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Immune checkpoint blockers (ICBs) have demonstrated substantial efficacy across various malignancies, yet the benefits of ICBs are limited to a subset of patients. Therefore, it is essential to identify novel therapeutic targets. By integrating multi-omics data from cohorts of patients with melanoma treated with ICBs, a positive correlation is observed between tumor CD137L expression and the efficacy of PD-1 blockade. Functionally, CD137L induction in cancer cells significantly enhances anti-tumor immunity by promoting CD8 + T cell survival, both in vivo and in vitro. Mechanistically, helicase-like transcription factor (HLTF) is identified as a pivotal transcriptional regulator of CD137L , controlling its expression through phosphorylation of serine at position 398. Therapeutically, the AMPK agonist AICAR (acadesine) as an inducer of CD137L , exhibiting synergistic effects with PD-1 or CTLA-4 blockade. In summary, our findings elucidate a mechanism controlling CD137L expression and highlight a promising combination therapy to enhance the efficacy of ICBs in melanoma. One Sentence Summary: Inducing co-stimulatory immune checkpoint CD137L expression in melanoma cells enhances T cell-mediated anti-tumor immunity. Subject terms: Tumour immunology, Cancer immunotherapy

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of ER, PR, and HER2 expression. Its aggressive behavior, high degree of tumor heterogeneity, and immunosuppressive tumor microenvironment (TME) are associated with poor clinical outcomes, rapid disease progression, and limited therapeutic options. Although chimeric antigen receptor (CAR)-engineered T cell therapy has shown certain promise, its applicability in TNBC is hindered by antigen escape, TME-mediated suppression, and the logistical constraints of autologous cell production. In this study, we employed hematopoietic stem and progenitor cell (HSPC) gene engineering and a feeder-free HSPC differentiation culture to generate allogeneic IL-15-enhanced, mesothelin-specific CAR-engineered invariant natural killer T ( Allo15 MCAR-NKT) cells. These cells demonstrated robust and multifaceted antitumor activity against TNBC, mediated by CAR- and NK receptor-dependent cytotoxicity, as well as selective targeting of CD1d + TME immunosuppressive cells through their TCR. In both orthotopic and metastatic TNBC xenograft models, Allo15 MCAR-NKT cells demonstrated potent antitumor activity, associated with robust effector and cytotoxic phenotypes, low exhaustion, and a favorable safety profile without inducing graft-versus-host disease. Together, these results support Allo15 MCAR-NKT cells as a next-generation, off-the-shelf immunotherapy with strong therapeutic potential for TNBC, particularly in the context of metastasis, immune evasion, and treatment resistance. The online version contains supplementary material available at 10.1186/s13045-025-01736-9.

The utility of measuring real-time cellular bioenergetics of peripheral blood mononuclear cells (PBMCs) as biomarkers in disease monitoring, such as the bioenergetic health index, is of emerging interest. However, various experimental factors can impact the accuracy and reproducibility of these measurements. Methods: : PBMC bioenergetics were probed in real-time using extracellular flux analysis to identify optimal seeding density and injection protocol. Using a modified protocol, we assessed the extent to which blood processing time and isolation method (SepMate™ vs. EasySep™ Direct) influence PBMC bioenergetics under basal and stimulated conditions. Advanced metabolic control analysis including mitochondrial and glycolytic ATP supply flux, respiratory control ratio, bioenergetic health index, and mitochondrial toxicity index were used to identify and quantify PBMC bioenergetics. Results: Measures of metabolic profiling such as mitochondrial respiration, glycolytic activity, ATP supply flux, and respiratory control ratio were significantly diminished in PBMCs due to blood processing delay (48–72 hours) and were influenced by isolation method. Extended blood processing time significantly lowered T cell activation capacity in PBMCs, evidenced by decreased responses of mitochondrial and glycolytic ATP supply to CD3/CD28 activation. Discussion/Conclusion: This study demonstrates that key experimental variables including blood processing time and isolation method critically affect the reliability and biological relevance of PBMC metabolic assessments, highlighting the importance of protocol standardisation for accurate bioenergetic biomarker measurements.