Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia.

Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

SBT-272 improves TDP-43 pathology in ALS upper motor neurons by modulating mitochondrial integrity, motility, and function.

Mitochondrial defects are one of the common underlying causes of neuronal vulnerability in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most commonly observed proteinopathy. Disrupted inner mitochondrial membrane (IMM) reported in the upper motor neurons (UMNs) of ALS patients with TDP-43 pathology is recapitulated in the UMNs of well-characterized hTDP-43 mouse model of ALS. The construct validity, such as shared and common cellular pathology in mice and human, offers a unique opportunity to test treatment strategies that may translate to patients. SBT-272 is a well-tolerated brain-penetrant small molecule that stabilizes cardiolipin, a phospholipid found in IMM, thereby restoring mitochondrial structure and respiratory function. We investigated whether SBT-272 can improve IMM structure and health in UMNs diseased with TDP-43 pathology in our well-characterized UMN reporter line for ALS. We found that SBT-272 significantly improved mitochondrial structural integrity and restored mitochondrial motility and function. This led to improved health of diseased UMNs in vitro. In comparison to edaravone and AMX0035, SBT-272 appeared more effective in restoring health of diseased UMNs. Chronic treatment of SBT-272 for sixty days starting at an early symptomatic stage of the disease in vivo led to a significant reduction in astrogliosis, microgliosis, and TDP-43 pathology in the ALS motor cortex. Our results underscore the therapeutic potential of SBT-272, especially within the context of TDP-43 pathology and mitochondrial dysfunction.

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in convalescent COVID-19 patients.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including convalescent COVID-19 and sero-negative controls. Flow cytometry analyses revealed reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells within the patients who have recovered from severe COVID-19. sc-RNA seq analysis identifies seven heterogeneous clusters of monocytes and nine Treg clusters featuring distinct molecular signatures in association with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocytes and Tregs expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters featuring S100 family genes: one monocyte cluster of S100A8 & A9 coupled with high HLA-I and another cluster of S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, as well as a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-lived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (≥ 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

Feeding mode influences dynamic gut microbiota signatures and affects susceptibility to anti-CD3 mAb-induced intestinal injury in neonatal mice.

Feeding modes influence the gut microbiome, immune system, and intestinal barrier homeostasis in neonates; how feeding modes impact susceptibility to neonatal gastrointestinal (GI) diseases is still uncertain. Here, we investigated the impact of dam feeding (DF) and formula feeding (FF) on features of the gut microbiome and physiological inflammation during the first 2 days of postnatal development and on the susceptibility to intestinal injury related to the inflammatory state in neonatal mouse pups. 16S rRNA sequencing data revealed microbiome changes, lower α-diversity, and a distinct pattern of ß-diversity including expansion of f_Enterobacteriaceae and f_Enterococcaceae in the ileum of FF pups compared with DF pups by postnatal day (P)2. Together with gut dysbiosis, the FF cohort also had greater ileal mucosa physiological inflammatory activity compared with DF pups by P2 but maintained normal histological features. Interestingly, FF but not DF mouse pups developed necrotizing enterocolitis (NEC)-like intestinal injury within 24 h after anti-CD3 mAb treatment, suggesting that FF influences the susceptibility to intestinal injury in neonates. We further found that NEC-like incidence in anti-CD3 mAb-treated FF neonatal pups was attenuated by antibiotic treatment. Collectively, our data suggest that FF predisposes mouse pups to anti-CD3 mAb-induced intestinal injury due to abnormal f_Enterobacteriaceae and f_Enterococcaceae colonization. These findings advance our understanding of FF-associated microbial colonization and intestinal inflammation, which may help inform the development of new therapeutic approaches to GI diseases like NEC in infants.NEW & NOTEWORTHY This report shows that a feeding mode profoundly affects gut colonization in neonatal mice. Furthermore, our results demonstrate that formula feeding predisposes mouse pups to anti-CD3 mAb-induced necrotizing enterocolitis (NEC)-like intestinal injury upon inadequate microbial colonization. The study suggests the role of the combined presence of formula feeding-associated dysbiosis and mucosal inflammation in the pathogenesis of NEC and provides a new mouse model to study this disease.

Improving the Quality and Reproducibility of Flow Cytometry in the Lung. An Official American Thoracic Society Workshop Report.

Defining responses of the structural and immune cells in biologic systems is critically important to understanding disease states and responses to injury. This requires accurate and sensitive methods to define cell types in organ systems. The principal method to delineate the cell populations involved in these processes is flow cytometry. Although researchers increasingly use flow cytometry, technical challenges can affect its accuracy and reproducibility, thus significantly limiting scientific advancements. This challenge is particularly critical to lung immunology, as the lung is readily accessible and therefore used in preclinical and clinical studies to define potential therapeutics. Given the importance of flow cytometry in pulmonary research, the American Thoracic Society convened a working group to highlight issues and technical challenges to the performance of high-quality pulmonary flow cytometry, with a goal of improving its quality and reproducibility.

A novel murine model of differentiation-mediated cytomegalovirus reactivation from latently infected bone marrow haematopoietic cells.

CD34+ myeloid lineage progenitor cells are an important reservoir of latent human cytomegalovirus (HCMV), and differentiation to macrophages or dendritic cells (DCs) is known to cause reactivation of latent virus. Due to its species-specificity, murine models have been used to study mouse CMV (MCMV) latency and reactivation in vivo. While previous studies have shown that MCMV genomic DNA can be detected in the bone marrow (BM) of latently infected mice, the identity of these cells has not been defined. Therefore, we sought to identify and enrich for cellular sites of MCMV latency in the BM haematopoietic system, and to explore the potential for establishing an in vitro model for reactivation of latent MCMV. We studied the kinetics and cellular characteristics of acute infection and establishment of latency in the BM of mice. We found that while MCMV can infect a broad range of haematopoietic BM cells (BMCs), latent virus is only detectable in haematopoietic stem cells (HSCs), myeloid progenitor cells, monocytes and DC-enriched cell subsets. Using three separate approaches, MCMV reactivation was detected in association with differentiation into DC-enriched BMCs cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) followed by lipopolysaccharide (LPS) treatment. In summary, we have defined the kinetics and cellular profile of MCMV infection followed by the natural establishment of latency in vivo in the mouse BM haematopoietic system, including the haematopoietic phenotypes of cells that are permissive to acute infection, establish and harbour detectable latent virus, and can be stimulated to reactivate following DC enrichment and differentiation, followed by treatment with LPS.

Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas.

The success of programmed cell death 1 (PD-1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)-positive; 14 HPV-negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4+ and CD8+ T-cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD-1, TIM-3 and CTLA-4. Next-generation sequencing of the T-cell receptor (TCR) ß chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV-negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8+ T cells expressing CTLA-4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV-positive patients, as well as higher expression levels of TIM-3+ PD-1+ CD8+ T cells (P = 0.0072) than controls. For all patients, PD-1 expression on CD8+ T cells - particularly in HPV-negative HNSCC cases - strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8+ TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV-positive patients had frequently expanded TCR clones that were more hydrophobic - and potentially more immunogenic - than those from HPV-negative patients. Collectively, our findings demonstrate, for the first time, that high-stage HPV-negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA-4+ CD8+ T-cell levels, that tumour-familiar CD8+ T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV-positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Microparticle levels after arterial injury and NO therapy in diabetes.

BACKGROUND: Little is known about how arterial injury, nitric oxide (NO), or the diabetic milieu impact microparticle (MP) levels in the vasculature. We hypothesized that MP levels would increase following local arterial injury, and that NO would modify MP levels differently based on the metabolic environment. MATERIALS AND METHODS: Type 1 diabetes was induced in male Lean Zucker (LZ) rats with streptozotocin, and type 2 diabetes was induced in male Zucker diabetic fatty rats through diet. Lean Zucker rats served as nondiabetic controls. The rat carotid balloon injury was performed ± NO (n > 4/group). Blood was obtained at intervals from baseline to 14 d after injury and analyzed for platelet MP (PMP), leukocyte MP (LMP), and endothelial MP (EMP) using fluorescence-activated cell sorting (FACS) analysis. RESULTS: At baseline, type 1 diabetic rats had the highest EMP levels (P < 0.05). After arterial injury, type 1 and type 2 diabetic rats had a transient increase in EMP levels (P < 0.05) before decreasing below baseline levels. Both LMP and PMP levels generally declined after injury in all three animal models but were the lowest in both type 1 and type 2 diabetic rats. NO therapy had little impact on MP levels in nondiabetic and type 1 diabetic rats after injury. Conversely, NO caused a dramatic increase in EMP, LMP, and PMP levels in type 2 diabetic animals at early time points after injury (P < 0.05). CONCLUSIONS: These data demonstrate that the diabetic milieu impacts MP levels at baseline, after arterial injury and with NO treatment.

FLT3 and NPM-1 mutations in a cohort of acute promyelocytic leukemia patients from India.

BACKGROUND: Acute promyelocytic leukemia (APL) with t (15;17) is a distinct category of acute myeloid leukemia (AML) and is reported to show better response to anthracyclin based chemotherapy. A favorable overall prognosis over other subtypes of AML has been reported for APL patients but still about 15% patients relapse. METHODS: This study evaluated the presence of Famus like tyrosine kinase-3 (FLT3) and nucleophosmin-1 (NPM1) gene mutations in a cohort of 40 APL patients. Bone marrow/peripheral blood samples from patients at the time of diagnosis and follow-up were processed for immunophenotyping, cytogenetic markers and isolation of DNA and RNA. Samples were screened for the presence of mutations in FLT3 and NPM1 genes using polymerase chain reaction followed by sequencing. RESULTS: Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45%) in the present population of APL patients.

Physics-driven structural docking and protein language models accelerate antibody screening and design for broad-spectrum antiviral therapy.

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. These viruses infect human host cells via the viral spike protein binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). Using over 1300 IgG sequences derived from convalescent patient B cells that bind with spike's receptor binding domain (RBD), we first established protein structural docking models in assessing the RBD-IgG-ACE2 interaction interfaces and predicting the virus-neutralizing activity of each IgG with a confidence score. Additionally, employing Gaussian process regression (also known as Kriging) in a latent space of an antibody language model, we predicted the landscape of IgGs' activity profiles against individual coronaviral variants of concern. With functional analyses and experimental validations, we efficiently prioritized IgG candidates for neutralizing a broad spectrum of viral variants (wildtype, Delta, and Omicron) to prevent the infection of host cells in vitro and hACE2 transgenic mice in vivo. Furthermore, the computational analyses enabled rational redesigns of selective IgG clones with single amino acid substitutions at the RBD-binding interface to improve the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screening and re-design even in low-data regimes combining protein language models and Kriging for antibody sequence analysis, activity prediction, and efficacy improvement, in synergy with physics-driven protein docking models for antibody-antigen interface structure analyses and functional optimization.

Normal bone marrow signal-transduction profiles: a requisite for enhanced detection of signaling dysregulations in AML.

Molecular and cytogenetic alterations are involved in virtually every facet of acute myeloid leukemia (AML), including dysregulation of major signal-transduction pathways. The present study examines 5 phosphoproteins (pErk, pAkt, pS6, pStat3, and pStat5) in response to 5 cytokine/growth factors (stem cell factor [SCF], Flt-3/Flk-2 ligand [FL], granulocyte/macrophage-colony stimulating factor [GM-CSF], interleukin-3 [IL-3], and granulocyte-CSF [G-CSF]) within 7 immunophenotypically defined populations, spanning progenitor to mature myeloid/myelomonocytic cells in normal bone marrows with further comparison to AML samples. The normal cohort showed pathway-specific responses related to lineage, maturation, and stimulus. Heterogeneous-signaling responses were seen in homogeneous immunophenotypic subsets emphasizing the additive information of signaling. These profiles provided a critical baseline for detection of dysregulated signaling in AML falling into 4 broad categories, viz lack of response, increased activation, altered constitutive expression, and dysregulated response kinetics, easily identified in 10 of 12 AMLs. These studies clearly show robust and reproducible flow cytometry phosphoprotein analyses capable of detecting abnormal signal-transduction responses in AML potentially contributing to definitive reliable identification of abnormal cells. As functional correlates of underlying genetic abnormalities, signal-transduction abnormalities may provide more stable indicators of abnormal cells than immunophenotyping which frequently changes after therapy and disease recurrence.

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies.

BACKGROUND: Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression. MATERIALS AND METHODS: A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously. RESULTS: A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro. CONCLUSION: Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.

Development of a novel humanized mouse model to study bronchopulmonary dysplasia.

Rationale: The role of circulating fetal monocytes in bronchopulmonary dysplasia is not known. We utilized a humanized mouse model that supports human progenitor cell engraftment (MISTRG) to test the hypothesis that prenatal monocyte programming alters early lung development and response to hyperoxia. Methods: Cord blood-derived monocytes from 10 human infants were adoptively transferred into newborn MISTRG mice at p0 (1 × 106 cells/mouse, intrahepatic injection) followed by normoxia versus hyperoxia (85% oxygen × 14 days). Lungs were harvested at p14 for alveolar histology (alveolar count, perimeter and area) and vascular parameters (vWF staining for microvessel density, Fulton's index). Human CD45 staining was conducted to compare presence of hematopoietic cells. Murine lung parameters were compared among placebo and monocyte-injected groups. The individual profiles of the 10 patients were further considered, including gestational age (GA; n = 2 term, n = 3 moderate/late preterm, and n = 5 very preterm infants) and preeclampsia (n = 4 patients). To explore the monocyte microenvironment of these patients, 30 cytokines/chemokines were measured in corresponding human plasma by multiplex immunoassay. Results: Across the majority of patients and corresponding mice, MISTRG alveolarization was simplified and microvessel density was decreased following hyperoxia. Hyperoxia-induced changes were seen in both placebo (PBS) and monocyte-injected mice. Under normoxic conditions, alveolar development was altered modestly by monocytes as compared with placebo (P < 0.05). Monocyte injection was associated with increased microvessel density at P14 as compared with placebo (26.7 ± 0.73 vs. 18.8 ± 1.7 vessels per lung field; P < 0.001). Pooled analysis of patients revealed that injection of monocytes from births complicated by lower GA and preeclampsia was associated with changes in alveolarization and vascularization under normoxic conditions. These differences were modified by hyperoxia. CD45+ cell count was positively correlated with plasma monocyte chemoattractant protein-1 (P < 0.001) and macrophage inflammatory protein-1ß (P < 0.01). Immunohistochemical staining for human CD206 and mouse F4/80 confirmed absence of macrophages in MISTRG lungs at P14. Conclusions: Despite the inherent absence of macrophages in early stages of lung development, immunodeficient MISTRG mice revealed changes in alveolar and microvascular development induced by human monocytes. MISTRG mice exposed to neonatal hyperoxia may serve as a novel model to study isolated effects of human monocytes on alveolar and pulmonary vascular development.

Inflammatory Cell Dynamics after Murine Femoral Artery Wire Injury: A Multi-Parameter Flow Cytometry-Based Analysis.

An acute inflammatory response following arterial surgery for atherosclerosis, such as balloon angioplasty, stenting, and surgical bypass, is an important driver of neointimal hyperplasia after arterial injury, which leads to recurrent ischemia. However, a comprehensive understanding of the dynamics of the inflammatory infiltrate in the remodeling artery is difficult to attain due to the shortcomings of conventional methods such as immunofluorescence. We developed a 15-parameter flow cytometry method to quantitate leukocytes and 13 leukocyte subtypes in murine arteries at 4 time points after femoral artery wire injury. Live leukocyte numbers peaked at 7 days, which preceded the peak neointimal hyperplasia lesion at 28 days. Neutrophils were the most abundant early infiltrate, followed by monocytes and macrophages. Eosinophils were elevated after 1 day, while natural killer and dendritic cells gradually infiltrated over the first 7 days; all decreased between 7 and 14 days. Lymphocytes began accumulating at 3 days and peaked at 7 days. Immunofluorescence of arterial sections demonstrated similar temporal trends of CD45+ and F4/80+ cells. This method allows for the simultaneous quantitation of multiple leukocyte subtypes from small tissue samples of injured murine arteries and identifies the CD64+Tim4+ macrophage phenotype as being potentially important in the first 7 days post-injury.

A distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia.

Pathogen clearance and resolution of inflammation in patients with pneumonia require an effective local T cell response. Nevertheless, local T cell activation may drive lung injury, particularly during prolonged episodes of respiratory failure characteristic of severe SARS-CoV-2 pneumonia. While T cell responses in the peripheral blood are well described, the evolution of T cell phenotypes and molecular signatures in the distal lung of patients with severe pneumonia caused by SARS-CoV-2 or other pathogens is understudied. Accordingly, we serially obtained 432 bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia and respiratory failure, including 74 unvaccinated patients with COVID-19, and performed flow cytometry, transcriptional, and T cell receptor profiling on sorted CD8+ and CD4+ T cell subsets. In patients with COVID-19 but not pneumonia secondary to other pathogens, we found that early and persistent enrichment in CD8+ and CD4+ T cell subsets correlated with survival to hospital discharge. Activation of interferon signaling pathways early after intubation for COVID-19 was associated with favorable outcomes, while activation of NF-κB-driven programs late in disease was associated with poor outcomes. Patients with SARS-CoV-2 pneumonia whose alveolar T cells preferentially targeted the Spike and Nucleocapsid proteins tended to experience more favorable outcomes than patients whose T cells predominantly targeted the ORF1ab polyprotein complex. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize patients who recover, yet these responses progress to NF-κB activation against non-structural proteins in patients who go on to experience poor clinical outcomes.

FLT3 and NPM1 mutations in a cohort of AML patients and detection of a novel mutation in tyrosine kinase domain of FLT3 gene from Western India.

Acute myeloid leukemia (AML) is an aggressive hematological disorder characterized by the loss of ability of the hematopoietic progenitor cells to differentiate and proliferate normally leading to an accumulation of immature myeloid cells in the bone marrow. Several novel molecular genetic aberrations in FLT3 and NPM1 have been shown to have a prognostic impact in AML, particularly in those having normal karyotype. Though there is substantial amount of data on these mutations from western literature, there is surprisingly little data from Indian subcontinent on the frequency of this mutation in AML patients from India. The present study screens a large cohort of non-acute promyelocytic leukemia (APL) AML patients (207 patients) for the presence of FLT3 and NPM1 mutations and further correlates with cytogenetics, immunophenotypic characteristics and with follow-up data wherever available. During the course of study, 56 APL patients were also studied. Briefly, both FLT3 (internal tandem duplication (ITD) in 19.4% and tyrosine kinase domain (TKD) in 9%) and NPM1 mutations were detected in 28.4% of the total non-APL AML patients screened showing distinct correlations with hematologic, immunophenotypic, cytogenetics characteristics and follow-up. With regards to adult APL patients, 22.2 and 32.6% of the patients showed FLT3 and NPM1 mutation, respectively. In the pediatrics age group (<15 years), 23 and 16% of patients with APL showed FLT3 and NPM1 mutation, respectively, while in non-APL patient is this age group, 23% of patients showed both FLT3 and NPM1 mutation. NPM1 mutation was distinctly uncommon in younger age group of patients. In contrast to report elsewhere, most of our FLT3 mutation was in exon 11 rather than in exon 12. FLT3 mutation due to ITD or TKD mutation was detected in 2:1 ratio in our patients and a new TKD mutation was also detected S840G in an M5 patient who did not go into remission and had a short survival of 3 months from diagnosis. Generally, patients with NPM1 mutation had a very high white cell count but they went into remission more often than those with wild (Wt)-type allele (written as NPM1- and FLT3-, respectively) and FLT3 mutation. These patients also tended to have significantly lower expression of CD34 antigen on flowcytometry. Distinct prognostic subclasses of adult AML patients were identified based on the presence of NPM1 and FLT3 mutations.

First-in-human Intravesical Delivery of Pembrolizumab Identifies Immune Activation in Bladder Cancer Unresponsive to Bacillus Calmette-Guérin.

BACKGROUND: Intravenous immune checkpoint inhibition is an effective anticancer strategy for bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC) but may be associated with greater systemic toxicity compared with localized therapies. OBJECTIVE: We assessed the safety and antitumor activity of intravesical pembrolizumab combined with BCG. DESIGN, SETTING, AND PARTICIPANTS: A 3 + 3 phase 1 trial of pembrolizumab + BCG was conducted in patients with BCG-unresponsive NMIBC (NCT02808143). INTERVENTION: Pembrolizumab was given intravesically (1-5 mg/kg for 2 h) beginning 2 weeks prior to BCG induction until recurrence. Urine profiling during treatment and spatial transcriptomic profiling of pre- and post-treatment tumors were conducted to identify biomarkers that correlated with response. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Safety and tolerability of immune checkpoint inhibition were assessed, and Kaplan-Meier survival analysis was performed. RESULTS AND LIMITATIONS: Nine patients completed therapy. Median follow-up was 35 months for five patients still alive at the end of the trial. The trial was closed due to the COVID-19 pandemic. Grade 1-2 urinary symptoms were common. The maximum tolerated dose was not reached; however, one dose-limiting toxicity was reported (grade 2 diarrhea) in the only patient who reached 52 weeks without recurrence. One death occurred from myasthenia gravis that was deemed potentially related to treatment. The 6-mo and 1-yr recurrence-free rates were 67% (95% confidence interval [CI]: 42-100%) and 22% (95% CI: 6.5-75%), respectively. Pembrolizumab was detected in the urine and not in blood. CD4+ T cells were significantly increased in the urine after treatment, and a transcriptomic analysis identified decreased expression of T-cell exhaustion markers in late recurrences. CONCLUSIONS: We demonstrate that intravesical pembrolizumab is safe, feasible, and capable of eliciting strong immune responses in a clinical setting and should be investigated further. PATIENT SUMMARY: Direct application of pembrolizumab to the bladder is a promising alternative for non-muscle-invasive bladder cancer unresponsive to Bacillus Calmette-Guérin and should be investigated further.

Placental dysfunction influences fetal monocyte subpopulation gene expression in preterm birth.

The placenta is the primary organ for immune regulation, nutrient delivery, gas exchange, protection against environmental toxins, and physiologic perturbations during pregnancy. Placental inflammation and vascular dysfunction during pregnancy are associated with a growing list of prematurity-related complications. The goal of this study was to identify differences in gene expression profiles in fetal monocytes - cells that persist and differentiate postnatally - according to distinct placental histologic domains. Here, by using bulk RNA-Seq, we report that placental lesions are associated with gene expression changes in fetal monocyte subsets. Specifically, we found that fetal monocytes exposed to acute placental inflammation upregulate biological processes related to monocyte activation, monocyte chemotaxis, and platelet function, while monocytes exposed to maternal vascular malperfusion lesions downregulate these processes. Additionally, we show that intermediate monocytes might be a source of mitogens, such as HBEGF, NRG1, and VEGFA, implicated in different outcomes related to prematurity. This is the first study to our knowledge to show that placental lesions are associated with unique changes in fetal monocytes and monocyte subsets. As fetal monocytes persist and differentiate into various phagocytic cells following birth, our study may provide insight into morbidity related to prematurity and ultimately potential therapeutic targets.

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in Convalescent COVID-19 patients.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.