Objective: To develop a workflow that transforms electronic health record data into machine learning-ready features for molecular endotype assignment and to evaluate whether clinician-informed feature engineering improves model performance and interpretability. Materials and Methods: We developed parallel clinician-informed and clinician-agnostic feature engineering pipelines to prepare raw EHR data from mechanically ventilated patients with respiratory failure. Molecular endotype labels derived from paired deep lung and blood profiling of subjects with acute lung injury were used to train candidate machine learning classifiers. Champion models from each pipeline were compared on predefined performance metrics. Results: Bayesian network classifiers were the top-performing models in both pipelines. The clinician-informed pipeline generated fewer features than the clinician-agnostic pipeline (645 vs 1,127) and produced a lower misclassification rate in the final Bayesian network model (0.047 vs 0.14). In an independent cohort of subjects with acute lung injury, the clinician-informed model better distinguished corticosteroid-responsive from non-responsive subgroups. Discussion: Clinical context improved feature engineering efficiency, model interpretability, and classification performance. These findings support the integration of domain expertise into machine learning workflows intended for critical care implementation. Conclusions: Clinician-informed feature engineering can simplify machine learning models while improving performance and preserving clinical relevance. AI tools developed for healthcare should incorporate subject matter expertise early in the feature engineering and analytic workflow. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was supported by the North Carolina Collaboratory at The University of North Carolina at Chapel Hill with funding appropriated by the North Carolina General Assembly (V.V. and M.C.W) and by the Rapidly Emerging Antiviral Drug Development Initiative at the University of North Carolina at Chapel Hill with funding from the North Carolina Coronavirus State and Local Fiscal Recovery Funds program, appropriated by the North Carolina General Assembly (J.C.S., R.S.H., and M.C.W.) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the institutional review board at the University of North Carolina at Chapel Hill with a waiver of informed consent (IRB 22-3196, January 11, 2023). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Immune cell populations are dysregulated in COVID-19 for currently unknown reasons: Plasmacytoid dendritic cell (pDC) populations are reduced, thus hampering antiviral responses. CD8+ T cell populations are reduced, the level of which has emerged as an index of disease severity. Recent work has shown that the proteome of SARS-CoV-2 is a rich reservoir of antimicrobial peptide-like sequence motifs (xenoAMPs) which can chaperone and organize dsRNA for amplified Toll-Like Receptor 3 (TLR3)-mediated inflammation in vitro and in vivo. Here, we demonstrate that proteolytic digestion of the SARS-CoV-2 spike protein by host trypsin-like serine proteases directly produces xenoAMPs. Synchrotron Small Angle X-ray Scattering, mass spectrometry, and a theoretical analysis based on continuum membrane elasticity show that proteolytically generated xenoAMPs from SARS-CoV-2 proteins in vitro and machine learning-predicted high-scoring xenoAMPs all induce negative Gaussian curvature (NGC) necessary for pore formation in membranes. We find that xenoAMPs alone as well as xenoAMPs synergistically with endogenous AMP LL-37 can induce NGC in membranes. A computational analysis of immune cells with morphologically complex shapes (e.g., pDC, CD8+, and CD4+ T cells) suggests that surfaces with high local NGC can concentrate AMP-like sequences and promote selective membrane disruption. Consistent with this hypothesis, experiments with freshly isolated human peripheral blood mononuclear cells confirm that viable pDCs, DCs, and T cells are significantly depleted after xenoAMP exposure, in contrast to monocytes and neutrophils, the immune cell subsets with spheroidal morphology. Structural data from Omicron variant xenoAMP homologs indicate reduced pore formation, consistent with clinical observations of reduced T cell cytopenia in Omicron variant infections.

Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with preexisting lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model ( Scnn1b- Tg), Scnn1b -Tg and WT mice were inoculated with a mouse-adapted SARS-CoV-2 virus (SARS-CoV-2 MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry, single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b -Tg mice inoculated with SARS-CoV-2 MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b -Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b- Tg mice. However, Scnn1b -Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were given a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia, while enteral prednisone suppressed IL-33 expression and disease. The eosinophilic pneumonia in Scnn1b -Tg mice after SARS-CoV-2 MA10 infection mimics reports of eosinophilic pneumonia in humans after SARS-CoV-2, suggesting that targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.

Background:Corticosteroids reduce mortality in severe COVID-19 requiring oxygen or invasive mechanical ventilation, yet emerging data suggest that SARS-CoV-2-associated acute lung injury is biologically heterogeneous and that treatment response may vary across molecularly defined disease states. Lung-derived molecular endotypes of severe COVID-19-associated acute lung injury have been described, but direct molecular profiling is not routinely available at the bedside. We evaluated whether a clinical predictor of previously defined lung molecular endotype identifies heterogeneity in corticosteroid treatment effect among mechanically ventilated patients with COVID-19. Methods:We utilized a single-center cohort of 5,000 patients with COVID-19 treated at the University of North Carolina Hospital between January 1, 2020, and December 31, 2022, to emulate a target trial assessing the effect of corticosteroid receipt on mortality, length of stay, and incident organ support. Confounding was addressed through inverse probability of treatment weighting (IPTW). Outcomes for severely ill patients requiring mechanical ventilation were compared to the RECOVERY trial results, with subsequent moderation analysis and stratified analysis by clinically predicted lung molecular endotype and vaccination status. The primary outcome was 28-day mortality. Secondary Outcomes were time to discharge alive and progression to additional organ support. Results:This emulated target trial showed a directionally favorable but non-statistically significant association between corticosteroid treatment and reduced 28-day mortality in patients requiring mechanical ventilation for SARS-CoV-2 infection. A clinical predictor of lung molecular endotype moderated the effect of corticosteroids on 28-day mortality (p-value for interaction 0.038) and identified distinct predicted endotype-specific treatment effect. Corticosteroid treatment was associated with lower 28-day mortality in the predicted Hyper-Inflammatory endotype (OR 0.62, 95% CI 0.39, 0.99) but not in the predicted Metabolic Dysregulation endotype (OR 1.15, 95% CI 0.82, 1.61). We did not detect significant effect modification by vaccination status (p-value for interaction 0.65), although inference was limited by the small, vaccinated subgroup (28-mortality OR 0.78, 95% CI 0.37, 1.65 in vaccinated vs 0.94, 95% CI 0.70, 1.26 in unvaccinated). Conclusions:In this target trial emulation of mechanically ventilated patients with severe COVID-19, corticosteroid treatment showed a directionally favorable but non-statistically significant association with reduced 28-day mortality in the overall cohort. However, a clinical predictor of lung molecular endotype identified significant heterogeneity in treatment effect, with benefit concentrated in the predicted Hyper-Inflammatory endotype and no apparent benefit in the predicted Metabolic Dysregulation endotype. These findings support prospective validation of clinically deployable endotype-guided corticosteroid treatment strategies in acute lung injury and ARDS.

Research using lower respiratory tract (LRT) sampling may lead to improved understanding and management of patients with acute respiratory failure (ARF). Research bronchoscopy is a valuable tool for sampling the LRT during ARF. However, bronchoscopy may be limited by challenges with repeated sampling, the inability to sample the most severely ill patients, and increased resource utilization. "Noninvasive" sampling strategies, such as nonbronchoscopic bronchoalveolar lavage, endotracheal aspirate collection, and heat moisture exchange filter fluid collection, may expand the opportunity to collect LRT samples for research. Upper respiratory tract and sputum sampling may enable sampling in nonintubated patients with ARF, for whom there is no direct noninvasive access to the LRT. We convened a workshop with 32 experts from diverse continents, scientific backgrounds, and healthcare professions, all with experience in clinical or research practice during ARF. The Workshop goals were to review existing noninvasive sampling methods, evaluate their respective potential benefits and limitations, and identify future research priorities aimed at improving standardization and integration of these approaches into practice. Findings were generated by review of literature, expert presentations, meeting discussions, and electronic surveys using a modified Delphi approach. Participants agreed that each sampling method provides biologically meaningful data during ARF while also acknowledging that each method has benefits and limitations. Various potential advantages to noninvasive methods include reduced cost, enhanced speed, and ease of adoption compared with standard fiberoptic bronchoscopy. Despite the potential advantages, rigorous head-to-head comparisons between noninvasive methods and/or standard bronchoscopy are limited, which is a priority for future research. Additional research priorities include examination of the feasibility and benefit of serial sampling of the LRT and investigation of how LRT biomarkers are related to lung pathophysiology and patient-centered clinical outcomes during ARF. This Workshop Report provides guidance for investigators to integrate LRT sampling into their research and highlights key priorities, such as rigorous head-to-head comparison of sampling methods for research, to improve the standardization of noninvasive sampling methods and use of LRT sampling in research and clinical practice.

Tumor-associated macrophages (TAMs) are the predominant immune cells in the tumor microenvironment that promote breast cancer brain metastasis (BCBM). Here, we identify TANK-binding kinase (TBK1) as a critical signaling molecule enriched and activated in TAMs of BCBM tumors, playing an indispensable role in BCBM development and metastatic outgrowth in the brain. Mechanistically, BCBM cell-secreted matrix metalloproteinase 1 binds to protease-activated receptor 1 and integrin αVβ5 on macrophages, leading to TBK1 activation mediated by the nuclear factor-kappa B pathway. Reciprocally, TBK1-regulated TAMs produce granulocyte-macrophage colony-stimulating factor (GM-CSF) to drive breast cancer cell epithelial-mesenchymal transition, migration, and invasion, ultimately contributing to BCBM development and brain metastatic outgrowth. Inhibition of TBK1 signaling in TAMs or GM-CSF receptor in cancer cells impedes BCBM development and brain metastatic outgrowth. Correspondingly, the TBK1-GM-CSF signaling axis correlates with lower overall survival in patients with BCBM. Thus, TBK1-mediated tumor-TAM symbiotic interaction provides a promising therapeutic target for patients with BCBM.

CD4+ forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are essential in maintaining immune tolerance and suppressing excessive immune responses. Tregs also contribute to tissue repair processes distinct from their roles in immune suppression. For these reasons, Tregs are candidates for targeted therapies for inflammatory and autoimmune diseases, and in diseases where tissue damage occurs. MT-2 cells, an immortalized Treg-like cell line, offer a model to study Treg biology and their therapeutic potential. In the present study, we use clustered regularly interspaced palindromic repeats (CRISPR)-mediated knockdown of FOXP3 in MT-2 cells to understand the transcriptional and functional changes that occur when FOXP3 is lost and to compare MT-2 cells with primary human Tregs. We demonstrate that loss of FOXP3 affects the transcriptome of MT-2 cells and that FOXP3's potential downstream targets include a wide range of transcripts that participate in the cell cycle, promote growth and contribute to inflammatory processes, but do not wholly simulate previously reported human primary Treg transcriptional changes in the absence of FOXP3. We also demonstrate that FOXP3 regulates cell cycling and proliferation, expression of molecules crucial to Treg function and MT-2 cell-suppressive activities. Thus, MT-2 cells offer opportunities to address regulatory T-cell functions in vitro.

CD4 + forkhead box P3 (FOXP3) + regulatory T cells (Tregs) are essential in maintaining immune tolerance and suppressing excessive immune responses. Tregs also contribute to tissue repair processes distinct from their roles in immune suppression. For these reasons, Tregs are candidates for targeted therapies for inflammatory and autoimmune diseases, and in diseases where tissue damage occurs. MT-2 cells, an immortalized Treg-like cell line, offer a model to study Treg biology and their therapeutic potential. In the present study, we use clustered regularly interspaced palindromic repeats (CRISPR)-mediated knockdown of FOXP3 in MT-2 cells to understand the transcriptional and functional changes that occur when FOXP3 is lost and to compare MT-2 cells with primary human Tregs. We demonstrate that loss of FOXP3 affects the transcriptome of MT-2 cells and that FOXP3's potential downstream targets include a wide range of transcripts that participate in the cell cycle, promote growth and contribute to inflammatory processes, but do not wholly simulate previously reported human primary Treg transcriptional changes in the absence of FOXP3. We also demonstrate that FOXP3 regulates cell cycling and proliferation, expression of molecules crucial to Treg function and MT-2 cell–suppressive activities. Thus, MT-2 cells offer opportunities to address regulatory T-cell functions in vitro .

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS- CoV- 2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID- 19), with amplified immune activation in diverse cell types, including cells without angiotensin- converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS- CoV- 2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self- organization in the infected host environment. Interestingly, a machine learning analysis to SARS- CoV- 2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL- 37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS- CoV- 2 relative to low- pathogenicity coronaviruses. Moreover, xenoAMPs from SARS- CoV- 2 but not low- pathogenicity homologs assemble double- stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll- like receptor (TLR)- 3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID- 19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin- 6 and CXCL1 levels as observed in COVID- 19 patients.

The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus -induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head -to -head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3 ' end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.

Glioblastoma (GBM) is one of the most aggressive tumors in the adult central nervous system. We previously revealed that circadian regulation of glioma stem cells (GSCs) affects GBM hallmarks of immunosuppression and GSC maintenance in a paracrine and autocrine manner. Here, we expand the mechanism involved in angiogenesis, another critical GBM hallmark, as a potential basis underlying CLOCK’s pro-tumor effect in GBM. Mechanistically, CLOCK-directed olfactomedin like 3 (OLFML3) expression results in hypoxia-inducible factor 1-alpha (HIF1α)-mediated transcriptional upregulation of periostin (POSTN). As a result, secreted POSTN promotes tumor angiogenesis via activation of the TANK-binding kinase 1 (TBK1) signaling in endothelial cells. In GBM mouse and patient-derived xenograft models, blockade of the CLOCK-directed POSTN-TBK1 axis inhibits tumor progression and angiogenesis. Thus, the CLOCK-POSTN-TBK1 circuit coordinates a key tumor-endothelial cell interaction and represents an actionable therapeutic target for GBM.

Type 1 IFN expression is critical in the innate immune response, but aberrant expression is associated with autoimmunity and cancer. Here, we identify N-[4-(1H46 pyrazolo[3,4-b] pyrazin-6-yl)-phenyl]-sulfonamide (Sanofi-14h), a compound with preference for inhibition of the AGC family kinase SGK3, as an inhibitor of Ifnb1 gene expression in response to STING stimulation of macrophages. Sanofi-14h abrogated SGK activity and also impaired activation of the critical TBK1/IRF3 pathway downstream of STING activation, blocking interaction of STING with TBK1. Deletion of SGK1/3 in a macrophage cell line did not block TBK1/IRF3 activation but decreased expression of transcription factors, such as IRF7 and STAT1, required for the innate immune response. Other AGC kinase inhibitors blocked TBK1 and IRF3 activation suggesting common action on a critical regulatory node in the STING pathway. These studies reveal both SGK-dependent and SGK-independent mechanisms in the innate immune response and indicate an approach to block aberrant Ifnb1 expression.

Genomic sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to provide valuable insight into the ever-changing variant makeup of the COVID-19 pandemic. More than three million SARS-CoV-2 genome sequences have been deposited in Global Initiative on Sharing All Influenza Data (GISAID), but contributions from the United States, particularly through 2020, lagged the global effort.

Background Interleukin-6 (IL-6) is elevated in SARS-CoV-2 infection. IL-6 regulates acute-phase proteins, such as alpha 1 antitrypsin (AAT), a key lung anti-protease. We investigated the protease-anti-protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS) compared to non-SARS-CoV-2 ARDS (nsARDS) and the effects of tocilizumab (IL-6 receptor antagonist) on anti-protease defence in SARS-CoV-2 infection.Methods Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma, airway tissue and tracheal secretions (TA) of people with SARS-CoV-2 ARDS or nsARDS. AAT and IL-6 levels were evaluated in people with moderate SARS-CoV-2 infection who received standard of care +/-tocilizumab.Findings AAT plasma levels doubled in SARS-CoV-2 ARDS. In lung parenchyma AAT levels were increased, as was the percentage of neutrophils involved in NET formation. A protease-anti-protease imbalance was detected in TA with active NE and no active AAT. The airway anti-protease, secretory leukoprotease inhibitor was decreased in SARS-CoV-2-infected lungs and cleaved in TA. In nsARDS, plasma AAT levels were elevated but TA samples had less AAT cleavage, with no detectable active NE in most samples. Induction of AAT in ARDS occurred mainly through IL-6. Tocilizumab down-regulated AAT during SARS-CoV-2 infection.Interpretation There is a protease-anti-protease imbalance in the airways of SARS-CoV-2-ARDS patients. This imbalance is a target for anti-protease therapy.

Bacterial pneumonia induces the rapid recruitment and activation of neutrophils and macrophages into the lung, and these cells contribute to bacterial clearance and other defense functions. TBK1 (TANK-binding kinase 1) performs many functions, including activation of the type I IFN pathway and regulation of autophagy and mitophagy, but its contribution to antibacterial defenses in the lung is unclear. We previously showed that lung neutrophils upregulate mRNAs for TBK1 and its accessory proteins during Streptococcus pneumoniae pneumonia, despite low or absent expression of type I IFN in these cells. We hypothesized that TBK1 performs key antibacterial functions in pneumonia apart from type I IFN expression. Using TBK1 null mice, we show that TBK1 contributes to antibacterial defenses and promotes bacterial clearance and survival. TBK1 null mice express lower concentrations of many cytokines in the infected lung. Conditional deletion of TBK1 with LysMCre results in TBK1 deletion from macrophages but not neutrophils. LysMCre TBK1 mice have no defect in cytokine expression, implicating a nonmacrophage cell type as a key TBK1-dependent cell. TBK1 null neutrophils have no defect in recruitment to the infected lung but show impaired activation of p65/NF-kappa B and STAT1 and lower expression of reactive oxygen species, IFN gamma, and IL12p40. TLR1/2 and 4 agonists each induce phosphorylation of TBK1 in neutrophils. Surprisingly, neutrophil TBK1 activation in vivo does not require the adaptor STING. Thus, TBK1 is a critical component of STING-independent antibacterial responses in the lung, and TBK1 is necessary for multiple neutrophil functions.

Abstract Background Global efforts are needed to elucidate the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the underlying cause of coronavirus disease 2019 (COVID-19), including seroprevalence, risk factors, and long-term sequelae, as well as immune responses after vaccination across populations and the social dimensions of prevention and treatment strategies. Methods In the United States, the National Cancer Institute in partnership with the National Institute of Allergy and Infectious Diseases, established the SARS-CoV-2 Serological Sciences Network (SeroNet) as the nation’s largest coordinated effort to study coronavirus disease 2019. The network comprises multidisciplinary researchers bridging gaps and fostering collaborations among immunologists, epidemiologists, virologists, clinicians and clinical laboratories, social and behavioral scientists, policymakers, data scientists, and community members. In total, 49 institutions form the SeroNet consortium to study individuals with cancer, autoimmune disease, inflammatory bowel diseases, cardiovascular diseases, human immunodeficiency virus, transplant recipients, as well as otherwise healthy pregnant women, children, college students, and high-risk occupational workers (including healthcare workers and first responders). Results Several studies focus on underrepresented populations, including ethnic minorities and rural communities. To support integrative data analyses across SeroNet studies, efforts are underway to define common data elements for standardized serology measurements, cellular and molecular assays, self-reported data, treatment, and clinical outcomes. Conclusions In this paper, we discuss the overarching framework for SeroNet epidemiology studies, critical research questions under investigation, and data accessibility for the worldwide scientific community. Lessons learned will help inform preparedness and responsiveness to future emerging diseases.

Introduction: CD19-targeted CAR-T cells are highly active in B-cell malignancies. While early use of corticosteroids (CS) and tocilizumab have improved the management of cytokine release syndrome (CRS), CS are the mainstay of ICANS management. Though patients with low-grade ICANS often do not require therapy, severe ICANS that does not respond to CS is life-threatening. Rimiducid (RIM) is a dimerizing drug that induces apoptosis in cells engineered with an inducible caspase-9 switch. We reported the first case of the administration of a potentially ablative dose of RIM (0.4 mg/kg) to a patient with B-lymphoblastic leukemia (B-ALL) with severe, prolonged ICANS (Foster MC et al. Blood 2021 Jun 10;137(23):3306-3309). Given this patient's rapid resolution of steroid-nonresponsive ICANS and reduction in iC9 CAR.19 numbers, we sought to explore the effects of lower, potentially non-ablative doses of RIM in patients with CS-nonresponsive ICANS before the development of life threatening complications, with a goal of clinical resolution of ICANS without ablation of CAR T cells. Here, we report the clinical and pharmacodynamic courses of ICANS for the four patients treated with RIM for ICANS to date, in an ongoing expansion cohort of a phase I/II study of iC9 CAR.19 cells in patients with B-ALL. Methods: Adults with B-ALL in 2nd or greater bone marrow (BM) relapse, relapse >100 days after allogeneic stem cell transplant, disease refractory to ≥2 induction therapies, or with measurable residual disease (MRD) persistence/recurrence were enrolled. T-lymphocytes were collected from the subjects, and CAR-T cell products generated by gene modification with a g-retroviral vector encoding for iC9, ΔNGFR (for selection and tracking purposes) and CAR.CD19 (encoding 4-1BB) genes (Diaconu I et al Mol Ther 2017; 25:580). Subjects underwent lymphodepletion with fludarabine and cyclophosphamide followed by infusion of iC9 CAR.19 cells at one of two dose levels (DL1: 5 x 105 CAR-T cells/kg; DL2: 1 x 106 CAR-T cells/kg). Toxicities were graded by CTCAE v5 or ASBMT consensus grading for CRS and ICANS. CAR-T cell expansion in peripheral blood (PB) was measured by flow cytometry (FC) and droplet digital PCR (ddPCR). Cytokines were measured by commercially available multiplex (Luminex®) cytokine assays. Leukemia response was determined by NCCN criteria. Subjects who experienced grade 3-4 ICANS despite CS for >72 hours received RIM 0.4 mg/kg IV over 2 hours. After protocol amendment, subjects with ≥ grade 2 ICANS for >24 hours despite CS received RIM 0.1 mg/kg IV over 2 hours. FC, ddPCR and cytokine levels were measured prior to RIM, at 4 hours after the end of RIM infusion and daily until ICANS resolution. Results: 13 subjects were treated, 3 on DL1 and 10 on DL2, which was the expansion cohort cell dose. Median age was 31 years (range 21-69). CRS developed in 11, with median duration of 5 days (range 1-8). Maximum CRS grade was 2 in 5 and 1 in 6. ICANS developed in 5 subjects, with maximum grade 4 in 2, and grade 3 in 3. One patient with CS-responsive grade 3 ICANS did not meet criteria for RIM. Two subjects with grade 3-4 ICANS for >72 hours received RIM 0.4 mg/kg, both achieving improvement by at least 1 grade within 12 hours and with resolution within 5 days (FIGURE 1). ddPCR for ΔNGFR showed reduction in transcripts by 4 hours after the end of RIM infusion (by 83.6% and 98.6% respectively, FIGURE 2). In two subjects with grade 3 ICANS for 24 hours treated with RIM 0.1 mg/kg, ICANS improved to grade 1 in 24 hours and grade 0 in 48 hours (FIGURE 1). In these subjects, ddPCR for ΔNGFR showed reduction in transcripts at 4 hours after end of RIM infusion by 88.6% and 88.1% respectively (FIGURE 2). Rates of overall response (CR+CRi+MLFS at 4 weeks) were 3/4 RIM subjects versus 9/9 in other subjects. The subject with grade 3 ICANS who did not receive RIM demonstrated peak ΔNGFR transcripts, IL-6, TNF-α and IFNγ two weeks after iC9 CAR.19 infusion, whereas peak transcripts and cytokine levels occurred at one week, and prior to RIM for RIM-treated subjects. Conclusion: RIM administration to patients with ICANS is associated with abrupt reduction of circulating iC9 CAR.19 cells and ICANS grade. Despite detection of CAR-T cells at 3 weeks post infusion and the observance of remissions after treatment with RIM, further investigation of the effects of RIM on iC9 CAR.19 persistence and antileukemic activity is warranted. Lower, potentially less ablative doses of RIM are being explored. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The inhibitor of kappa B kinase (IKK) family consists of IKK alpha, IKK beta, and the IKK-related kinases TBK1 and IKKE. These kinases are considered master regulators of inflammation and innate immunity via their control of the transcription factors NF-kappa B, IRF3, and IRF7. Novel phosphorylated substrates have been attributed to these kinases, a subset of which is not directly related to either inflammation or innate immunity. These findings have greatly expanded the perspectives on the biological activities of these kinases. In this review we highlight some of the novel substrates for this kinase family and discuss the biological implications of these phosphorylation events.

ABSTRACT Non-small cell lung cancers demonstrate intrinsic resistance to cell death even in response to chemotherapy. Previous work suggested that defective nuclear translocation of active caspase 3 may play a role in resistance to cell death. Separately, our group has identified that mitogen activated protein kinase activated protein kinase 2 (MK2) is required for nuclear translocation of active caspase 3 in the execution of apoptosis. This study demonstrates a relatively low expression of MK2 in non-small cell lung carcinoma cell lines compared to small cell carcinoma cell lines. Further, overexpression of MK2 in non-small cell lung carcinoma cell lines results in increased caspase 3 activity and caspase 3 mediated cell death. Higher MK2 transcript levels were observed in patients with earlier-stage non-small cell lung cancer. Higher expression of MK2 is associated with better survival in patients with early stage non-small cell lung cancer across two independent clinical datasets. Using data sets spanning multiple cancer types, we observed improved survival with higher MK2 expression was unique to lung adenocarcinoma. Mechanistically, MK2 promotes nuclear translocation of caspase 3 leading to PARP1 cleavage and execution of cell death. While MK2 can directly phosphorylate caspase 3, neither phosphorylation status of caspase 3 nor the kinase activity of MK2 impacts caspase 3 activation, nuclear translocation and execution of cell death. Rather, a non-kinase function of MK2, specifically trafficking via its nuclear localization sequence, is required for caspase 3 mediated cell death. In summary this study highlights the importance of a non-enzymatic function of MK2 in the execution of apoptosis, which may be leveraged in the adjunctive treatment of NSCLC or other conditions where regulation of apoptosis is crucial.