murine PCK2WT and PCK2KO CD8+ T cells (OT-I background) were evaluated in antigen-driven proliferation assays, serial co-cultures with tumor cells, and adoptive-transfer tumor models (B16.F10SIINFEKL, MC38SIINFEKL).Results CD28 co-stimulation enriched metabolic and inflammatory pathways of activated CD8 + T cells; PCK2 was among the most significantly upregulated proteins (0.719 = log2 fold change, p = 1.3 × 10−8). CD28, but not 4-1BB, induced upregulation of PCK2 in murine and human CD8+ T cells, suggesting specificity for CD28-associated signaling. CD28-co-stimulation resulted in increased abundance of PCK2 protein but not mRNA. Disruption of intracellular CD28 signaling motifs reduced PCK2 levels, suggesting regulation downstream of CD28. Rapamycin-induced inhibition of mTOR, a key mediator of CD28 signaling, decreased PCK2 protein abundance after CD28 co-stimulation (figure 1). After in vivo ovalbumin stimulation, PCK2KO T cells proliferated comparably to PCK2WT cells, with no significant difference in proliferation index, and, in serial co-culture assays, tumor control by PCK2WT and PCK2KO T cells was comparable. However, in both tumor models, in vivo adoptive transfer of PCK2WT T cells significantly delayed tumor progression and improved host survival, whereas PCK2KO T cells did not (figure 2). Consistent with this context-dependent requirement, metabolomic profiling revealed that PCK2KO T cells had decreased levels of phosphoenolpyruvate, suggesting that PCK2 supports biosynthetic metabolism.Conclusions We showed that CD28 co-stimulation upregulates mitochondrial metabolic enzyme PCK2, primarily at the protein level. Although dispensable for antigen-driven proliferation and cytotoxicity in vitro, PCK2 is required for optimal CD8+ T-cell function within the solid-tumor microenvironment. This work links CD28 co-stimulation to post-transcriptional regulation of mitochondrial carbon metabolism and defines PCK2 as a key determinant of T-cell fitness in metabolically hostile tumors.Abstract 718 Figure 1CD28 co-stimulation selectively induces post-transcriptional PCK2 upregulation in CD8+ T cells. Murine CD8+ T cells were stimulated with CD3±CD28 and analyzed at day 4. (A–C) proteomic changes and pathway enrichment; (D–E) PCK2 immunoblot validation; (F–H) PCK2 mRNA/protein levels; I, CD28 motif editing; (J) rapamycin/mTOR inhibitionAbstract 718 Figure 2PCK2 supports sustained T-cell tumor control without altering antigen-driven proliferation. (A) immunoblot confirms PCK2 WT/OE/KO CD8+ T cells; (B) serial B16-SIINFEKL killing after repeated tumor rechallenge; (C) in vivo OVA-driven proliferation is unchanged; (D–E) ACT with WT, but not KO, CD8+ T cells improves B16 and MC38 control/survival

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0718