NEDDylation is a post-translational modification whereby the ubiquitin-like molecule NEDD8 is attached to protein substrates in a process dependent on NEDD8 activating enzyme regulatory subunit (NAE1). NEDDylation is emerging as a regulator of cancer biology, but its precise role in antitumor immunity has not been thoroughly characterized. Here, we study the impact of NEDDylation in CD8+ T cell–mediated antitumor responses. Analysis of publicly available single-cell RNA sequencing databases revealed that CD8+ tumor-infiltrating lymphocytes (TILs) showed increased expression of NEDD8 during their differentiation into effector memory cells. In vitro activation of mouse and human CD8+ T cells drove the upregulation of the NEDDylation enzymatic pathway, resulting in an enrichment of NEDDylated proteins. In vivo tumor challenge assays demonstrated that CD8+ T cells lacking NAE1 (NAE1-KO) exhibited reduced antitumor capability and a less activated phenotype with compromised differentiation into effector cells. Upregulating NEDDylation by knocking out deNEDDylase sentrin-specific protease 8 (SENP8) increased the in vitro cytotoxic capability of CD8+ CAR T cells. In addition, LC MS/MS proteomic analyses of NAE1-KO CD8+ T cells and CD8+ T cells treated with the NEDDylation inhibitor MLN4924, showed a pronounced impairment in metabolic pathways, including glycolysis and oxidative phosphorylation. In this context, we validated lactate dehydrogenase A, α-enolase and hexokinase 1, which are relevant glycolytic enzymes, as NEDD8 targets. In line with this, NEDDylation-deficient CD8+ T cells demonstrated reduced transcription, protein expression and enzymatic activity of lactate dehydrogenase. In summary, we uncover NEDDylation as a critical regulator of CD8+ T cell–mediated antitumor immunity.

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First page of NEDDylation regulates CD8+ T cell metabolism and anti-tumor immunity<alt-title alt-title-type="left-running">NEDDylation regulates CD8+ T-cell function</alt-title>

Supplementary data

Supplementary Figure S1

T cell activation triggers the upregulation of NAE1 expression and NEDDylation activity.

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Supplementary Figure S2

Analysis of thymus, spleen and lymph node populations in NAE1-KO mice.

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Supplementary Figure S3

Inhibition of NEDDylation induces a viability impairment in mouse and human CD8+ T cells, as well as an accumulation of cell cycle regulators WEE-1, P21 and P27.

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Supplementary Figure S4

In vivo tumor-challenge assays show a defect in the anti-tumor function of NAE1-KO CD8+ T cells.

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Supplementary Figure S5

NEDDylation is a relevant agent in exhausted CD8+ T cells.

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Supplementary Figure S6

The reduced anti-tumor function of NAE1-KO CD8+ T cells is confirmed by additional tumor-challenge experiments and antigen-specific assays.

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Supplementary Figure S7

T cell receptor signaling, migration, MYC activity, metabolic pathways and HIF-1α signaling are impaired in NAE1-KO CD8+ T cells.

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Supplementary Figure S8

Densitometric analysis of bands corresponding to LDHA, ENO1 and HK-1 in the indicated conditions.

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Supplementary Table S1

List of Western Blot antibodies, Flow Cytometry antibodies, and primers used in the study.

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Supplementary Table S2

Raw proteomics data.

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Supplementary Table S3

IPA analysis of proteomic data.

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Supplementary Table S4

NEDD8 targets in CD8+ T cells: proteomic analysis.

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