Background: Dendritic cells (DCs) are central for the initiation and regulation of innate and adaptive immunity in the tumor microenvironment. As such, many kinds of DC-targeted vaccines have been developed to improve cancer immunotherapy in numerous clinical trials. Targeted delivery of antigens and adjuvants to DCs in vivo represents an important approach for the development of DC vaccines. However, nonspecific activation of systemic DCs and the preparation of optimal immunodominant tumor antigens still represent major challenges. Methods: We loaded the immunogenic cell death (ICD) inducers human neutrophil elastase (ELANE) and Hiltonol (TLR3 agonist) into α-lactalbumin (α-LA)-engineered breast cancer-derived exosomes to form an in situ DC vaccine (HELA-Exos). HELA-Exos were identified by transmission electron microscopy, nanoscale flow cytometry, and Western blot analysis. The targeting, killing, and immune activation effects of HELA-Exos were evaluated in vitro. The tumor suppressor and immune-activating effects of HELA-Exos were explored in immunocompetent mice and patient-derived organoids. Results: HELA-Exos possessed a profound ability to specifically induce ICD in breast cancer cells. Adequate exposure to tumor antigens and Hiltonol following HELA-Exo-induced ICD of cancer cells activated type one conventional DCs (cDC1s) in situ and cross-primed tumor-reactive CD8⁺ T cell responses, leading to potent tumor inhibition in a poorly immunogenic triple negative breast cancer (TNBC) mouse xenograft model and patient-derived tumor organoids. Conclusions: HELA-Exos exhibit potent antitumor activity in both a mouse model and human breast cancer organoids by promoting the activation of cDC1s in situ and thus improving the subsequent tumor-reactive CD8⁺ T cell responses. The strategy proposed here is promising for generating an in situ DC-primed vaccine and can be extended to various types of cancers. Graphic Scheme 1. Schematic illustration of HELA-Exos as an in situ DC-primed vaccine for breast cancer. (A) Allogenic breast cancer-derived exosomes isolated from MDA-MB-231 cells were genetically engineered to overexpress α-LA and simultaneously loaded with the ICD inducers ELANE and Hiltonol (TLR3 agonist) to generate HELA-Exos. (B) Mechanism by which HELA-Exos activate DCs in situ in a mouse xenograft model ofTNBC. HELA-Exos specifically homed to the TME and induced ICD in cancer cells, which resulted in the increased release of tumor antigens, Hiltonol, and DAMPs, as well as the uptake of dying tumor cells by cDC1s. The activated cDC1s then cross-primed tumor-reactive CD8+ T cell responses. (C) HELA-Exos activated DCs in situ in the breast cancer patient PBMC-autologous tumor organoid coculture system. Abbreviations: DCs: dendritic cells; α-LA: α-lactalbumin; HELA-Exos: Hiltonol-ELANE-α-LA-engineered exosomes; ICD: immunogenic cell death; ELANE: human neutrophil elastase; TLR3: Toll-like receptor 3; TNBC: triple-negative breast cancer; TME: tumor microenvironment; DAMPs: damage-associated molecular patterns; cDC1s: type 1 conventional dendritic cells; PBMCs: peripheral blood mononuclear cells

Trained immunity refers to the long-term memory of the innate immune cells. However, little is known about how environmental nutrient availability influences trained immunity. This study finds that physiologic carbon sources impact glucose contribution to the tricarboxylic acid (TCA) cycle and enhance cytokine production of trained monocytes. Our experiments demonstrate that trained monocytes preferentially employe lactate over glucose as a TCA cycle substrate, and lactate metabolism is required for trained immune cell responses to bacterial and fungal infection. Except for the contribution to the TCA cycle, endogenous lactate or exogenous lactate also supports trained immunity by regulating histone lactylation. Further transcriptome analysis, ATAC-seq, and CUT&Tag-seq demonstrate that lactate enhance chromatin accessibility in a manner dependent histone lactylation. Inhibiting lactate-dependent metabolism by silencing lactate dehydrogenase A (LDHA) impairs both lactate fueled the TCA cycle and histone lactylation. These findings suggest that lactate is the hub of immunometabolic and epigenetic programs in trained immunity.