CD28 costimulation is organized by signaling condensates and counteracted by PD-1

PNAS, 21 October, 2026, DOI：https://doi.org/10.1073/pnas.2612742123

CD28 costimulation is organized by signaling condensates and counteracted by PD-1

Hui Chen, Songfang Wu, Yong Zhang, Wei Hu, Changjie Lou, Wei Chen, and Jizhong Lou

Abstract

T cell activation relies on the coordinated integration of T cell receptor (TCR) and cosignaling pathways, yet how these signals are physically organized remains unclear. Here, we demonstrate that CD28 co-stimulation is driven by a condensation mechanism in which the CD28 intracellular domain (ICD) forms signaling condensates with the Src-family kinase Lck. Ligand engagement promotes CD28 ICD accessibility and triggers rapid CD28/Lck condensation, enhancing CD28 phosphorylation, cytoskeletal polarization, and cytokine production. The downstream effectors of CD28, including PI3K and GRB2, can also be recruited into the CD28-Lck condensates in our reconstituted system. We further identify PD-1 as a graded regulator of this process: Monophosphorylated PD-1 is recruited into CD28/Lck condensates, whereas diphosphorylated 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 condensation mechanism, we engineer CD28 ICD mutants that preserve Lck condensation while resisting PD-1-mediated disruption. Incorporation of these condensation-selective mutants into Chimeric antigen receptor (CAR) constructs confers resistance to inhibitory signaling and enhances antitumor efficacy in vivo. These findings establish condensation as a central organizing principle of CD28 costimulation and provide a biophysical framework for engineering more resilient therapeutic T cells.

文章链接：

https://www.pnas.org/doi/10.1073/pnas.2612742123

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