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Tumor patients often exhibit limited responses to immunotherapy owing to the low immunogenicity and immunosuppressive environment of tumors. Our previous studies revealed that cryo-thermal therapy caused tumor cell rupture due to mechanical compression, notably causing the release of a substantial amount of DAMPs (danger-associated molecular patterns), such as heat shock protein 70, calreticulin and high-mobility group box protein 1; the release of these DAMPs increased myeloid cell maturation, thereby reshaping the systemic immune environment and ultimately inducing durable CD4+ T helper type 1 (Th1) cell-dominated antitumor immunity. In fact, under conditions of mechanical stress and rapid temperature changes, the disruption of tumor cells caused by cryo-thermal therapy results in extensive deoxyribonucleic acid (DNA) damage and the rapid release of substantial amounts of DNA. Consequently, tumor-derived DNA, which potently activates innate immunity by engaging multiple DNA sensors, plays a pivotal role in orchestrating antitumor immunity. We hypothesized that cryo-thermal therapy induces the transient release of high levels of DNA, which modulates CD11b+ myeloid cell function, subsequently influencing CD4+ Th1-cell dominated antitumor immune responses. In this study, a B16F10 melanoma model was established, and DNA concentrations were measured at different time points after cryo-thermal therapy. Deoxyribonuclease I (DNase I) was subsequently administered immediately following cryo-thermal therapy to deplete extracellular DNA, allowing an investigation of the role of DNA in regulating CD11b+ myeloid cell function and CD4+ T cell differentiation. The phenotype and function of CD11b+ myeloid cells and CD4+ T cells were assessed by flow cytometry, RNA sequencing, and cell culture in vitro. Our studies confirmed that cryo-thermal therapy triggered a transient release of high levels of DNA, which was internalized by CD11b+ myeloid cells via C-type lectin receptors and subsequently sensed by inflammasomes. Then, the intracellular sensing of DNA induced the production of the mature form of interleukin (IL)-18, ultimately promoting the Th1 differentiation of CD4+ T cells. This study highlights the pivotal role of DNA release after cryo-thermal therapy in driving CD4+ Th1 cell-dominant antitumor immunity.

CD4+ T cells have been well-regarded as ¡°helper¡± cells in activating the cytotoxicity of CD8+ T cells for effective tumor eradication, while few studies have focused on whether CD8+ T cells regulate CD4+ T cells. Our previous studies provided evidence for an interaction between CD4+ and CD8+ T cells after cryo-thermal therapy, but the mechanism remains unclear, especially pertaining to how CD8+ T cells promote the Th1 differentiation of CD4+ T cells. This study revealed that activated CD4+ and CD8+ T cells are critical for CTT-induced antitumor immunity, and the interaction between activated T cells is enhanced. The reciprocal regulation of activated CD8+ and CD4+ T cells was through LFA-1/ICAM-1 interactions, in which CD8+ T cells facilitate Notch1-dependent CD4+ Th1-dominant differentiation and promote IL-2 secretion of CD4+ T cells. Meanwhile, IL-2 derived from CD4+ T cells enhances the cytotoxicity of CD8+ T cells and establishes a positive feedback loop via increasing the expression of LFA-1 and ICAM-1 on T cells. Clinical analyses further validated that LFA-1/ICAM interactions between CD4+ and CD8+ T cells are correlated with clinical outcomes. Our study extends the functions of the LFA-1/ICAM-1 adhesion pathway, indicating its novel role in the interaction of CD4+ and CD8+ T cells.

IntroductionRecently, more and more research illustrated the importance of inducing CD4+ T helper type (Th)-1 dominant immunity for the success of tumor immunotherapy. Our prior studies revealed the crucial role of CD4+ Th1 cells in orchestrating systemic and durable antitumor immunity, which contributes to the satisfactory outcomes of the novel cryo-thermal therapy in the B16F10 tumor model. However, the mechanism for maintaining the cryo-thermal therapy-mediated durable CD4+ Th1-dominant response remains uncovered. Additionally, cryo-thermal-induced early-stage CD4+ Th1-dominant T cell response showed a correlation with the favorable prognosis in patients with colorectal cancer liver metastasis (CRCLM). We hypothesized that CD4+ Th1-dominant differentiation induced during the early stage post cryo-thermal therapy would affect the balance of CD4+ subsets at the late phase.MethodsTo understand the role of interferon (IFN)-¦Ã, the major effector of Th1 subsets, in maintaining long-term CD4+ Th1-prone polarization, B16F10 melanoma model was established in this study and a monoclonal antibody was used at the early stage post cryo-thermal therapy for interferon (IFN)-¦Ã signaling blockade, and the influence on the phenotypic and functional change of immune cells was evaluated.ResultsIFN¦Ã at the early stage after cryo-thermal therapy maintained long-lasting CD4+ Th1-prone immunity by directly controlling Th17, Tfh, and Tregs polarization, leading to the hyperactivation of Myeloid-derived suppressor cells (MDSCs) represented by abundant interleukin (IL)-1¦Â generation, and thereby further amplifying Th1 response.DiscussionOur finding emphasized the key role of early-phase IFN¦Ã abundance post cryo-thermal therapy, which could be a biomarker for better prognosis after cryo-thermal therapy.