Bovis Bacillus Calmette-Guérin Research Articles

Abstract P29: GSTT2 AND THE RESPONSE TO BCG IMMUNOTHERAPY

Abstract Whilst M. bovis Bacillus Calmette-Guérin (BCG) therapy remains the gold-standard for treatment of high-risk non-muscle invasive bladder cancer (BC), 30-40% of patients fail therapy, resulting in disease recurrence and progression. A better understanding of the underlying mechanisms of BCG therapy can aid in revealing key response pathways, which may lead to the discovery of new targets, and in turn new approaches to treat BC. The glutathione-S-transferase theta 2 (GSTT2) gene is a member of the GST family. Loss of GSTT2 expression has been associated with modulation of intracellular ROS and BCG survival in BC cell lines. Retrospective analysis of BC patients revealed that patients with the GSTT2B deletion responded better to fewer instillations of BCG. To understand these responses, wild-type (WT) and GSTT2-knockout (KO) mice were implanted with MB49-PSA BC cells, in subcutaneous and orthotopic settings. Once tumors were established, the mice were treated with four weekly BCG instillations, after which the subcutaneous tumors and bladders were harvested for downstream analysis. Single-cell RNA sequencing of whole-bladders was used to identify specific cell clusters within the bladder, and to analyze differential gene expression. Hmga2, Ahnak and Peak1 were found to be differentially expressed in bladders from WT and GSTT2-KO mice. These genes are known to be involved in pro- and anti-tumorigenic pathways such as epithelial-mesenchymal transition and inflammation. Additionally, quantitative real-time PCR analysis in the subcutaneous and orthotopic tumors revealed differences in the expression of immune-related genes, especially markers of immune exhaustion such as PD-L1 and CTLA-4. Flow cytometry analysis of subcutaneous tumors also revealed differences in T-cell infiltration. Thus, loss of GSTT2 may influence the response to BCG therapy, through modulating downstream signaling pathways and immune responses. Elucidating such pathways can lead to a better understanding of the mechanism of BCG and its role as a therapy for BC. Citation Format: Mugdha Patwardhan, Kane Toh, Edmund Chiong, Ratha Mahendran. GSTT2 AND THE RESPONSE TO BCG IMMUNOTHERAPY [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P29.

Mycobacterium bovis BCG as immunostimulating agent prevents the severe form of chronic experimental Chagas disease.

There is currently no vaccine against Chagas disease (ChD), and the medications available confer multiple side effects. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produces balanced Th1, Th2, and Th17 modulatory immune responses and has improved efficacy in controlling chronic infections through nonspecific immunity. We aimed to improve the response to infection by inducing a stronger immune response and greater protection against the parasite by trained immunity. BALB/c mice were immunized with BCG subcutaneously, and 60 days later, they were infected with Trypanosoma cruzi intraperitoneally. An evaluation of the progression of the disease from the acute to the chronic stage, analyzing various aspects such as parasitemia, survival, clinical status, and humoral and cellular immune response, as well as the appearance of visceral megas and the histopathological description of target organs, was performed. Vaccination reduced parasitemia by 70%, and 100% survival was achieved in the acute stage; although the presentation of clinical signs was reduced, there was no increase in the antibody titer or in the differential production of the isotypes. Serum cytokine production indicated a proinflammatory response in infected animals, while in those who received BCG, the response was balanced by inducing Th1/Th2-type cytokines, with a better prognosis of the disease in the chronic stage.

Host Genetic Background Influences BCG-Induced Antibodies Cross-Reactive to SARS-CoV-2 Spike Protein.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) protects against childhood tuberculosis; and unlike most vaccines, BCG broadly impacts immunity to other pathogens and even some cancers. Early in the COVID-19 pandemic, epidemiological studies identified a protective association between BCG vaccination and outcomes of SARS-CoV-2, but the associations in later studies were inconsistent. We sought possible reasons and noticed the study populations often lived in the same country. Since individuals from the same regions can share common ancestors, we hypothesized that genetic background could influence associations between BCG and SARS-CoV-2. To explore this hypothesis in a controlled environment, we performed a pilot study using Diversity Outbred mice. First, we identified amino acid sequences shared by BCG and SARS-CoV-2 spike protein. Next, we tested for IgG reactive to spike protein from BCG-vaccinated mice. Sera from some, but not all, BCG-vaccinated Diversity Outbred mice contained higher levels of IgG cross-reactive to SARS-CoV-2 spike protein than sera from BCG-vaccinated C57BL/6J inbred mice and unvaccinated mice. Although larger experimental studies are needed to obtain mechanistic insight, these findings suggest that genetic background may be an important variable contributing to different associations observed in human randomized clinical trials evaluating BCG vaccination on SARS-CoV-2 and COVID-19.

Comparative transcriptomics reveals common and strain-specific responses of human macrophages to infection with Mycobacterium tuberculosis and Mycobacterium bovis BCG

Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (M. bovis) are closely related pathogenic mycobacteria known to cause chronic pulmonary infections in both humans and animals. Despite sharing nearly identical genomes and virulence factors, these two bacteria display variations in host tropism, epidemiology, and clinical presentations. M. bovis Bacillus Calmette–Guérin (BCG) is an attenuated strain of M. bovis commonly utilized as a vaccine for tuberculosis (TB). Nevertheless, the molecular underpinnings of these distinctions and the intricacies of host-pathogen interactions remain areas of ongoing research. In this study, a comparative transcriptomic analysis was conducted on human leukemia macrophages (THP-1) infected with either MTB H37Rv or M. bovis BCG (Tokyo strain) to elucidate common and strain-specific responses at the transcriptional level. RNA sequencing was utilized to characterize the transcriptomes of human primary macrophages infected with MTB or BCG at 6 and 24 h post-infection. The findings indicate that both MTB and BCG induce substantial and dynamic alterations in the transcriptomes of THP-1, with a notable overlap in the quantity and extent of differentially expressed genes (DEGs). Moreover, gene ontology (GO) enrichment analysis unveiled shared pathways related to immune response, cytokine signaling, and apoptosis. The immune response of macrophages to bacterial infections at 6 h exhibited significantly greater intensity compared to that at 24 h. Furthermore, distinct gene sets displaying notable variances between MTB and BCG infections were identified. The profound impact of MTB infection on macrophage gene expression, particularly within the initial 6 h, was evident. Additionally, downregulation of pathways such as Focal adhesion, Rap1 signaling pathway, and Regulation of actin cytoskeleton was observed. The pathways associated with inflammation reactions and cell apoptosis exhibited significant differences, with BCG triggering macrophage apoptosis and MTB enhancing the survival of intracellular bacteria. Our findings reveal that MTB and BCG provoke similar yet distinct transcriptional responses in human macrophages, indicating variations in their pathogenesis and ability to adapt to host environments. These results offer novel insights into the molecular mechanisms governing host-pathogen interactions and may contribute to a deeper understanding of TB pathogenesis.

Caveolin-1 affects early mycobacterial infection and apoptosis in macrophages and mice

Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the deadliest infections in humans. Because Mycobacterium bovis Bacillus Calmette-Guérin (BCG) share genetic similarities with Mycobacterium tuberculosis, it is often used as a model to elucidate the molecular mechanisms of more severe tuberculosis infection. Caveolin-1 has been implied in many physiological processes and diseases, but it's role in mycobacterial infections has barely been studied. We isolated macrophages from Wildtype or Caveolin-1 deficient mice and analyzed hallmarks of infection, such as internalization, induction of autophagy and apoptosis. For in vivo assays we intravenously injected mice with BCG and investigated tissues for bacterial load with colony-forming unit assays, bioactive lipids with mass spectrometry and changes of protein expressions by Western blotting. Our results revealed that Caveolin-1 was important for early killing of BCG infection in vivo and in vitro, controlled acid sphingomyelinase (Asm)-dependent ceramide formation, apoptosis and inflammatory cytokines upon infection with BCG. In accordance, Caveolin-1 deficient mice and macrophages showed higher bacterial burdens in the livers. The findings indicate that Caveolin-1 plays a role in infection of mice and murine macrophages with BCG, by controlling cellular apoptosis and inflammatory host response. These clues might be useful in the fight against tuberculosis.

Heparin-binding hemagglutinin of Mycobacterium tuberculosis inhibits autophagy via TLR4 and drives M2 polarization in macrophages.

Tuberculosis (TB), predominantly caused by Mycobacterium tuberculosis (MTB) infection, remains a prominent global health challenge. Macrophages are the frontline defense against MTB, relying on autophagy for intracellular bacterial clearance. However, MTB can combat and evade autophagy, and it influences macrophage polarization, facilitating immune evasion and promoting infection. We previously found that heparin-binding hemagglutinin (HBHA) inhibits autophagy in A549 cells; however, its role in macrophage autophagy and polarization remains unclear. Bacterial cultures, cell cultures, western blotting, immunofluorescence, macrophage infection assays, siRNA knockdown, and ELISA were used to investigate HBHA's impact on macrophages and its relevance in Mycobacterium infection. HBHA inhibited macrophage autophagy. Expression of recombinant HBHA in Mycobacterium smegmatis (rMS-HBHA) inhibited autophagy, promoting bacterial survival within macrophages. Conversely, HBHA knockout in the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mutant (BCG-ΔHBHA) activated autophagy and reduced bacterial survival. Mechanistic investigations revealed that HBHA may inhibit macrophage autophagy through the TLR4-dependent PI3K-AKT-mTOR signaling pathway. Furthermore, HBHA induced macrophage M2 polarization. Mycobacterium may exploit HBHA to suppress the antimicrobial immune response in macrophages, facilitating intracellular survival, and immune evasion through autophagy inhibition and M2 polarization induction. Our findings may help identify novel therapeutic targets and develop more effective treatments against MTB infection.

Cutting Edge: Cytosolic Receptor AIM2 Is Induced by Peroxisome Proliferator-activated Receptor γ following Mycobacterium tuberculosis Infection of Human Macrophages but Does Not Contribute to IL-1β Release.

AIM2 (absent in melanoma 2), an inflammasome component, mediates IL-1β release in murine macrophages and cell lines. AIM2 and IL-1β contribute to murine control of Mycobacterium tuberculosis (M.tb) infection, but AIM2's impact in human macrophages, the primary niche for M.tb, remains unclear. We show that M.tb, Mycobacterium bovis bacillus Calmette-Guérin (BCG), and M. smegmatis induce AIM2 expression in primary human macrophages. M.tb-induced AIM2 expression is peroxisome proliferator-activated receptor γ (PPARγ)-dependent and M.tb ESX-1-independent, whereas BCG- and M. smegmatis-induced AIM2 expression is PPARγ-independent. PPARγ and NLRP3, but not AIM2, are important for IL-1β release in response to M.tb, and NLRP3 colocalizes with M.tb. This is in contrast to the role for AIM2 in inflammasome activation in mice and peritoneal macrophages. Altogether, we show that mycobacteria induce AIM2 expression in primary human macrophages, but AIM2 does not contribute to IL-1β release during M.tb infection, providing further evidence that AIM2 expression and function are regulated in a cell- and/or species-specific manner.

Saponin TQL1055 adjuvant-containing vaccine confers protection upon Mycobacterium tuberculosis challenge in mice.

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), affects the lungs of infected individuals (pulmonary TB) but can also affect other sites (extrapulmonary TB). The only licensed vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) protects infants and young children but exhibits variable efficacy in protecting against adult pulmonary TB. Poor compliance and prolonged treatment regimens associated with the use of chemotherapy has contributed to the development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Thus, there is an urgent need for the design of more effective vaccines against TB. The development of safe and novel adjuvants for human use is critical. In this study, we demonstrate that saponin-based TQL1055 adjuvant when formulated with a TLR4 agonist (PHAD) and Mtb specific immunodominant antigens (ESAT-6 and Ag85B) and delivered intramuscularly in mice, the SA-TB vaccine induced potent lung immune responses. Additionally, the SA-TB vaccine conferred significant protection against Mtb infection, comparable with levels induced by BCG. These findings support the development of a SA-TB vaccine comprising TQL1055, as a novel, safe and effective TB vaccine for potential use in humans.

Culture filtrate proteins from BCG act as adjuvants for cytotoxic T lymphocyte induction.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is a licensed vaccine against tuberculosis. It requires attenuated live bacteria to be effective, possibly because actively secreted proteins play a critical role in inducing anti-tuberculosis immunity. BCG also functions as an effective adjuvant. Moreover, the effects of BCG components as adjuvants are not important as those of attenuated live BCG, which is used in cancer immunotherapy. However, the BCG secreted proteins have not been paid attention in anticancer immunity. To understand mycobacterial secreted proteins' function, we investigate immune responses to BCG culture filtrate proteins (CFP). Here, CFP strongly induce both antigen-specific CD4+ T cells and specific CD8+ T cells, which may be functional cytotoxic T lymphocytes (CTLs). In this study, we clearly demonstrate that CFP acts as an adjuvant for CTL induction against specific co-administered proteins and propose CFP as a new protein adjuvant. The CTL response shows potent anticancer effects in mice. These findings could provide insight into the contribution of mycobacterial secreted proteins in both anticancer and antimycobacterial immunity.

Specific and non-specific effects of Mycobacterium bovis BCG vaccination in dairy calves.

Bovine tuberculosis (bTB) is a chronic disease mainly caused by Mycobacterium bovis, a zoonotic pathogen with economic significance as it leads to reduced milk and meat production, and high costs for control measures. The Bacillus Calmette-Guérin (BCG) vaccine, primarily used to prevent tuberculosis in humans, has also been studied for controlling bTB. While showing effectiveness in preventing M. bovis infection and disease in cattle, the BCG vaccine can induce non-specific effects on the immune system, enhancing responses to infections caused by unrelated pathogens, and also having non-specific effects on lactation. The aim of this study is to describe both the specific and non-specific effects of BCG vaccination in calves from a commercial dairy herd in central Chile. Diagnosis of M. bovis infection was performed through the IFNγ release assay (IGRA) using ESAT6/CFP-10 and Rv3615c antigens. The records of milk production, somatic cell count (SCC), clinical mastitis (CM) and retained placenta (RP) during the first lactation were compared between vaccinated and non-vaccinated animals. The breed (Holstein Friesian [HF] v/s HF × Swedish Red crossbred [HFSR]) and the season (warm v/s cold) were also analyzed as categorical explanatory variables. Results of IGRA showed significant differences between vaccinated and control groups, indicating a vaccine efficacy of 58.5% at 18 months post vaccination in HFSR crossbred animals. Although milk production did not vary, SCC and CM showed differences between groups, associated to the breed and the season, respectively. When analyzing CM and RP as a whole entity of disease, BCG showed protection in all but the cold season variables. Overall, the BCG vaccine induced protective specific and non-specific effects on health parameters, which may be influenced by the breed of animals and the season. These results provide new features of BCG protection, supporting initiatives for its implementation as a complementary tool in bTB control.

Case of Mycobacterium bovis Prosthetic Joint Infection from Intravesicular BCG

An 89-year-old man was seen for worsening left hip pain. He had a history of bladder cancer status post-transurethral resection of bladder tumor followed by intravesicular instillations of bacillus Calmette-Guérin (BCG). About one year later, he developed worsening pain in his left prosthetic hip that continued to worsen over the next three months. A computed tomography scan of the hip revealed loosening hardware and periprosthetic fluid collection. Aspiration and operative cultures revealed Mycobacterium bovis BCG. Susceptibility testing revealed resistance to pyrazinamide. The patient underwent a surgical washout with retention of hardware. Treatment with isoniazid, rifampin, ethambutol, and pyridoxine was initiated. This case highlights an extremely rare and delayed complication of intravesicular BCG leading to prosthetic joint infection.

Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin.

Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb is a typical intracellular parasite, and macrophages are its main host cells. NLRP3 inflammasome-mediated pyroptosis is a form of programmed cell death implicated in the clearance of pathogenic infections. The bidirectional regulatory effect of endoplasmic reticulum stress (ERS) plays a crucial role in determining cell survival and death. Whether ERS is involved in macrophage pyroptosis with Mtb infection remains unclear. This article aims to explore the regulation of the NLRP3 inflammasome and pyroptosis by ERS in THP-1 macrophages infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The results showed that BCG infection induced THP-1 macrophage ERS, NLRP3 inflammasome activation and pyroptosis, which was inhibited by ERS inhibitor TUDCA. NLRP3 inhibitor MCC950 inhibited THP-1 macrophage NLRP3 inflammasome activation and pyroptosis caused by BCG infection. Compared with specific Caspase-1 inhibitor VX-765, pan-Caspase inhibitor Z-VAD-FMK showed a more significant inhibitory effect on BCG infection-induced pyroptosis of THP-1 macrophages. Taken together, this study demonstrates that ERS mediated NLRP3 inflammasome activation and pyroptosis after BCG infection of THP-1 macrophages, and that BCG infection of THP-1 macrophages induces pyroptosis through canonical and noncanonical pathways.

BCG vaccine-induced mucosal humoral immunity in human nasal associated lymphoid tissue

ObjectivesMycobacterium tuberculosis (Mtb) is the causative pathogen of tuberculosis (TB). TB vaccine studies have long been conducted. Since the discovery of the successful intradermally administered Mycobacterium bovis Bacillus Calmette–Guérin (BCG) vaccine against TB, no licensed TB mucosal vaccine has yet been approved. Our research aims to investigate the suitability of human NALT-derived MNCs as an in vitro human cell culture model to explore the mucosal humoral immune responses to the BCG vaccine. Moreover, to assure that BCG vaccine able to induce the different antibody isotypes including IgG, IgM and IgA. MethodsTonsils from 20 patients who underwent elective tonsillectomies at Madinah Germany Hospital were recruited. Tonsillar mononuclear cells (MNCs) were separated and co-cultured in the presence and absence of the BCG vaccine. ELISA was used to measure BCG-induced IgG, IgM and IgA antibodies in the cell culture supernatants following stimulation and culture. ResultsNasal-associated lymphoid tissue (NALT) cell culture as an in vitro model was successfully used to evaluate the BCG-induced humoral immune response. Significant (p = 0.001, n = 20) levels of specific, anti-TB IgG, IgM, and IgA antibody responses were detected in MNCs stimulated with the BCG vaccine compared with unstimulated MNCs. Significant differences were found between anti-TB antibody classes. Anti-TB IgG antibody was significantly higher (mean = 2.33) than anti-TB IgM (mean = 1.37) antibody (p = 0.0001, n = 20). Anti-TB IgM antibody was significantly higher than IgA (mean = 0.93) antibody (p = 0.0018, n = 20). ConclusionsThe current human model will be beneficial for the future comprehensive study of other immune components such as cellular immune responses to the BCG vaccine. The model would be ideal to design, improve and study the possible intranasal use of the BCG vaccine.

Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.

The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3'-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.

Repetitive vaccination with intravenous BCG is safe and immunogenic in naïve and SIV-infected macaques

Abstract Tuberculosis (TB) is the leading cause of death in people with HIV (PWH) on or off antiretroviral therapy (ART); thus, new TB vaccine candidates should be evaluated in the setting of HIV. Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the live attenuated bacterial TB vaccine given intradermally (ID) at birth, is contraindicated in PWH. We recently demonstrated that intravenous (IV) delivery of BCG elicits superior immunity and protection compared to ID delivery against Mycobacterium tuberculosis (Mtb) challenge in rhesus macaques. To assess IV BCG in an animal model of HIV infection, we immunized SIVmac251-infected macaques on suppresive ART (6m) and uninfected controls. Rhesus macaques received three doses of IV BCG, 8wks apart, and were monitored for the presence of BCG colony forming units (CFU) in peripheral blood and bronchoalveolar lavage (BAL) and Mtb-specific T cell responses in BAL. Repetitive IV BCG immunization was well-tolerated in SIV-infected and uninfected animals, with no difference in clinical parameters or BCG CFU in blood or BAL. T cell responses after IV BCG were similar, with both groups displaying a 10–50 fold increase in BAL T cell abundance after initial dose. Mtb-specific CD4 and CD8 responses peaked after the initial IV BCG dose and were maintained by subsequent immunizations for at least 6m. These data indicate that IV BCG is safe and immunogenic in SIV-infected, ART-suppressed macaques and that IV BCG boosting maintains T cell responses at the site of infection. NIH Intramural Program

Abstract 5648: Single cell spatial phenotyping of bladder tumors with a novel mechanism of NKG2A and HLA-E mediated resistance to BCG immunotherapy

Abstract Background: ~75% of diagnosed bladder tumors are non-muscle-invasive, requiring instillation of M. bovis Bacillus Calmette-Guérin (BCG) with recurrence rates observed in ~50% of patients. Treatments for BCG-resistant bladder tumors have lagged because few studies have tried to understand the relationship between timing of tumor recurrence and the state of immune system at the time of recurrence. Natural Killer (NK) cells are very early responders to tumor cells. HLA-E, like PD-L1, is sensitive to IFN-γ and strongly inhibits NKG2A+ NK and CD8 T cells and is commonly upregulated on tumors. We observed that BCG exposure results in chronic activation of NK and CD8 T cells and their acquisition of NKG2A and PD-1 phenotypes. Upon tumor recurrence in all patients sampled, a subset of the tumor cells aggregated in nests, were activated in response to NK/CD8 T cell-derived IFN-γ, and upregulate HLA-E and PD-L1. Spatial sequencing revealed HLA-EBright tumor nests with high levels of CXCL9/10/11 and in significantly closer proximity to NK/CD8 T cells (and Tregs) compared with HLA-EDim tumor nests that lacked CXCL9/10/11. Here, we apply single cell spatial phenotyping to study major subsets of NK cells in the TME of bladder tumors obtained from patient matched tumor sections before and after BCG therapy. Purpose of the study: The goal of this study is to understand the underlying cell-to-cell interactions that promote BCG resistance in non-muscle-invasive bladder cancer (NMIBC). Methods: The PhenoCycler technology centers around an oligo-based barcoding strategy, in which oligonucleotide-labeled antibodies are imaged in iterative cycles of hybridized/dehybridized complementary oligonucleotide fluorescent reporters. The platform enables (1) whole-slide imaging, (2) single-cell and subcellular resolution, (3) compatibility with a wide range of FFPE tissues, (4) customized content and plexing capability and (5) sophisticated analysis of cell phenotypes and cellular neighborhoods. Results and conclusions: We developed a comprehensive antibody panel for in-depth analysis of the TME of bladder tumors. Our panel centers around a core of 67 markers for cell lineage, immune checkpoints, immune activation markers, tissue structure and vasculature. This core was supplemented with a module of additional antibodies that define complex NK cell phenotypes, including markers of maturation and differentiation, metabolism, activation and inhibition, trafficking, cytolytic killing, as well as cytokines/chemokines that shape the TME. The power of single cell spatial biology is the ability to visualize all major subsets of NK cells and their associated microenvironment simultaneously in single tissue sections. In doing so, we are defining novel functional interactions as new correlates of BCG resistance and identifying pathways that can be exploited for next-gen immunotherapies. Citation Format: Dmytro Klymyshyn, Niyati Jhaveri, Nadine Nelson, Michael Prater, Zhenqin Wu, Steven Hamel, Matthew D. Galsky, Nina Bhardwaj, John P. Sfakianos, Subham Basu, Oliver Braubach, Amir Horowitz. Single cell spatial phenotyping of bladder tumors with a novel mechanism of NKG2A and HLA-E mediated resistance to BCG immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5648.

Dimethyl itaconate is effective in host-directed antimicrobial responses against mycobacterial infections through multifaceted innate immune pathways

BackgroundItaconate, a crucial immunometabolite, plays a critical role in linking immune and metabolic functions to influence host defense and inflammation. Due to its polar structure, the esterified cell-permeable derivatives of itaconate are being developed to provide therapeutic opportunities in infectious and inflammatory diseases. Yet, it remains largely uncharacterized whether itaconate derivatives have potentials in promoting host-directed therapeutics (HDT) against mycobacterial infections. Here, we report dimethyl itaconate (DMI) as the promising candidate for HDT against both Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria by orchestrating multiple innate immune programs.ResultsDMI per se has low bactericidal activity against Mtb, M. bovis Bacillus Calmette–Guérin (BCG), and M. avium (Mav). However, DMI robustly activated intracellular elimination of multiple mycobacterial strains (Mtb, BCG, Mav, and even to multidrug-resistant Mtb) in macrophages and in vivo. DMI significantly suppressed the production of interleukin-6 and -10, whereas it enhanced autophagy and phagosomal maturation, during Mtb infection. DMI-mediated autophagy partly contributed to antimicrobial host defenses in macrophages. Moreover, DMI significantly downregulated the activation of signal transducer and activator of transcription 3 signaling during infection with Mtb, BCG, and Mav.ConclusionTogether, DMI has potent anti-mycobacterial activities in macrophages and in vivo through promoting multifaceted ways for innate host defenses. DMI may bring light to new candidate for HDT against Mtb and nontuberculous mycobacteria, both of which infections are often intractable with antibiotic resistance.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.

Mycobacterium bovis BCG increase the selected determinants of monocyte/macrophage activity, which were diminished in response to gastric pathogen Helicobacter pylori

High antibiotic resistance of gastric pathogen Helicobacter pylori (Hp) and the ability to escape the host immune response prompt searching for therapeutic immunomodulators. Bacillus Calmette–Guerin (BCG) vaccine with Mycobacterium bovis (Mb) is a candidate for modulation the activity of immunocompetent cells, and onco-BCG formulation was successfully used in immunotherapy of bladder cancer. We determined the influence of onco-BCG on the phagocytic capacity of human THP-1 monocyte/macrophage cells, using the model of Escherichia coli bioparticles and Hp fluorescently labeled. Deposition of cell integrins CD11b, CD11d, CD18, membrane/soluble lipopolysaccharide (LPS) receptors, CD14 and sCD14, respectively, and the production of macrophage chemotactic protein (MCP)-1 were determined. Furthermore, a global DNA methylation, was also assessed. Human THP-1 monocytes/macrophages (TIB 202) primed or primed and restimulated with onco-BCG or Hp, were used for assessment of phagocytosis towards E. coli or Hp, surface (immunostaining) or soluble activity determinants, and global DNA methylation (ELISA). THP-1 monocytes/macrophages primed/restimulated with BCG showed increased phagocytosis capacity towards E. coli fluorescent particles, elevated expression of CD11b, CD11d, CD18, CD14, sCD14, increased MCP-1 secretion and DNA methylation. Preliminary results indicate that BCG mycobacteria may also induce the phagocytosis of H. pylori by THP-1 monocytes. Priming or priming and restimulation of monocytes/macrophages with BCG resulted in an increased activity of these cells, which was negatively modulated by Hp.

DUSP1 mediates BCG induced apoptosis and inflammatory response in THP-1 cells via MAPKs/NF-κB signaling pathway

Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Apoptosis and necrosis caused by the interaction between the host and the pathogen, as well as the host’s inflammatory response, play an important role in the pathogenesis of TB. Dual-specificity phosphatase 1 (DUSP1) plays a vital role in regulating the host immune responses. However, the role of DUSP1 in the regulation of THP-1 macrophage apoptosis induced by attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection remains unclear. In the present study, we report that infection with BCG significantly induces macrophage apoptosis and induces the production of DUSP1, TNF-α and IL-1β. DUSP1 knockdown significantly inhibited BCG-induced macrophage apoptosis and activation of MAPKs/NF-κB signaling pathway. In addition, DUSP1 knockdown suppressed BCG-induced inflammation in vivo. Taken together, this study demonstrates that DUSP1, as a regulator of MAPKs/NF-κB signaling pathway, plays a novel role in BCG-induced macrophage apoptosis and inflammatory response.