921 The NAE1-mediated Neddylation operates as an essential post-translational modification checkpoint for effector CD8+ T cells

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Background Optimal activation of CD8+ T cells is crucial for immunity-mediated destruction of cancer, requiring the participation of a substantial amount of proteins involving in metabolism, proliferation, and effector function. Despite extensive studies emphasizing the role of transcriptional regulation in this process, paired RNA sequencing and proteomics analyses reveal that the RNA profile is poorly correlated with protein levels. This discrepancy underscores the importance of post-translational modifications (PTMs) in controlling protein abundance during activation. However, the impact of PTMs on the CD8+ T cell protein dynamic remains underexplored. Methods To determine the intrinsic role of NAE1 in CD8+ T cells, we conditionally deleted Nae1 in the T cell (Nae1T-CKO) by crossing Nae1flox/flox mice with dLckCre mice. We analyzed the proteomic and transcriptomic profiles of polyclonally activated CD8+ T cell isolated from Nae1T-CKO and Nae1T-WT mice, which were subject to differentially expression assay and gene set enrichment analysis (GSEA). We assessed the activation, proliferation, apoptosis, and mitochondria function of activated Nae1-deficient CD8+ T cell by spectral flow cytometry. To evaluate the antitumoral function, murine colon cancer cell line MC38 was subcutaneously injected to C57BL/6 mice, and tumor volume were tracked and tumor-infiltrating lymphocytes were profiled with spectral flow cytometry. We employed qPCR in combination with CRISPR/Cas9-mediated gene knockout approach to determine the regulator of Nae1. Results Our proteomics analysis identifies that neddylation, a recently discovered PTM, is activated in response to T-cell receptor (TCR) stimulation and enriched in effector CD8+ T cells from colon cancer patients. Mechanistically, we found the rate-limiting enzyme of neddylation, NEDD8 activating enzyme E1 (NAE1), is induced by the nuclear factor of activated T cells 1 (NFATc1), a critical transcription factor downstream of TCR signaling. Our observation revealed that genetic ablation of NAE1 significantly disturbed the proteomic landscape related to activation and mitochondrial function with minimal impact on the transcriptome. As a result, CD8+ T cells lacking NAE1 exhibited severely impaired activation, proliferation, and survival. Functional analyses further revealed that NAE1 deficiency impaired mitochondrial function and led to the accumulation of depolarized mitochondria. Consistently, deletion of NAE1 in CD8+ T cells abolished their anti-tumor function and promoted tumor progression. By contrast, the overexpression of NAE1 significantly improved the function of tumor-infiltrating CD8+ T cells. Conclusions Overall, we uncovered neddylation, a previously underappreciated PTM, as a proteomic checkpoint for mitochondrial function and CD8+ T cell activation. Enforced expression of NAE1 offers promising therapeutic potential for boosting the anti-tumor CD8+ T cell responses. Ethics Approval All mouse handling conformed to the requirements of the Institutional Animal Care and Use Guidelines of Ohio State University under protocol 2020A00000066-R1.

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