Daniel L. Barber

Pulmonary tuberculosis (TB), caused by lung infection with Mycobacterium tuberculosis ( Mtb ), often manifests both alveolar pneumonia and aggregation of immune cells containing Mtb , termed granulomas, within the same lung. Here, we integrate spatial transcriptomics, single-cell RNA sequencing, high-resolution imaging, and functional assays of lung biopsies from four men with pulmonary TB to compare alveolar pneumonia with adjacent granulomas. We find that alveolar TB pneumonia is enriched for TREM2⁺ lipid-laden macrophages characterized by increased lipid metabolism gene expression, reduced T cell infiltration, attenuated antimicrobial gene expression, and abundant Mtb transcripts and antigens. By contrast, neighboring granulomas exhibit organized lymphoid architecture and robust antimicrobial programs. In vitro, Mtb lipids induce TREM2 expression, activate TREM2–DAP12 signaling, and promote lipid droplet accumulation in primary human monocyte-derived macrophages (MDM). Furthermore, TREM2 ⁺ MDMs are more susceptible to in vitro Mtb infection. When treating MDMs with 1,25-dihydroxyvitamin D₃, which induces antimicrobial macrophage programs, TREM2 ⁺ MDM numbers, lipid droplet abundance and Mtb viability are decreased. Thus, we propose a spatially localized, alveolar pneumonia-intrinsic TREM2⁺ macrophage program that potentially contributes to Mtb persistence in pulmonary TB infection in humans.

IFNγ is considered the primary mediator of adaptive immunity to Mycobacterium tuberculosis (Mtb) infection. In mice, control of Mtb requires IFNγ. In humans, IFNγ is critical for resistance to infection with non-tuberculous mycobacteria (NTM), but its relative requirement for control of pulmonary tuberculosis (TB) is less clear. Here we block IFNγR1 signaling in macaques at different times following Mtb infection. IFNγ blockade from day 45 to 49 post-infection rapidly reduced ¹⁸ FDG-PET/CT scores and broadly enhanced anti-viral-like inflammatory responses. Strikingly, IFNγR1 blockade for the first three months of infection had no impact on bacterial loads despite suppression of bioactive IFNγ in granulomas and changes to host immune responses and granuloma structure. We find individuals from Mtb endemic regions with anti-IFNγ neutralizing autoantibodies who develop NTM disease and not TB despite evidence of previous exposure to Mtb. Lastly, we show that mice over-estimate the importance IFNγ in host resistance to TB due to a species-specific induction of iNOS by IFNγ. Thus, while IFNγ has immunoregulatory effects in granulomas, normal resistance to TB in macaques and humans likely requires little IFNγR1 signaling during infection, indicating that the major mechanisms of adaptive immunity to Mtb infection remain unknown.

Immune dysregulation contributes to death and disability in tuberculous meningitis. People living with HIV have the least evidence that anti-inflammatory therapy improves the poor outcome. Improving therapy relies on a more refined understanding of the host immune response. Single-cell RNA sequencing of 188,983 CSF cells from 25 adults with HIV-associated TBM revealed a predominance of cytotoxic CD8 T cells with low cytokine expression. In microbiologically-confirmed TBM, there was greater cytotoxicity in T, NK and γδ cells, and higher type 1 interferon stimulation in T and B lymphocytes. Neutrophils expressed markers suggesting heightened cytokine stimulation, enhanced effector function, and IL-8-mediated neutrophil recruitment. In a longitudinal cohort, type 1 interferon signaling increased in blood and CSF following treatment initiation. Overall, findings indicate a hyper-inflammatory immune response in the CSF of HIV-associated TBM patients characterised by an accumulation of granzyme-rich cytotoxic CD8 T cells, highly activated neutrophils and host-detrimental type 1 interferon signaling.

In severe cases of COVID-19, glucocorticoid treatment improves clinical outcomes. However, in non-hospitalized patients, glucocorticoids have limited benefit and may impair viral clearance. Here, we used the rhesus macaque model of acute SARS-CoV-2 infection to investigate the impact of glucocorticoids on host responses and viral control in a setting of mild disease. Rhesus macaques were pre-treated with intravenous methylprednisolone for 5 days prior to SARS-CoV-2 (Delta) infection and maintained on a daily oral prednisolone until necropsy at day 13 post infection. Glucocorticoid (GC) treatment decreased local lung inflammation measured with ¹⁸ FDG-PET/CT imaging. GC treated animals also had evidence of elevated SARS-CoV-2 viral titers in the lower airways and pulmonary draining lymph nodes. Glucocorticoid treatment blunted plasmacytoid dendritic cell (pDC), eosinophil, gamma delta T cell and early SARS-CoV-2 specific CD8 T cell responses in the airways. These data reveal the cell types that are directly impacted by immunosuppression with glucocorticoids and provide insights into the mechanism of delayed viral clearance observed with glucocorticoid administration during SARS-CoV-2 infection.

Tuberculosis (TB) remains a leading infectious cause of morbidity and mortality, and the development of a new, highly effective vaccine would have a tremendous beneficial impact on global health. Although conventional memory CD4 and CD8 T cells will likely be key mediators of long‐lived, vaccine‐elicited protection, a potent T cell–inducing vaccine against TB has been elusive. Protection by Mycobacterium tuberculosis (Mtb)‐specific T cells is mediated primarily through their communication with Mtb‐infected macrophages. Here, we discuss emerging evidence of multiple structural and immunoregulatory factors that limit effective T cell–macrophage interactions in TB granulomas, posing a unique challenge to vaccine‐induced protection. Developing new TB vaccination strategies will require a better understanding of the crosstalk between T cells and infected pulmonary macrophages and strategies to enhance these interactions.

The prevalence of pulmonary disease due to nontuberculous mycobacteria (NTM) has been increasing globally. Though NTM-induced pulmonary disease often presents with bronchiectasis, lung nodules, and cavitary disease, the host response associated with these distinct pulmonary injury patterns has not been well characterized in situ. We sought to evaluate mechanisms of NTM-induced pathology by performing deep phenotypic analysis of immune cell populations in lung tissue from individuals with NTM disease in comparison to a gold standard of granulomatous inflammation, tuberculous (TB) lung disease. Formalin-fixed, paraffin-embedded (FFPE) lung blocks from patients with either disseminated NTM, pulmonary NTM, or pulmonary TB disease were sectioned and stained following a modified version of the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method. Optimization of the IBEX method was first required to address the significant endogenous fluorescence of lung sections due to the high density of collagen, elastin, and erythrocytes native to pulmonary tissue that is worsened by FFPE preservation and the age of the FFPE blocks. In this protocol, we detail a photoirradiation protocol and modified antigen retrieval method that reduces the autofluorescence of clinically archived samples. Furthermore, we provide guidance on antibody panel design, including common challenges such as cross-reactivity, epitope loss following photoirradiation, spectral overlap between adjacent fluorophores, and low signal-to-noise for certain targets. To facilitate efficient imaging of whole lung tissue sections, slides were imaged with a widefield microscope and postprocessed with open-source software to improve image quality. Together, this workflow addresses a critical need by documenting how to obtain highly multiplexed, high-resolution, whole-slide images from challenging samples using widely available instrumentation and reagents. Beyond quantitative imaging of clinical samples, these techniques can be applied to diverse sample types to overcome both endogenous and exogenous sources of fluorescence.

Pulmonary tuberculosis (TB) exhibits marked spatial heterogeneity, with alveolar pneumonia and organized granulomas frequently coexisting within the same lung. While granulomas have long dominated conceptual models of TB pathogenesis, the immune programs operating within alveolar TB pneumonia in humans remain incompletely defined. Here, we integrate spatial transcriptomics, single-cell RNA sequencing, high-resolution imaging, and functional assays of human lung biopsies to directly compare alveolar pneumonia with adjacent granulomas from the same individuals. We demonstrate that alveolar TB pneumonia is enriched for TREM2⁺ lipid-laden macrophages characterized by lipid metabolic reprogramming, sparse T-cell infiltration, attenuated antimicrobial gene expression, and abundant Mycobacterium tuberculosis ( Mtb ) transcripts and antigens. In contrast, neighboring granulomas exhibit organized lymphoid architecture and robust antimicrobial programs. Mechanistically, the mycobacterial virulence lipid phthiocerol dimycocerosate (PDIM) and free mycolic acids induce TREM2 expression and activate TREM2–DAP12 signaling, promoting lipid droplet accumulation, suppressing autophagy, and enhancing intracellular Mtb survival in human macrophages. This immunometabolic state is pharmacologically reversible: 1,25-dihydroxyvitamin D₃ downregulates TREM2, restores autophagy, reduces lipid droplets, and limits bacterial viability. Together, these findings define a spatially localized TREM2⁺ foamy macrophage program within alveolar pneumonia that contrasts sharply with adjacent granulomatous immunity, establishing an niche permissive for bacillary persistence and potentially transmission, as well as identifying a tractable host pathway in human TB pathogenesis.

The prevalence of pulmonary disease due to nontuberculous mycobacteria (NTM) has been increasing globally. Though NTM-induced pulmonary disease often presents with bronchiectasis, lung nodules, and cavitary disease, the host response associated with these distinct pulmonary injury patterns has not been well characterized in situ. We sought to evaluate mechanisms of NTM-induced pathology by performing deep phenotypic analysis of immune cell populations in lung tissue from individuals with NTM disease in comparison to a gold standard of granulomatous inflammation, tuberculous (TB) lung disease. Formalin-fixed, paraffin-embedded (FFPE) lung blocks from patients with either disseminated NTM, pulmonary NTM, or pulmonary TB disease were sectioned and stained following a modified version of the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method. Optimization of the IBEX method was first required to address the significant endogenous fluorescence of lung sections due to the high density of collagen, elastin, and erythrocytes native to pulmonary tissue that is worsened by FFPE preservation and the age of the FFPE blocks. In this protocol, we detail a photoirradiation protocol and modified antigen retrieval method that reduces the autofluorescence of clinically archived samples. Furthermore, we provide guidance on antibody panel design, including common challenges such as cross-reactivity, epitope loss following photoirradiation, spectral overlap between adjacent fluorophores, and low signal-to-noise for certain targets. To facilitate efficient imaging of whole lung tissue sections, slides were imaged with a widefield microscope and postprocessed with open-source software to improve image quality. Together, this workflow addresses a critical need by documenting how to obtain highly multiplexed, high-resolution, whole-slide images from challenging samples using widely available instrumentation and reagents. Beyond quantitative imaging of clinical samples, these techniques can be applied to diverse sample types to overcome both endogenous and exogenous sources of fluorescence.