The MARK2 kinase acts as a gatekeeper of CD28-dependent co-stimulation in T cells

Naïve T cell activation requires not only antigen recognition through the TCR but also a co-stimulatory signal, mainly provided by CD28. Here, using a T cell–specific conditional knockout (cKO) model, we identify the microtubule-affinity kinase 2 (MARK2) as a key intracellular checkpoint that limits CD28-mediated co-stimulation. In vivo, MARK2 deficiency promotes the development of central memory T cells, enhances basal glycolysis activity in naïve CD8 T cells, and leads to the development of systemic autoimmunity in aged mice. In MARK2-deficient CD8 T cells, TCR engagement alone drives sustained proliferation, cytokine production, and glycolysis, processes that normally require CD28 co-stimulation. Single-cell transcriptomic analysis reveals that MARK2 regulates the expression of genes involved in CD28 signaling and metabolic switch. We show that MARK2 restrains the PI3K–AKT–mTORC1 pathway by limiting CD28-driven transcriptional and metabolic programs. Mechanistically, we demonstrate that MARK2 phosphorylates CREB regulated transcription coactivator 2 (CRTC2) and suppresses CREB-mediated transcription and mTOR activation, whereas CD28 engagement lifts this inhibition. Together, our results redefine the role of CD28 that not only amplifies TCR signaling but also relieves a MARK2-dependent inhibitory signal. This work provides new insights into T cell activation, metabolism and immune tolerance with potential implications for immunotherapeutic strategies in cancer and autoimmunity.