Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation.

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

An observational cohort study of bronchoalveolar lavage fluid galactomannan and Aspergillus culture positivity in patients requiring mechanical ventilation.

Rationale: Critically ill patients who develop invasive pulmonary aspergillosis (IPA) have high mortality rates despite antifungal therapy. Diagnosis is difficult in these patients. Bronchoalveolar lavage (BAL) fluid galactomannan (GM) is a helpful marker of infection, although the optimal cutoff for IPA is unclear. We aimed to evaluate the BAL fluid GM and fungal culture results, demographics, and outcomes among a large cohort of mechanically ventilated patients with suspected pneumonia. Methods: A single-center cohort study of patients enrolled in the Successful Clinical Response in Pneumonia Therapy (SCRIPT) study from June 2018 to March 2023. Demographics, BAL results, and outcomes data were extracted from the electronic health record and compared between groups of patients who grew Aspergillus on a BAL fluid culture, those who had elevated BAL fluid GM levels (defined as >0.5 or >0.8) but did not grow Aspergillus on BAL fluid culture, and those with neither. Results: Of over 1700 BAL samples from 688 patients, only 18 BAL samples grew Aspergillus. Patients who had a BAL sample grow Aspergillus (n=15) were older (median 71 vs 62 years, p=0.023), had more days intubated (29 vs 11, p=0.002), and more ICU days (34 vs 15, p=0.002) than patients whose BAL fluid culture was negative for Aspergillus (n=672). The BAL fluid galactomannan level was higher from samples that grew Aspergillus on culture than those that did not (median ODI 7.08 vs 0.11, p<0.001), though the elevation of BAL fluid GM varied across BAL samples for patients who had serial sampling. Patients who grew Aspergillus had a similar proportion of underlying immunocompromise compared with the patients who did not, and while no statistically significant difference in overall unfavorable outcome, had longer duration of ventilation and longer ICU stays. Conclusions: In this large cohort of critically ill patients with a high number of BAL samples with GM levels, we found a relatively low rate of Aspergillus growth. Patients who eventually grew Aspergillus had inconsistently elevated BAL fluid GM, and many patients with elevated BAL fluid GM did not grow Aspergillus. These data suggest that the pre-test probability of invasive pulmonary aspergillosis should be considered low in a general ICU population undergoing BAL evaluation to define the etiology of pneumonia. Improved scoring systems are needed to enhance pre-test probability for diagnostic test stewardship purposes.

Transcriptional profiling of peripheral blood mononuclear cells identifies inflammatory phenotypes in Ataxia Telangiectasia.

INTRODUCTION: Ataxia telangiectasia (A-T) is an autosomal recessive neurodegenerative disease with widespread systemic manifestations and marked variability in clinical phenotypes. In this study, we sought to determine whether transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) defines subsets of individuals with A-T beyond mild and classic phenotypes, enabling identification of novel features for disease classification and treatment response to therapy. METHODS: Participants with classic A-T (n = 77), mild A-T (n = 13), and unaffected controls (n = 15) were recruited from two outpatient clinics. PBMCs were isolated and bulk RNAseq was performed. Plasma was also isolated in a subset of individuals. Affected individuals were designated mild or classic based on ATM mutations and clinical and laboratory features. RESULTS: People with classic A-T were more likely to be younger and IgA deficient and to have higher alpha-fetoprotein levels and lower % forced vital capacity compared to individuals with mild A-T. In classic A-T, the expression of genes required for V(D)J recombination was lower, and the expression of genes required for inflammatory activity was higher. We assigned inflammatory scores to study participants and found that inflammatory scores were highly variable among people with classic A-T and that higher scores were associated with lower ATM mRNA levels. Using a cell type deconvolution approach, we inferred that CD4 + T cells and CD8 + T cells were lower in number in people with classic A-T. Finally, we showed that individuals with classic A-T exhibit higher SERPINE1 (PAI-1) mRNA and plasma protein levels, irrespective of age, and higher FLT4 (VEGFR3) and IL6ST (GP130) plasma protein levels compared with mild A-T and controls. CONCLUSION: Using a transcriptomic approach, we identified novel features and developed an inflammatory score to identify subsets of individuals with different inflammatory phenotypes in A-T. Findings from this study could be used to help direct treatment and to track treatment response to therapy.

AMP-activated protein kinase is necessary for Treg cell functional adaptation to microenvironmental stress.

CD4+FOXP3+ regulatory T (Treg) cells maintain self-tolerance, suppress the immune response to cancer, and protect against tissue injury in the lung and other organs. Treg cells require mitochondrial metabolism to exert their function, but how Treg cells adapt their metabolic programs to sustain and optimize their function during an immune response occurring in a metabolically stressed microenvironment remains unclear. Here, we tested whether Treg cells require the energy homeostasis-maintaining enzyme AMP-activated protein kinase (AMPK) to adapt to metabolically aberrant microenvironments caused by malignancy or lung injury, finding that AMPK is dispensable for Treg cell immune-homeostatic function but is necessary for full Treg cell function in B16 melanoma tumors and during acute lung injury caused by influenza virus pneumonia. AMPK-deficient Treg cells had lower mitochondrial mass and exhibited an impaired ability to maximize aerobic respiration. Mechanistically, we found that AMPK regulates DNA methyltransferase 1 to promote transcriptional programs associated with mitochondrial function in the tumor microenvironment. In the lung during viral pneumonia, we found that AMPK sustains metabolic homeostasis and mitochondrial activity. Induction of DNA hypomethylation was sufficient to rescue mitochondrial mass in AMPK-deficient Treg cells, linking DNA methylation with AMPK function and mitochondrial metabolism. These results define AMPK as a determinant of Treg cell adaptation to metabolic stress and offer potential therapeutic targets in cancer and tissue injury.

ELOA3: A primate-specific RNA polymerase II elongation factor encoded by a tandem repeat gene cluster.

The biological role of the repetitive DNA sequences in the human genome remains an outstanding question. Recent long-read human genome assemblies have allowed us to identify a function for one of these repetitive regions. We have uncovered a tandem array of conserved primate-specific retrogenes encoding the protein Elongin A3 (ELOA3), a homolog of the RNA polymerase II (RNAPII) elongation factor Elongin A (ELOA). Our genomic analysis shows that the ELOA3 gene cluster is conserved among primates and the number of ELOA3 gene repeats is variable in the human population and across primate species. Moreover, the gene cluster has undergone concerted evolution and homogenization within primates. Our biochemical studies show that ELOA3 functions as a promoter-associated RNAPII pause-release elongation factor with distinct biochemical and functional features from its ancestral homolog, ELOA. We propose that the ELOA3 gene cluster has evolved to fulfil a transcriptional regulatory function unique to the primate lineage that can be targeted to regulate cellular hyperproliferation.

Prolonged exposure to lung-derived cytokines is associated with inflammatory activation of microglia in patients with COVID-19.

Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia1-4. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans5. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-α and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-196, was associated with lower levels of CXCL10, CCL8, and CCL2-molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells7. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.

Differential roles of regulatory T cells in acute respiratory infections.

Acute respiratory infections trigger an inflammatory immune response with the goal of pathogen clearance; however, overexuberant inflammation causes tissue damage and impairs pulmonary function. CD4+FOXP3+ regulatory T cells (Tregs) interact with cells of both the innate and the adaptive immune system to limit acute pulmonary inflammation and promote its resolution. Tregs also provide tissue protection and coordinate lung tissue repair, facilitating a return to homeostatic pulmonary function. Here, we review Treg-mediated modulation of the host response to respiratory pathogens, focusing on mechanisms underlying how Tregs promote resolution of inflammation and repair of acute lung injury. We also discuss potential strategies to harness and optimize Tregs as a cellular therapy for patients with severe acute respiratory infection and discuss open questions in the field.

An Adjudication Protocol for Severe Pneumonia.

Background: Clinical end points that constitute successful treatment in severe pneumonia are difficult to ascertain and vulnerable to bias. The utility of a protocolized adjudication procedure to determine meaningful end points in severe pneumonia has not been well described. Methods: This was a single-center prospective cohort study of patients with severe pneumonia admitted to the medical intensive care unit. The objective was to develop an adjudication protocol for severe bacterial and/or viral pneumonia. Each episode of pneumonia was independently reviewed by 2 pulmonary and critical care physicians. If a discrepancy occurred between the 2 adjudicators, a third adjudicator reviewed the case. If a discrepancy remained after all 3 adjudications, consensus was achieved through committee review. Results: Evaluation of 784 pneumonia episodes during 593 hospitalizations achieved only 48.1% interobserver agreement between the first 2 adjudicators and 78.8% when agreement was defined as concordance between 2 of 3 adjudicators. Multiple episodes of pneumonia and presence of bacterial/viral coinfection in the initial pneumonia episode were associated with lower interobserver agreement. For an initial episode of bacterial pneumonia, patients with an adjudicated day 7-8 clinical impression of cure (compared with alternative impressions) were more likely to be discharged alive (odds ratio, 6.3; 95% CI, 3.5-11.6). Conclusions: A comprehensive adjudication protocol to identify clinical end points in severe pneumonia resulted in only moderate interobserver agreement. An adjudicated end point of clinical cure by day 7-8 was associated with more favorable hospital discharge dispositions, suggesting that clinical cure by day 7-8 may be a valid end point to use in adjudication protocols.

Research Bronchoscopies in Critically Ill Research Participants: An Official American Thoracic Society Workshop Report.

Bronchoscopy for research purposes is a valuable tool to understand lung-specific biology in human participants. Despite published reports and active research protocols using this procedure in critically ill patients, no recent document encapsulates the important safety considerations and downstream applications of this procedure in this setting. The objectives were to identify safe practices for patient selection and protection of hospital staff, provide recommendations for sample procurement to standardize studies, and give guidance on sample preparation for novel research technologies. Seventeen international experts in the management of critically ill patients, bronchoscopy in clinical and research settings, and experience in patient-oriented clinical or translational research convened for a workshop. Review of relevant literature, expert presentations, and discussion generated the findings presented herein. The committee concludes that research bronchoscopy with bronchoalveolar lavage in critically ill patients on mechanical ventilation is valuable and safe in appropriately selected patients. This report includes recommendations on standardization of this procedure and prioritizes the reporting of sample management to produce more reproducible results between laboratories. This document serves as a resource to the community of researchers who endeavor to include bronchoscopy as part of their research protocols and highlights key considerations for the inclusion and safety of research participants.

Machine learning links unresolving secondary pneumonia to mortality in patients with severe pneumonia, including COVID-19.

BACKGROUNDDespite guidelines promoting the prevention and aggressive treatment of ventilator-associated pneumonia (VAP), the importance of VAP as a driver of outcomes in mechanically ventilated patients, including patients with severe COVID-19, remains unclear. We aimed to determine the contribution of unsuccessful treatment of VAP to mortality for patients with severe pneumonia.METHODSWe performed a single-center, prospective cohort study of 585 mechanically ventilated patients with severe pneumonia and respiratory failure, 190 of whom had COVID-19, who underwent at least 1 bronchoalveolar lavage. A panel of intensive care unit (ICU) physicians adjudicated the pneumonia episodes and endpoints on the basis of clinical and microbiological data. Given the relatively long ICU length of stay (LOS) among patients with COVID-19, we developed a machine-learning approach called CarpeDiem, which grouped similar ICU patient-days into clinical states based on electronic health record data.RESULTSCarpeDiem revealed that the long ICU LOS among patients with COVID-19 was attributable to long stays in clinical states characterized primarily by respiratory failure. While VAP was not associated with mortality overall, the mortality rate was higher for patients with 1 episode of unsuccessfully treated VAP compared with those with successfully treated VAP (76.4% versus 17.6%, P < 0.001). For all patients, including those with COVID-19, CarpeDiem demonstrated that unresolving VAP was associated with a transitions to clinical states associated with higher mortality.CONCLUSIONSUnsuccessful treatment of VAP is associated with higher mortality. The relatively long LOS for patients with COVID-19 was primarily due to prolonged respiratory failure, placing them at higher risk of VAP.FUNDINGNational Institute of Allergy and Infectious Diseases (NIAID), NIH grant U19AI135964; National Heart, Lung, and Blood Institute (NHLBI), NIH grants R01HL147575, R01HL149883, R01HL153122, R01HL153312, R01HL154686, R01HL158139, P01HL071643, and P01HL154998; National Heart, Lung, and Blood Institute (NHLBI), NIH training grants T32HL076139 and F32HL162377; National Institute on Aging (NIA), NIH grants K99AG068544, R21AG075423, and P01AG049665; National Library of Medicine (NLM), NIH grant R01LM013337; National Center for Advancing Translational Sciences (NCATS), NIH grant U01TR003528; Veterans Affairs grant I01CX001777; Chicago Biomedical Consortium grant; Northwestern University Dixon Translational Science Award; Simpson Querrey Lung Institute for Translational Science (SQLIFTS); Canning Thoracic Institute of Northwestern Medicine.

Intranasal soluble ACE2 improves survival and prevents brain SARS-CoV-2 infection.

A soluble ACE2 protein bioengineered for long duration of action and high affinity to SARS-CoV-2 was administered either intranasally (IN) or intraperitoneally (IP) to SARS-CoV-2-inoculated k18hACE2 mice. This decoy protein (ACE2 618-DDC-ABD) was given either IN or IP, pre- and post-inoculation, or IN, IP, or IN + IP but only post-inoculation. Survival by day 5 was 0% in untreated mice, 40% in the IP-pre, and 90% in the IN-pre group. In the IN-pre group, brain histopathology was essentially normal and lung histopathology significantly improved. Consistent with this, brain SARS-CoV-2 titers were undetectable and lung titers reduced in the IN-pre group. When ACE2 618-DDC-ABD was administered only post-inoculation, survival was 30% in the IN + IP, 20% in the IN, and 20% in the IP group. We conclude that ACE2 618-DDC-ABD results in markedly improved survival and provides organ protection when given intranasally as compared with when given either systemically or after viral inoculation, and that lowering brain titers is a critical determinant of survival and organ protection.

DNA methyltransferase inhibition induces dynamic gene expression changes in lung CD4+ T cells of neonatal mice with E. coli pneumonia.

Bacterial pulmonary infections are a major cause of morbidity and mortality in neonates, with less severity in older children. Previous studies demonstrated that the DNA of CD4+ T cells in the mouse lung, whose primary responsibility is to coordinate the immune response to foreign pathogens, is differentially methylated in neonates compared with juveniles. Nevertheless, the effect of this differential DNA methylation on CD4+ T cell gene expression and response to infection remains unclear. Here we treated E. coli-infected neonatal (4-day-old) and juvenile (13-day-old) mice with decitabine (DAC), a DNA methyltransferase inhibitor with broad-spectrum DNA demethylating activity, and performed simultaneous genome-wide DNA methylation and transcriptional profiling on lung CD4+ T cells. We show that juvenile and neonatal mice experienced differential demethylation in response to DAC treatment, with larger methylation differences observed in neonates. By cross-filtering differentially expressed genes between juveniles and neonates with those sites that were demethylated in neonates, we find that interferon-responsive genes such as Ifit1 are the most down-regulated methylation-sensitive genes in neonatal mice. DAC treatment shifted neonatal lung CD4+ T cells toward a gene expression program similar to that of juveniles. Following lung infection with E. coli, lung CD4+ T cells in neonatal mice exhibit epigenetic repression of important host defense pathways, which are activated by inhibition of DNA methyltransferase activity to resemble a more mature profile.

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.

Superiority of intranasal over systemic administration of bioengineered soluble ACE2 for survival and brain protection against SARS-CoV-2 infection.

The present study was designed to investigate the effects of a soluble ACE2 protein termed ACE2 618-DDC-ABD, bioengineered to have long duration of action and high binding affinity to SARS-CoV-2, when administered either intranasally (IN) or intraperitoneally (IP) and before or after SARS-CoV-2 inoculation. K18hACE2 mice permissive for SARS-CoV-2 infection were inoculated with 2Ã-10 4 PFU wildtype SARS-CoV-2. In one protocol, ACE2 618-DDC-ABD was given either IN or IP, pre- and post-viral inoculation. In a second protocol, ACE2 618-DDC-ABD was given either IN, IP or IN+IP but only post-viral inoculation. In addition, A549 and Vero E6 cells were used to test neutralization of SARS-CoV-2 variants by ACE2 618-DDC-ABD at different concentrations. Survival by day 5 was 0% in infected untreated mice, and 40% in mice from the ACE2 618-DDC-ABD IP-pre treated group. By contrast, in the IN-pre group survival was 90%, histopathology of brain and kidney was essentially normal and markedly improved in the lungs. When ACE2 618-DDC-ABD was administered only post viral inoculation, survival was 30% in the IN+IP group, 20% in the IN and 0% in the IP group. Brain SARS-CoV-2 titers were high in all groups except for the IN-pre group where titers were undetectable in all mice. In cells permissive for SARS-CoV-2 infection, ACE2 618-DDC-ABD neutralized wildtype SARS-CoV-2 at high concentrations, whereas much lower concentrations neutralized omicron BA. 1. We conclude that ACE2 618-DDC-ABD provides much better survival and organ protection when administered intranasally than when given systemically or after viral inoculation and that lowering brain titers is a critical determinant of survival and organ protection.

Advancing Lung Immunology Research: An Official American Thoracic Society Workshop Report.

The mammalian airways and lungs are exposed to a myriad of inhaled particulate matter, allergens, and pathogens. The immune system plays an essential role in protecting the host from respiratory pathogens, but a dysregulated immune response during respiratory infection can impair pathogen clearance and lead to immunopathology. Furthermore, inappropriate immunity to inhaled antigens can lead to pulmonary diseases. A complex network of epithelial, neural, stromal, and immune cells has evolved to sense and respond to inhaled antigens, including the decision to promote tolerance versus a rapid, robust, and targeted immune response. Although there has been great progress in understanding the mechanisms governing immunity to respiratory pathogens and aeroantigens, we are only beginning to develop an integrated understanding of the cellular networks governing tissue immunity within the lungs and how it changes after inflammation and over the human life course. An integrated model of airway and lung immunity will be necessary to improve mucosal vaccine design as well as prevent and treat acute and chronic inflammatory pulmonary diseases. Given the importance of immunology in pulmonary research, the American Thoracic Society convened a working group to highlight central areas of investigation to advance the science of lung immunology and improve human health.