Background: M2-like tumor-associated macrophages (TAMs) promote an immunosuppressive microenvironment and contribute to tumor progression and metastasis. However, their molecular characterization and prognostic value have not been fully explored in the field of breast cancer.Methods: Weighted gene co-expression network analysis (WGCNA) was used to identify modules significantly associated with M2-like TAMs. Consensus clustering analysis identified three molecular subtypes with distinct clinical features, and we explored potential differences in genomic mutations, pathway enrichment, and immune infiltration in patients between subtypes. Machine learning algorithms were used to screen key genes and construct M2-like macrophage-associated prognostic models. Comprehensive transcriptomic analysis and in vitro phenotyping and polarization experiments were performed on the key gene DLG3.Results: M2-like TAMs infiltration was strongly associated with the prognosis of BC patients, and the associated gene characterization revealed three molecular subtypes, of which C2 has the worst prognosis with high M2 macrophages, immune desert phenotype, and immunotherapeutic resistance; C1 had the best prognosis, rich in stromal and immune cell infiltration, and metabolic pathway activation; and C3 had a high level of TILs and genomic mutations, with a high degree of immunogenicity and immunotherapeutic Potential. Risk scores can effectively predict the prognosis and immunotherapy response of BC patients, in which DLG3 is a key gene that may be involved in shaping the immunosuppressive microenvironment of breast cancer, and down-regulation of DLG3 can inhibit M2 polarization of macrophages.Discussion: We constructed and comprehensively solved a model of M2-like TAM-related molecular subtypes and prognosis, which helps stratify and customize treatment regimens for BC patients. We also explored the role of DLG3 in BC progression and macrophage polarization.

Introduction: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health threat. γδ T cells, critical innate immune responders, provide rapid anti-TB defenses and act as a bridge between innate and adaptive immunity. Studies have demonstrated that γδ T-cell activation by phosphoantigens is mediated by butyrophilin subfamily 3 member A1 (BTN3A1), leading to enhanced cytokine production and cytotoxicity. Mtb heat-resistant antigen (Mtb-HAg), extracted from Mtb H37Ra, specifically activates γδ T cells and induces cytokine secretion. However, the contribution of Mtb-HAg to γδ T cell-mediated cytotoxicity and its dependence on BTN3A1 remain unclear. Methods: This study explored the regulatory mechanism of Mtb-HAg on the cytotoxic function of γδ T cells through RNA-Seq analysis and functional validation methods. The RNA-Seq analysis to profile the transcriptome of Mtb-HAg-activated γδ T cells.The expression of key cytotoxic factors was analyzed using ELISA, and the capacity of activated γδ T cells to inhibit intracellular Mtb growth was assessed using a co-culture assay with Mtb-infected macrophages. The specific role of BTN3A1 was investigated using a blocking antibody to assess its impact on activation markers, cytotoxic factor secretion, and mycobacterial killing efficiency. Results: RNA-Seq analysis revealed that Mtb-HAg-activated γδ T cells are significantly enriched for genes associated with cytotoxic immune responses, with significant upregulation of key cytotoxic factors granzyme B (GzmB) and perforin (PFP), indicating that the cytotoxic function of these cells was activated at the transcriptional level. Subsequently, protein expression analysis revealed that the secretion of GzmB and PFP was increased in Mtb-HAg-activated γδ T cells. Meanwhile, intracellular Mtb growth inhibition assays demonstrated that activated γδ T cells lysed infected target cells and suppressed intracellular Mtb proliferation. We further found that BTN3A1 blockade significantly reduced the expression of CD69 and CD107a in γδ T cells, decreased the secretion of GzmB and PFP, and diminished the killing efficiency of γδ T cells against Mtb-infected macrophages. Conclusion: Our findings demonstrated that Mtb-HAg enhances the cytolytic activity of γδ T cells and inhibits intracellular Mtb growth, with BTN3A1 playing a regulatory role in these processes.

Purpose: The only World Health Organization (WHO)-approved vaccine for tuberculosis (TB), Bacillus Calmette–Guérin (BCG), shows limited efficacy, particularly against adult pulmonary TB. Moreover, traditional subunit vaccines often suffer from poor immunogenicity. To address these challenges, we aimed to enhance the immune activation potential of the early secreted antigenic target of 6 kDa (ESAT-6) by developing a novel nanoparticle-based vaccine platform. Methods: We engineered a nanovaccine using mi3 nanoparticles to display the ESAT-6 antigen on their surface. These nanoparticles self-assembled into uniform dodecahedral structures, enabling enhanced antigen presentation. To evaluate its immunogenicity, mice (n= 6 per group) were primed with BCG and subsequently boosted with the ESAT-6–mi3 nanovaccine. Control groups included mice receiving BCG alone or unconjugated nanoparticles. Results: Compared with mice that received BCG alone or unconjugated nanoparticles, those boosted with the ESAT-6–mi3 nanoparticle vaccine exhibited significantly stronger immune responses. Antigen-specific antibody titers increased by more than 10-fold compared to the BCG-only group. Additionally, elevated levels of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-2 were observed, indicating a robust Th1/Th17-biased cellular immune response. Our findings indicate that the vaccine candidate can elicit effective immune responses in vitro. However, it should be noted that this study has limitations, primarily including the lack of an in vivo pathogen challenge to assess actual protective efficacy and the evaluation of immune responses against only a single antigen. Therefore, these results require further validation in future studies involving challenge experiments and multi-antigen strategies. Conclusion: Our findings demonstrate that the ESAT-6–mi3 nanoparticle vaccine markedly enhances both humoral and cellular immune responses, outperforming traditional subunit vaccine approaches. This strategy shows strong potential as a next-generation booster vaccine to complement BCG and improve TB immunization outcomes.