STAT1 Serotonylation Remodels CD8+ T Cell Mitochondrial Function to Potentiate Antitumor Efficacy of PD-1 Blockade

Technical Summary: STAT1 Serotonylation Remodels CD8+ T Cell Mitochondrial Function to Potentiate Antitumor Efficacy of PD-1 Blockade

Problem Statement
Immune checkpoint inhibitors (ICIs), particularly anti-PD-1 therapies, have revolutionized cancer treatment but suffer from limited response rates (20–30%) in monotherapy settings. A primary driver of this resistance is T cell functional impairment within the tumor microenvironment (TME), often characterized by metabolic reprogramming and mitochondrial dysfunction. While mitochondrial fission-fusion homeostasis is critical for T cell persistence and effector function, the mechanisms regulating this balance in the context of immunotherapy remain incompletely understood. Furthermore, the role of the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) in anti-tumor immunity is complex; while often viewed as a tumor-promoting factor, recent evidence suggests it may also modulate immune cells via post-translational modifications (PTMs) known as serotonylation, a process catalyzed by tissue transglutaminase 2 (TGM2).

Methodology
The study employed a multi-faceted approach combining in vivo mouse models, in vitro cellular assays, proteomics, and clinical data analysis:

• In Vivo Models: Subcutaneous tumor models using SJT1601 (lung adenocarcinoma) and MC38 (colon carcinoma) cell lines in C57BL/6 mice. Treatments included PBS, anti-PD-1 antibodies, exogenous 5-HT, and combination therapy. CD8+ T cell depletion and adoptive transfer experiments using Tgm2−/− and Rag1−/− mice were conducted to establish cell-type specificity.
• In Vitro Assays: Primary murine CD8+ T cells and EL4 T lymphoma cells were stimulated with anti-CD3/anti-CD28. Serotonylation was inhibited using the TGM2 inhibitor LDN27219, and STAT1 function was blocked with fludarabine.
• Proteomics and Molecular Biology: Immunoprecipitation (IP) coupled with LC-MS/MS was used to identify serotonylated proteins. Site-directed mutagenesis (Q120A and Q285A) in STAT1 was performed to map serotonylation sites. Chromatin immunoprecipitation (ChIP) and qPCR were used to assess STAT1 binding to mitochondrial gene promoters.
• Metabolic and Functional Analysis: Mitochondrial function was assessed via transmission electron microscopy (TEM), mitochondrial membrane potential (MMP) staining, mtROS detection, and Seahorse XF analysis (OCR/ECAR). T cell cytotoxicity was measured via LDH release and flow cytometry for effector molecules (IFN-γ, GzmB).
• Clinical Correlation: Public single-cell RNA-seq datasets (GSE189357, GSE207422) were analyzed for TPH1 expression and T cell exhaustion. Additionally, serum 5-HT levels were measured in a cohort of 46 NSCLC patients receiving nivolumab to correlate with progression-free survival (PFS).

Key Contributions and Results

1. Synergistic Antitumor Efficacy: Exogenous 5-HT supplementation significantly enhanced the efficacy of anti-PD-1 therapy in reducing tumor growth and weight in both SJT1601 and MC38 models. This synergy was strictly CD8+ T cell-dependent, as depletion of CD8+ T cells abolished the therapeutic benefit. The combination therapy increased IFN-γ and GzmB production while reducing exhaustion markers (KLRG1, LAG3).
2. Serotonylation is Essential: The study demonstrated that 5-HT levels increase in CD8+ T cells upon stimulation and anti-PD-1 treatment. Inhibition of TGM2 (via LDN27219) or genetic deletion of Tgm2 impaired CD8+ T cell proliferation, cytokine production, and cytotoxicity. In vivo, Tgm2−/− CD8+ T cells failed to control tumor growth compared to wild-type cells, confirming that serotonylation is indispensable for antitumor function.
3. Mitochondrial Remodeling: Inhibition of serotonylation led to mitochondrial dysfunction, characterized by elevated mitochondrial ROS, reduced spare respiratory capacity, decreased ATP production, and altered mitochondrial morphology (reduced number and area). This indicated that serotonylation is required to maintain mitochondrial homeostasis.
4. Identification of STAT1 Q285 Serotonylation: Proteomic screening identified STAT1 as a direct substrate for TGM2-mediated serotonylation. Mass spectrometry and mutagenesis pinpointed Glutamine 285 (Q285) in the coiled-coil domain as the critical site. The Q285A mutation (mimicking non-serotonylated STAT1) recapitulated the mitochondrial defects and metabolic failure seen with TGM2 inhibition, whereas the Q120A mutant did not.
5. Mechanism of Action (STAT1 S727 Phosphorylation): STAT1 serotonylation at Q285 was found to promote phosphorylation at Serine 727 (S727). This pSTAT1S727 acts as a dual transcriptional regulator:
   • It represses Mtfr2 (Mitochondrial Fission Regulator 2) transcription. Since MTFR2 stabilizes DRP1 to promote fission, its repression inhibits excessive mitochondrial fission.
   • It activates Mfn2 (Mitochondrial Fusion Protein 2) transcription, promoting mitochondrial fusion.
   • Consequently, STAT1 serotonylation shifts the balance toward mitochondrial fusion and homeostasis, preventing the fragmentation and dysfunction associated with T cell exhaustion.
6. Clinical Relevance: Analysis of human NSCLC data revealed that high TPH1 expression (indicating 5-HT synthesis capacity) correlated with lower T cell exhaustion scores and higher proportions of memory CD8+ T cells. In a clinical cohort of 46 NSCLC patients, higher serum 5-HT levels post-treatment were significantly associated with longer progression-free survival and a lower proportion of LAG3+ CD8+ T cells.

Significance and Claims
The paper claims to uncover a previously underappreciated mechanism where 5-HT, via serotonylation of STAT1 at Q285, remodels mitochondrial dynamics to sustain CD8+ T cell effector function. This "serotonylation-mitochondria axis" provides a novel molecular explanation for how neurotransmitter signaling can directly enhance T cell metabolism and anti-tumor immunity.

The authors posit that this discovery offers a translational strategy: 5-HT supplementation could serve as a potential combination therapy with ICIs to overcome resistance. The study suggests that monitoring 5-HT levels or targeting the serotonylation pathway might improve clinical outcomes in patients receiving anti-PD-1 blockade. The authors maintain a modest tone, acknowledging limitations such as the need for larger prospective clinical trials to validate 5-HT as a biomarker and the possibility that other serotonylated proteins contribute to T cell function. They emphasize that their findings bridge the gap between neuro-immunology and mitochondrial metabolism in the context of cancer immunotherapy.