Imaging the Architecture of Granulomas Induced by Mycobacterium tuberculosis Infection with Single-molecule Fluorescence In Situ Hybridization.

Granulomas are an important hallmark of Mycobacterium tuberculosis infection. They are organized and dynamic structures created when immune cells assemble around the sites of infection in the lungs that locally restrict M. tuberculosis growth and the host's inflammatory responses. The cellular architecture of granulomas is traditionally studied by immunofluorescence labeling of surface markers on the host cells. However, very few Abs are available for model animals used in tuberculosis research, such as nonhuman primates and rabbits, and secreted immunological markers such as cytokines cannot be imaged in situ using Abs. Furthermore, traditional phenotypic surface markers do not provide sufficient resolution for the detection of the many subtypes and differentiation states of immune cells. Using single-molecule fluorescence in situ hybridization (smFISH) and its derivatives, amplified smFISH and iterative smFISH, we developed a platform for imaging mRNAs encoding immune markers in rabbit and macaque tuberculosis granulomas. Multiplexed imaging for several mRNA and protein markers was followed by quantitative measurement of the expression of these markers in single cells. An analysis of the combinatorial expressions of these markers allowed us to classify the cells into several subtypes, and to chart their densities within granulomas. For one mRNA target, hypoxia-inducible factor-1α, we imaged its mRNA and protein in the same cells, demonstrating the specificity of the probes. This method paves the way for defining granular differentiation states and cell subtypes from transcriptomic data, identifying key mRNA markers for these cell subtypes, and then locating the cells in the spatial context of granulomas.

S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

The innate memory response of macrophages to Mycobacterium tuberculosis is shaped by the nature of the antigenic stimuli.

Innate immune cells, such as macrophages, mount an immune response upon exposure to antigens and pathogens. Emerging evidence shows that macrophages exposed to an antigen can generate a "memory-like" response (a.k.a. trained immunity), which confers a non-specific and enhanced response upon subsequent stimulation with a second antigen/microbe. This trained immunity has been implicated in the enhanced response of macrophages against several invading pathogens. However, the association between the nature of the antigen and the corresponding immune correlate of elicited trained immunity is not fully understood. Similarly, the response of macrophages trained and restimulated with homologous stimulants to subsequent infection by pathogenic Mycobacterium tuberculosis (Mtb) remains unexplored. Here, we report the immune and metabolic profiles of trained immunity in human THP-1-derived macrophages after homologous training and restimulation with BCG, LPS, purified protein Derivative (PPD), heat-killed Mtb strains HN878 (hk-HN), and CDC1551 (hk-CDC). Furthermore, the impact of training on the autophagic and antimicrobial responses of macrophages with or without subsequent infection by clinical Mtb isolates HN878 and CDC1551 was evaluated. Results show that repeated stimulation of macrophages with different antigens displays distinct pro-inflammatory, metabolic, antimicrobial, and autophagy induction profiles. These macrophages also induce a differential antimicrobial response upon infection with clinical Mtb HN878 and CDC1551 isolates. A significantly reduced intracellular bacterial load was noted in the stimulated macrophages, which was augmented by the addition of rapamycin, an autophagy inducer. These observations suggest that the nature of the antigen and the mode of stimulation shape the magnitude and breadth of macrophage innate memory response, which impacts subsequent response to Mtb infection. IMPORTANCE: Trained immunity (a.k.a. innate memory response) is a novel concept that has been rapidly emerging as a mechanism underpinning the non-specific immunity of innate immune cells, such as macrophages. However, the association between the nature of the stimuli and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the kinetics of immunological and metabolic characteristics of macrophages upon "training" by the same antigen as primary and secondary stimuli (homologous stimulation) are not fully characterized. Furthermore, the ability of antigens such as purified protein derivative (PPD) and heat-killed-Mtb to induce trained immunity remains unknown. Similarly, the response of macrophages primed and trained by homologous stimulants to subsequent infection by pathogenic Mtb is yet to be reported. In this study, we evaluated the hypothesis that the nature of the stimuli impacts the depth and breadth of trained immunity in macrophages, which differentially affects their response to Mtb infection.