PD-1 disrupts CD28 signalling condensates, guiding redesign of CAR-T cells

Researchers in China have shown that the costimulatory receptor CD28 forms signalling condensates with the kinase Lck, and that the inhibitory receptor PD-1 can break these condensates apart. CAR-T cells carrying CD28 mutants that evade PD-1 were less suppressed in laboratory tests and performed better than wild-type CD28 CAR-T cells in a mouse tumour model. The work was published in Proceedings of the National Academy of Sciences.

Background: phase separation in T cell signalling

Earlier work established that, once an antigen is detected, the T cell receptor (TCR) undergoes phase separation with Lck. This helps trigger rapid T cell activation. How costimulatory and coinhibitory receptors influence this process, however, had remained unclear. The new study, led by Hui Chen and colleagues at the Institute of Biophysics of the Chinese Academy of Sciences, Zhejiang University and Harbin Medical University Cancer Hospital, addresses that gap for CD28 and PD-1.

One mechanism, two opposing outcomes

The team found that CD28 can itself undergo phase separation with Lck, forming condensates that support T cell activation. Phosphorylated PD-1 inhibits this process, and the effect depends strongly on PD-1's phosphorylation state.

According to the authors, PD-1 competes for binding to the intracellular domain (ICD) of CD28 and co-condenses with it. This disrupts the CD28/Lck condensates and dampens CD28 signalling. Phase separation therefore plays a dual role in this pathway: it amplifies CD28 signals in one configuration and suppresses them in another.

Designing mutants that escape PD-1

Using molecular dynamics simulations, the researchers mapped the interaction interface between CD28 and PD-1 and identified several key binding sites. They then designed a series of CD28 mutants based on these sites.

In a two-dimensional membrane reconstitution system, phase separation between the mutants and PD-1 was markedly reduced. Their capacity to phase-separate with Lck was unaffected, which suggests the mutations separate the activating function from the inhibitory interaction.

Testing in CAR-T cells

The mutants were then placed in second-generation CAR-T constructs. In vitro, when target cells did not express PD-L1, mutant and wild-type CD28 CAR-T cells behaved similarly. When target cells did express PD-L1, wild-type CD28 CAR-T cells were significantly inhibited, whereas CAR-T cells based on 4-1BB or on mutant CD28 were less affected.

In NSG mice carrying Raji cell-based tumours, mutant CD28 CAR-T cells had a therapeutic effect comparable to 4-1BB CAR-T cells. Both were markedly better than CAR-T cells with the wild-type CD28 intracellular domain.

Why it matters and what comes next

CD28 and 4-1BB are the two costimulatory domains most commonly used in second-generation CAR designs. The findings offer a molecular explanation for why CD28-based constructs may be more vulnerable to PD-1 signalling, and suggest a route to retaining CD28 function while limiting that vulnerability. More broadly, the study frames costimulatory and coinhibitory receptors as coupled components of one regulatory network rather than independent switches.

The evidence comes from in vitro systems and a single immunodeficient mouse model using a lymphoma cell line, so the results remain preclinical. The source does not report whether the mutants affect other aspects of T cell behaviour, such as persistence or exhaustion over longer periods, or how they perform against other tumour types. Those questions, along with testing in additional models, would be the natural next steps before any clinical consideration.

Source: Scientists reveal how cosignaling receptors dually regulate T cell activation via phase separation (Phys.org, 5 November 2026).

Image: AI-generated illustration.