Background:
Persistent PD-1 expression in tumor-infiltrating CD8⁺ T cells drives immune exhaustion and immune checkpoint blockade (ICB) resistance. Although post-translational modifications regulate PD-1 stability, the crosstalk between ubiquitination and deubiquitination remains unclear. This study identifies KLHL20 and USP24 as key IL-6–responsive regulators that oppositely control PD-1 protein stability in the tumor microenvironment.
Materials and Methods:
Using Jurkat T cells, human PBMCs, and murine CD8⁺ T cells, we assessed PD-1 ubiquitination, membrane presentation, and stability after manipulating USP24 or KLHL20. Mechanistic studies included immunoprecipitation, CHX chase, RNA-seq, and IL-6 stimulation. Transgenic mice and T cell cytotoxicity assays evaluated in vivo effects. Clinical lung cancer samples were analyzed for gene expression correlations with ICB response.
Results:
USP24 stabilized PD-1 by removing K48-linked polyubiquitin chains, thereby protecting PD-1 from proteasomal degradation. IL-6-driven STAT3/NF-κB signaling transcriptionally upregulated USP24 in T cells, promoting PD-1 accumulation and dysfunction. Conversely, KLHL20, an E3 ligase, interacted with the intracellular domain of PD-1 to mediate its ubiquitination and degradation. IL-6-induced JAK2 activation suppressed KLHL20 expression and PD-1 ubiquitination, further enhancing PD-1 stability. Targeting PD-1 stability with the novel inhibitor USP24-i-101 boosted cytotoxic T-cell activity and enhanced therapeutic efficacy when combined with anti-CTLA4 immunotherapy. Moreover, KLHL20 overexpression reduced PD-1 levels, restored T cell function, and improved antitumor immunity.
Conclusion:
This study uncovers a novel IL-6-driven regulatory axis in which USP24 and KLHL20 oppositely modulate PD-1 ubiquitination and stability. Targeting this USP24-KLHL20 balance may offer therapeutic potential to reverse T cell exhaustion and improve ICB efficacy in lung cancer.