Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that poses a severe threat to human health. A variety of highly immunogenic tuberculosis proteins have been used as targets in vaccine development to mitigate the spread of TB. Although Th1-type immunity has long been considered a crucial part of resistance to Mtb, γδ T cells, the predominant source of IL-17, are not negligible in controlling the early stages of TB infection. In addition to classical phosphoantigens, Mycobacterium tuberculosis heat-resistant antigens (HAg), a complex containing 564 proteins obtained from live tuberculosis bacteria after heat treatment at 121 °C for 20 min, have been confirmed to be highly effective γδ T cell stimulators as well. Several studies have demonstrated that HAg-activated γδ T cells can participate in TB immunity by secreting multiple cytokines against Mtb or by interacting with other innate immune cells. In this review, we present a possible mechanism of HAg stimulation of γδ T cells and the role of HAg-activated γδ T cells in anti-TB immunity. We also highlight the limitations of studies on HAg activation of γδ T cells and suggest further research directions on the relationship between HAg and γδ T cells.

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.

Background/Objectives: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major global health problem. Drug resistance and the limitations of sputum-based diagnostic methods highlight the need for additional host-response biomarkers. Protein tyrosine phosphatase receptor type O (PTPRO) has been implicated in inflammatory signaling and macrophage immune regulation, but its relationship with TB-related host transcriptional responses remains unclear. This study aimed to identify and preliminarily evaluate a PTPRO-related blood transcriptional signature with potential relevance to TB discrimination and treatment-response assessment. Methods: Genes correlated with PTPRO expression were first screened using TCGA-LUSC as a large human transcriptomic discovery resource. The resulting candidate genes were then filtered in TB-related whole-blood datasets by intersecting genes upregulated in TB compared with healthy controls, pneumonia, and lung cancer. This strategy yielded a five-gene PTPRO-related signature, termed PO5. The signature was evaluated in independent GEO cohorts and further explored by RT-qPCR in H37Ra-infected THP-1-derived macrophages and in a small clinical blood cohort. A PO5-derived TB risk score was calculated for each sample, and receiver operating characteristic analysis was used to assess discriminatory performance. Changes in TB risk scores during anti-TB treatment were also examined. Results: PTPRO expression was increased in TB whole-blood transcriptomic data and in H37Ra-infected macrophages. In public datasets, PO5 showed potential for distinguishing TB from healthy controls, latent TB, pneumonia, and lung cancer. PO5-derived TB risk scores also decreased after anti-TB treatment. In the exploratory clinical cohort, several PO5 genes showed expression changes in the same general direction as those observed in the public datasets, although the small sample size limited the strength of this evidence. Conclusions: PO5 represents a preliminary PTPRO-related blood transcriptional signature with potential relevance to TB discrimination and treatment-response assessment. These findings remain exploratory and require validation in larger prospective multicenter cohorts, together with further mechanistic studies.