CD28 Co-stimulation Is Organized by Signaling Condensates and Counteracted by PD-1

T cell activation relies on the coordinated integration of TCR and co-signaling pathways, yet how these signals are physically organized remains unclear. Here, we demonstrate that CD28 co-stimulation is driven by a phase-separation mechanism in which the CD28 intracellular domain (ICD) forms signaling condensates with the Src-family kinase Lck. Ligand engagement promotes the CD28 ICD accessibility, and triggers rapid CD28/Lck condensation, enhancing CD28 phosphorylation, cytoskeletal polarization, and cytokine production. We further identify PD-1 as a graded regulator of this process: monophosphorylated PD-1 is recruited into CD28/Lck condensates, whereas di-phosphorylated PD-1 competitively binds the CD28 ICD and dissolves these assemblies. CD28 and PD-1 ICDs also undergo condensation directly, revealing a dynamic and compositionally heterogeneous signaling system. Guided by this competitive phase-separation mechanism, we engineer CD28 ICD mutants that preserve Lck condensation while resisting PD-1-mediated disruption. Incorporation of these condensation-selective mutants into CAR constructs confers resistance to inhibitory signaling and enhances antitumor efficacy in vivo. These findings establish phase separation as a central organizing principle of CD28 co-stimulation and provide a biophysical framework for engineering more resilient therapeutic T cells.