Previous studies have shown that upon antigen stimulation, the T cell receptor (TCR) undergoes phase separation with the kinase Lck to mediate rapid T cell activation. However, the manner in which costimulating receptors regulate this process has remained unclear.

In a study published in PNAS on October 21, researchers from the Institute of Biophysics of the Chinese Academy of Sciences, Zhejiang University, and Harbin Medical University Cancer Hospital, have systematically elucidated how the costimulatory receptor CD28 and the coinhibitory receptor PD-1 regulate T cell activation through phase separation.

The researchers found that the costimulatory receptor CD28 can undergo phase separation with Lck. They also found that phosphorylated PD-1 inhibits CD28/Lck phase separation. Notably, this inhibitory effect depends highly on the phosphorylation state of PD-1.

The results reveal an elegant dual regulatory mode. On one hand, CD28 promotes T cell activation by undergoing phase separation with Lck to form signaling condensates. On the other hand, PD-1 competitively binds the CD28 intracellular domain (ICD) and co-condenses with it, thereby disrupting CD28/Lck condensates and inhibiting CD28 signaling. In other words, phase separation plays a "dual role" in the CD28 signaling pathway.

Using molecular dynamics simulations, the researchers characterized the interaction interface between CD28 and PD-1, identified several key binding sites, and designed a series of mutants accordingly. Experiments in a two-dimensional membrane system confirmed that phase separation between these mutants and PD-1 was significantly inhibited, while their ability to undergo phase separation with Lck remained unaffected.

The researchers then introduced the CD28 mutants into second-generation CAR-T constructs. In vitro experiments revealed that, in the absence of PD-L1, mutant and wild-type CD28 CAR-T cells exhibited similar functions. However, when target cells expressed PD-L1, wild-type CD28 CAR-T cells were significantly inhibited, while 4-1BB- and mutant CD28-based CAR-T cells were less impacted.

Furthermore, in a Raji cell-based tumor model in NSG mice, the therapeutic efficacy of mutant CD28 CAR-T cells was comparable to that of 4-1BB CAR-T cells and markedly superior to that of CAR-T cells carrying the wild-type CD28 intracellular domain.

By elucidating the coupled regulatory molecular mechanisms within cosignaling receptors, this study deepens the understanding of the complex cosignaling regulatory network and provides important guidance for the rational design and optimization of CAR-T cells.