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

Naive 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 costimulation. In vivo, MARK2 deficiency promotes the development of central memory T cells, enhances basal glycolysis activity in naive 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 costimulation. 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.