Performance and reliability assessment of a lower dose, task-based scoliosis radiography protocol in pediatric patients.

BACKGROUND: Follow-up scoliosis radiographs are performed to assess the degree of spinal curvature and skeletal maturity, which can be done at lower radiation exposures than those in standard-dose radiography. OBJECTIVE: Describe and evaluate a protocol that reduced the radiation in follow-up frontal-view scoliosis radiographs. MATERIALS AND METHODS: We implemented a postero-anterior lower dose modified-technique for scoliosis radiography with task-based definition of adequate image quality and use of technique charts based on target exposure index and patient's height and weight. We subsequently retrospectively evaluated 40 consecutive patients who underwent a follow-up radiograph using the modified-technique after an initial standard-technique radiograph. We evaluated comparisons of proportions for subjective assessment with chi-squared tests, and agreements of reader's scores with intraclass correlation coefficients and Bland-Altman plots. We determined incident air kerma, exposure index, deviation index/standard deviation, dose-area product (DAP), and effective dose for each radiograph. We set statistical significance at P<0.05. RESULTS: Forty patients (65% female), aged 4-17 years. Median effective dose was reduced from 39 to 10 µSv (P<0.001), incident air kerma from 139 to 29 µSv (P<0.001), and DAP from 266 to 55 mGy*cm2 (P<0.001). All modified-technique parameters were rated with a mean score of acceptable or above. All modified-technique measurements obtained inter- and intra-observer correlation coefficient agreements of 0.86 ("Good") or greater. CONCLUSION: Substantial dose reduction on follow-up scoliosis imaging with existing radiography units is achievable through task-based definition of adequate image quality and tailoring of radiation to each patient's height and weight, while still allowing for reliable assessment and reproducible measurements.

Tumor-associated myeloid cells provide critical support for T-ALL.

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells, from the thymic tumor microenvironment support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in 2 distinct mouse models of T-ALL and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of antitumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid-depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

The role of rumination and positive beliefs about rumination in eating pathology.

PURPOSE: General and eating disorder (ED)-specific ruminations have been identified as key factors that may contribute to eating pathology. Positive beliefs about rumination (e.g., "Ruminating helps me to prevent future mistakes") may impact this association. However, the effect of positive beliefs about rumination on the links between rumination and ED symptom severity has not been investigated. This study sought to clarify relations between rumination and ED symptom severity and to evaluate the potential moderating effect of positive beliefs about rumination on these associations. METHODS: During a laboratory visit, undergraduate participants (N = 473, MAge = 18.90 ± 2.27, MBMI = 23.45 kg/m2 ± 4.31, 54.8% female) completed an online battery of questionnaires assessing general and ED-specific ruminative processes (e.g., brooding, reflection), positive beliefs about rumination, and global ED symptoms. Hierarchical multiple regression analyses assessed the unique contributions of specific ruminative processes, and the moderating effect of positive beliefs on associations between ruminative processes and ED symptom severity. RESULTS: Hierarchical multiple regression results suggest that, after controlling for gender and BMI, ED-specific brooding, b = 1.32, SE = 0.13, ß = 0.46, p < 0.0001, and reflection, b = 1.44, SE = 0.33, ß = 0.19, p < 0.0001, accounted for unique variance in ED symptom severity. Moderation model results indicate that, at low levels of general reflection, b = - 0.06, SE = 0.02, ß = - 0.51, p = 0.003, and ED-specific reflection, b = - 0.15, SE = 0.03, ß = - 0.59, p < 0.0001, increased positive beliefs about rumination were associated with greater ED symptom severity. CONCLUSION: Findings suggest ED-specific rumination accounts for ED symptom severity above and beyond general rumination, and that rumination-related expectancies influence the association between reflection and ED symptom severity. LEVEL OF EVIDENCE: Level III, evidence obtained from a well-designed cohort study.

Chemokine-Mediated Choreography of Thymocyte Development and Selection.

As they differentiate, thymocytes encounter spatially restricted cues critical for differentiation and selection of a functional, self-tolerant T cell repertoire. Sequential migration of developing T cells through distinct thymic microenvironments is enforced by the ordered expression of chemokine receptors. Herein, we provide an updated perspective on T cell differentiation through the lens of recent advances that illuminate the dynamics of chemokine-driven thymocyte migration, localization, and interactions with stromal cells. We consider these findings in the context of earlier groundwork exploring the contribution of chemokines to T cell development, recent advances regarding the specificity of chemokine signaling, and novel techniques for evaluating the T cell repertoire. We suggest future research should amalgamate visualization of localized cellular interactions with downstream molecular signals.

Microglia depletion and alcohol: Transcriptome and behavioral profiles.

Alcohol abuse induces changes in microglia morphology and immune function, but whether microglia initiate or simply amplify the harmful effects of alcohol exposure is still a matter of debate. Here, we determine microglia function in acute and voluntary drinking behaviors using a colony-stimulating factor 1 receptor inhibitor (PLX5622). We show that microglia depletion does not alter the sedative or hypnotic effects of acute intoxication. Microglia depletion also does not change the escalation or maintenance of chronic voluntary alcohol consumption. Transcriptomic analysis revealed that although many immune genes have been implicated in alcohol abuse, downregulation of microglia genes does not necessitate changes in alcohol intake. Instead, microglia depletion and chronic alcohol result in compensatory upregulation of alcohol-responsive, reactive astrocyte genes, indicating astrocytes may play a role in regulation of these alcohol behaviors. Taken together, our behavioral and transcriptional data indicate that microglia are not the primary effector cell responsible for regulation of acute and voluntary alcohol behaviors. Because microglia depletion did not regulate acute or voluntary alcohol behaviors, we hypothesized that these doses were insufficient to activate microglia and recruit them to an effector phenotype. Therefore, we used a model of repeated immune activation using polyinosinic:polycytidylic acid (poly(I:C)) to activate microglia. Microglia depletion blocked poly(I:C)-induced escalations in alcohol intake, indicating microglia regulate drinking behaviors with sufficient immune activation. By testing the functional role of microglia in alcohol behaviors, we provide insight into when microglia are causal and when they are consequential for the transition from alcohol use to dependence.

Effect of Reader and Patient Parameters on the Performance of Last-Image-Hold for Fluoroscopic Grading of Vesicoureteric Reflux.

OBJECTIVE. The purpose of this study is to determine the effect of different reader and patient parameters on the degree of agreement and the rate of misclassification of vesicoureteric reflux grading on last-image-hold frames in relation to spot-exposed frames from voiding cystourethrography (VCUG) as well as to determine the nature of reflux misclassification on last-image-hold frames. MATERIALS AND METHODS. Blinded readers conducted a retrospective evaluation of last-image-hold and spot-exposed frames of the renal fossae from 191 sequential VCUG examinations performed during a five-year period. Kappa tests were used to determine the agreement between reflux gradings and to assess the impact of reader and patient parameters. Pearson product-moment correlations were used to evaluate the effect of patient parameters on reader level of certainty regarding reflux grading. RESULTS. We measured almost perfect overall agreement for more experienced readers and substantial overall agreement for less experienced readers. Point estimates of overall misclassification were less than 2% for more experienced readers and less than 4% for less experienced readers. The readers' level of certainty about reflux grading had a positive impact on agreement values and misclassification rates. Experienced readers' most common misclassification was assigning reflux a grade of 3 on a spot-exposed frame and a grade of 2 on an equivalent last-image-hold frame. Inexperienced readers' most common misclassification involved missing reflux altogether. CONCLUSION. Instances of grade 2 reflux on last-image-hold frames may warrant supplemental evaluation with spot-exposed frames. Otherwise, a reader's level of certainty regarding reflux grading on a last-image-hold frame may help determine whether a supplemental spot-exposed frame would be beneficial.

Endogenous dendritic cells from the tumor microenvironment support T-ALL growth via IGF1R activation.

Primary T-cell acute lymphoblastic leukemia (T-ALL) cells require stromal-derived signals to survive. Although many studies have identified cell-intrinsic alterations in signaling pathways that promote T-ALL growth, the identity of endogenous stromal cells and their associated signals in the tumor microenvironment that support T-ALL remains unknown. By examining the thymic tumor microenvironments in multiple murine T-ALL models and primary patient samples, we discovered the emergence of prominent epithelial-free regions, enriched for proliferating tumor cells and dendritic cells (DCs). Systematic evaluation of the functional capacity of tumor-associated stromal cells revealed that myeloid cells, primarily DCs, are necessary and sufficient to support T-ALL survival ex vivo. DCs support T-ALL growth both in primary thymic tumors and at secondary tumor sites. To identify a molecular mechanism by which DCs support T-ALL growth, we first performed gene expression profiling, which revealed up-regulation of platelet-derived growth factor receptor beta (Pdgfrb) and insulin-like growth factor I receptor (Igf1r) on T-ALL cells, with concomitant expression of their ligands by tumor-associated DCs. Both Pdgfrb and Igf1r were activated in ex vivo T-ALL cells, and coculture with tumor-associated, but not normal thymic DCs, sustained IGF1R activation. Furthermore, IGF1R signaling was necessary for DC-mediated T-ALL survival. Collectively, these studies provide the first evidence that endogenous tumor-associated DCs supply signals driving T-ALL growth, and implicate tumor-associated DCs and their mitogenic signals as auspicious therapeutic targets.

EBI2 contributes to the induction of thymic central tolerance in mice by promoting rapid motility of medullary thymocytes.

Maturing thymocytes enter the thymic medulla, where they encounter numerous self-antigens presented by antigen presenting cells (APCs). Those thymocytes that are strongly self-reactive undergo either negative selection or diversion into the regulatory T-cell lineage. Although the majority of the proteome is expressed in the medulla, many self-antigens are expressed by only a minor fraction of medullary APCs; thus, thymocytes must efficiently enter the medulla and scan APCs to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APCs in lymphoid organs. The chemokine receptor EBI2 governs localization of T cells, B cells, and dendritic cells (DCs) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4+ single positive (CD4SP) thymocytes and thymic DCs. EBI2 deficiency alters the TCR repertoire, but does not grossly impact thymocyte cellularity or subset distribution. EBI2 deficiency also impairs negative selection of OT-II TCR transgenic thymocytes responding to an endogenous self-antigen. Two-photon imaging revealed that EBI2 deficiency results in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient thymic central tolerance.

Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.

T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used two-photon microscopy to visualize migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4(-)8(-)) and double-positive (CD4(+)8(+)) thymocytes were confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were unable to migrate on medullary substrates. In contrast, CD4 single positive (SP) thymocytes migrated directionally toward the medulla, where they accumulated and moved very rapidly. Our results revealed a requisite two-step process governing CD4 SP cell medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus.

Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively.

Low ligand requirement for deletion and lack of synapses in positive selection enforce the gauntlet of thymic T cell maturation.

Immature double-positive (CD4(+)CD8(+)) thymocytes respond to negatively selecting peptide-MHC ligands by forming an immune synapse that sustains contact with the antigen-presenting cell (APC). Using fluorescently labeled peptides, we showed that as few as two agonist ligands could promote APC contact and subsequent apoptosis in reactive thymocytes. Furthermore, we showed that productive signaling for positive selection, as gauged by nuclear translocation of a green fluorescent protein (GFP)-labeled NFATc construct, did not involve formation of a synapse between thymocytes and selecting epithelial cells in reaggregate thymus cultures. Antibody blockade of endogenous positively selecting ligands prevented NFAT nuclear accumulation in such cultures and reversed NFAT accumulation in previously stimulated thymocytes. Together, these data suggest a "gauntlet" model in which thymocytes mature by continually acquiring and reacquiring positively selecting signals without sustained contact with epithelial cells, thereby allowing them to sample many cell surfaces for potentially negatively selecting ligands.

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

Clinical and imaging considerations in primary immunodeficiency disorders: an update.

Primary immunodeficiencies are a group of genetically determined disorders with diverse presentations. The purpose of this review is to provide a practical and brief description of a select number of these diseases and to discuss the important role the radiologist can have in making an early diagnosis and in detecting and following disease complications. The role of diagnostic imaging and informed performance and interpretation are vital in the diagnosis, surveillance and management of all primary immunodeficiency disorders.

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly.

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

Coactivator-associated arginine methyltransferase 1 regulates fetal hematopoiesis and thymocyte development.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.

Antithrombin activity and central venous catheter-associated thrombosis in critically ill children at high risk of bleeding.

BACKGROUND: Normalization of antithrombin activity may prevent catheter-associated thrombosis in critically ill children at high risk of bleeding. OBJECTIVES: To characterize the temporal pattern of antithrombin activity, assess its association with catheter-associated thrombosis and clinically relevant bleeding, and evaluate its relationship with thrombin generation in these children. METHODS: In this prospective cohort study, critically ill children <18 years old at high risk of bleeding with central venous catheter were eligible. Antithrombin activity and thrombin generation were measured from platelet-poor plasma and after in vitro antithrombin supplementation. Systematic surveillance ultrasound was performed to diagnose thrombosis. Children were followed for bleeding. RESULTS: We enrolled 8 infants (median age: 0.2 years, IQR: 0.2, 0.3 years) and 72 older children (median age: 14.3 years, IQR: 9.1, 16.1 years). Mean antithrombin on the day of catheter insertion was 64 IU/dL (SD: 32 IU/dL) in infants and 83 IU/dL (SD: 35 IU/dL) in older children. Antithrombin normalized by the day of catheter removal. Thrombosis developed in 27 children, while 31 children bled. Thrombosis (regression coefficient: 0.008, 95% CI: -0.01, 0.03) and bleeding (regression coefficient: -0.0007, 95% CI: -0.02, 0.02) were not associated with antithrombin. Antithrombin was not correlated with in vivo change in endogenous thrombin potential (correlation coefficient: -0.07, 95% CI: -0.21, 0.08). In vitro supplementation reduced endogenous thrombin potential (correlation coefficient: -0.78; 95% CI: -0.95, -0.23). CONCLUSION: These findings may not support normalization of antithrombin activity to prevent catheter-associated thrombosis in critically ill children at high risk of bleeding.

Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Features of acute COVID-19 associated with post-acute sequelae of SARS-CoV-2 phenotypes: results from the IMPACC study.

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

Integrated longitudinal multiomics study identifies immune programs associated with acute COVID-19 severity and mortality.

BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors.

The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials.