Cullin-5–RING E3 Ligase Limits CD8+ T-cell Persistence and Effector Responses during Chronic Antigen Stimulation

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Abstract
T-cell exhaustion limits durable antitumor responses and remains a barrier to adoptive cell therapies (ACT) in solid tumors. To identify regulators of T-cell persistence, we performed genome-wide CRISPR knockout screens in primary human CD8+ T cells expressing an NY-ESO-1–specific T-cell receptor and subjected them to repeat tumor cell rechallenges. These screens revealed an enrichment of single-guide RNAs targeting components of cullin–RING E3 ubiquitin ligases, particularly cullin-5–RING E3 ligase (CRL5) assemblies comprising CUL5, RNF7, and ARIH2. Disruption of CRL5 genes in T cells preserved cytotoxic activity and enhanced cytokine production while sustaining effector-associated transcriptional programs and suppressing exhaustion-related signatures. Pharmacologic inhibition of cullin neddylation with the NEDD8-activating enzyme inhibitor MLN4924 recapitulated CRL5 loss and enhanced T-cell effector function in vitro. In a human melanoma xenograft model, ACT with CRL5-deficient T cells improved tumor control. Together, these findings identify CRL5 as a negative regulator of T-cell fitness and a potential therapeutic target to enhance ACT durability in solid tumors. Significance: The CRL5 complex negatively regulates CD8+ T-cell persistence during chronic antigen stimulation, identifying CRL5 disruption as a potential strategy to sustain T-cell effector function and improve ACT. See related commentary by Hanina and Maus, p. XX . See related article by Bachl et al., p. XX .
Funding Information
  • National Cancer Institute (R35 CA197633)
  • National Cancer Institute (P01 CA168585)
  • Parker Institute for Cancer Immunotherapy