Introduction Clearance of damaged cells and debris is beneficial for the functional recovery after ischemic brain injury. However, the specific phagocytic receptor that mediates microglial phagocytosis after ischemic stroke is unknown. Aim To investigate whether P2Y6 receptor‐mediated microglial phagocytosis is beneficial for the debris clearance and functional recovery after ischemic stroke. Results The expression of the P2Y6 receptor in microglia increased within 3 days after transient middle cerebral artery occlusion. Inhibition of microglial phagocytosis by the selective inhibitor MRS2578 enlarged the brain atrophy and edema volume after ischemic stroke, subsequently aggravated neurological function as measured by modified neurological severity scores and Grid walking test. MRS2578 treatment had no effect on the expression of IL‐1α, IL‐1β, IL‐6, IL‐10, TNF‐α, TGF‐β, and MPO after ischemic stroke. Finally, we found that the expression of myosin light chain kinase decreased after microglial phagocytosis inhibition in the ischemic mouse brain, which suggested that myosin light chain kinase was involved in P2Y6 receptor‐mediated phagocytosis. Conclusion Our results indicate that P2Y6 receptor‐mediated microglial phagocytosis plays a beneficial role during the acute stage of ischemic stroke, which can be a therapeutic target for ischemic stroke.

BackgroundTraditional tumor thermal ablations, such as radiofrequency ablation (RFA) and cryoablation, can result in good local control of tumor, but traditional tumor thermal ablations are limited by poor long-term survival due to the failure of control of distal metastasis. Our previous studies developed a novel cryo-thermal therapy to treat the B16F10 melanoma mouse model. Long-term survival and T-cell-mediated durable antitumor immunity were achieved after cryo-thermal therapy, but whether tumor antigen-specific T-cells were augmented by cryo-thermal therapy was not determined.MethodsThe long-term antitumor therapeutic efficacy of cryo-thermal therapy was performed in B16F10 murine melanoma models. Splenocytes derived from mice treated with RFA or cryo-thermal therapy were coincubated with tumor antigen peptides to detect the frequency of antigen specific CD4+ and CD8+ T-cells by flow cytometry. Splenocytes were then stimulated and expanded by αCD3 or peptides and adoptive T-cell therapy experiments were performed to identify the antitumor efficacy of T-cells induced by RFA and cryo-thermal therapy. Naïve mice and tumor-bearing mice were used as control groups.ResultsLocal cryo-thermal therapy generated a stronger systematic antitumor immune response than RFA and a long-lasting antitumor immunity that protected against tumor rechallenge. In vitro studies showed that the antigen-specific CD8+ T-cell response was induced by both cryo-thermal therapy and RFA, but the strong neoantigen-specific CD4+ T-cell response was only induced by cryo-thermal therapy. Cryo-thermal therapy-induced strong antitumor immune response was mainly mediated by CD4+ T-cells, particularly neoantigen-specific CD4+ T-cells.ConclusionCryo-thermal therapy induced a stronger and broader antigen-specific memory T-cells. Specifically, cryo-thermal therapy, but not RFA, led to a strong neoantigen-specific CD4+ T-cell response that mediated the resistance to tumor challenge.

BackgroundMyeloid‐derived suppressor cells (MDSCs) can potently inhibit T-cell activity, promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive activities is intimately associated with cancer immunotherapy. Natural killer (NK) cells can engage in crosstalk with multiple myeloid cells to alter adaptive immune responses, triggering T-cell immunity. However, whether the NK-cell-MDSC interaction can modulate the T-cell immune response requires further study. Cryo-thermal therapy could induce the maturation of MDSCs by creating an acute inflammatory environment to elicit a CD4+Th1-dominant immune response, but the mechanism regulating this process remains unclear.MethodsNK cells were depleted and NKG2D was blocked with monoclonal antibodies in vivo. MDSCs, NK cells and T cells were assessed by flow cytometry and isolated by magnetic-activated cell sorting (MACS). MDSCs and NK cells were cocultured with T cells to determine their immunological function. The transcriptional profiles of MDSCs were measured by qRT-PCR and RNA-sequencing. Isolated NK cells and MDSCs by MACS were cocultured to study the viability and maturation of MDSCs regulated by NK cells. TIMER was used to comprehensively examine the immunological, clinical, and genomic features of tumors.ResultsNK-cell activation after cryo-thermal therapy decreased MDSC accumulation and reprogrammed immunosuppressive MDSCs toward a mature phenotype to promote T cell antitumor immunity. Furthermore, we discovered that NK cells could kill MDSCs via the NKG2D-NKG2DL axis and promote MDSC maturation by interferon gamma (IFN-γ) in response to NKG2D. In addition, CD4+Th1-dominant antitumor immune response was dependent on NKG2D, which promoted the major histocompatibility complex Ⅱ pathway of MDSCs. High activated NK-cell infiltration and NKG2D level in tumors were positively correlated with better clinical outcomes.ConclusionsCryo-thermal therapy induces effective CD4+Th1-dominant antitumor immunity by activating NK cells to reprogram MDSCs, providing a promising therapeutic strategy for cancer immunotherapy.

Macrophages play critical roles in both innate and adaptive immunity and are known for their high plasticity in response to various external signals. Macrophages are involved in regulating systematic iron homeostasis and they sequester iron by phagocytotic activity, which triggers M1 macrophage polarization and typically exerts antitumor effects. We previously developed a novel cryo-thermal therapy that can induce the mass release of tumor antigens and damage-associated molecular patterns (DAMPs), promoting M1 macrophage polarization. However, that study did not examine whether iron released after cryo-thermal therapy induced M1 macrophage polarization; this question still needed to be addressed. We hypothesized that cryo-thermal therapy would cause the release of a large quantity of iron to augment M1 macrophage polarization due to the disruption of tumor cells and blood vessels, which would further enhance antitumor immunity. In this study, we investigated iron released in primary tumors, the level of iron in splenic macrophages after cryo-thermal therapy and the effect of iron on macrophage polarization and CD4+ T cell differentiation in metastatic 4T1 murine mammary carcinoma. We found that a large amount of iron was released after cryo-thermal therapy and could be taken up by splenic macrophages, which further promoted M1 macrophage polarization by inhibiting ERK phosphorylation. Moreover, iron promoted DC maturation, which was possibly mediated by iron-induced M1 macrophages. In addition, iron-induced M1 macrophages and mature DCs promoted the differentiation of CD4+ T cells into the CD4 cytolytic T lymphocytes (CTL) subset and inhibited differentiation into Th2 and Th17 cells. This study explains the role of iron in cryo-thermal therapy-induced antitumor immunity from a new perspective.