Expanding the PRAAS spectrum: De novo mutations of immunoproteasome subunit ß-type 10 in six infants with SCID-Omenn syndrome.

Mutations in proteasome ß-subunits or their chaperone and regulatory proteins are associated with proteasome-associated autoinflammatory disorders (PRAAS). We studied six unrelated infants with three de novo heterozygous missense variants in PSMB10, encoding the proteasome ß2i-subunit. Individuals presented with T-B-NK± severe combined immunodeficiency (SCID) and clinical features suggestive of Omenn syndrome, including diarrhea, alopecia, and desquamating erythematous rash. Remaining T cells had limited T cell receptor repertoires, a skewed memory phenotype, and an elevated CD4/CD8 ratio. Bone marrow examination indicated severely impaired B cell maturation with limited V(D)J recombination. All infants received an allogeneic stem cell transplant and exhibited a variety of severe inflammatory complications thereafter, with 2 peri-transplant and 2 delayed deaths. The single long-term transplant survivor showed evidence for genetic rescue through revertant mosaicism overlapping the affected PSMB10 locus. The identified variants (c.166G>C [p.Asp56His] and c.601G>A/c.601G>C [p.Gly201Arg]) were predicted in silico to profoundly disrupt 20S immunoproteasome structure through impaired ß-ring/ß-ring interaction. Our identification of PSMB10 mutations as a cause of SCID-Omenn syndrome reinforces the connection between PRAAS-related diseases and SCID.

Defective neutrophil development and specific granule deficiency caused by a homozygous splice-site mutation in SMARCD2.

BACKGROUND: SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2. OBJECTIVE: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells. METHODS: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34+ cells were then isolated, phenotyped, and assessed functionally. RESULTS: Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34+ cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later. CONCLUSIONS: This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency.

BCG lymphadenitis: a potential complication of immune reconstitution following haematopoietic stem cell transplant.

An MHC class II deficient 2-year-old boy presented with fever and an enlarging left neck mass 100 days post allogeneic haematopoietic stem cell transplant (HSCT). Fever persisted despite treatment with broad-spectrum ß-lactam antibiotics. His BCG vaccination site at presentation was quiescent. Ultrasound showed enlarged cervical lymph nodes. An incisional biopsy of the large nodal mass yielded acid-fast bacilli, identified as Mycobacterium bovis by genome sequencing. Treatment with rifampicin, isoniazid and pyridoxine was started. The mass suppurated (figure 1), before healing concurrently with T-lymphocyte reconstitution at approximately day 130 post-HSCT.

Zap70 is essential for long-term survival of naive CD8 T cells.

Survival of naive T cells requires engagement of TCR with self-peptide major histocompatibility Ags. The signaling pathways required to transmit this survival signal are poorly understood. In this study, we asked whether the tyrosine kinase Zap70 is required to transmit survival signals in naive CD8 T cells. In the absence of Zap70 expression, thymic development is completely blocked. Using a tetracycline-inducible Zap70 transgene (TetZap70), thymic development of Zap70-deficient TCR transgenic F5 mice was restored. Feeding mice doxycycline to induce Zap70 expression resulted in repopulation of the peripheral naive compartment. Zap70 transgene expression was then ablated by withdrawal of doxycycline. Survival of Zap70-deficient naive CD8 T cells depended on host environment. In hosts with a replete T cell compartment, naive T cells died rapidly in the absence of Zap70 expression. In lymphopenic hosts, Zap70-deficient T cells survived far longer, in an IL-7-dependent manner, but failed to undergo lymphopenia-induced proliferation. Analyzing mixed bone marrow chimeras revealed that intact Zap70-dependent signaling was important for integration of recent thymic emigrants into the mature naive compartment. Finally, we asked whether adaptor function conferred by Zap70 tyrosines 315 and 319 was necessary for transmission of homeostatic TCR signals. This was done by analyzing F5 mice expressing mutant Zap70 in which these residues had been mutated to alanines (Zap70(YYAA)). Inducible Zap70 expression rescued thymic development in F5 TetZap70 Zap70(YYAA) mice. However, in the absence of wild-type Zap70 expression, the Zap70(YYAA) mutant failed to transmit either survival or proliferative homeostatic signals.

NUDCD3 deficiency disrupts V(D)J recombination to cause SCID and Omenn syndrome.

Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for a single deleterious missense variant in the gene NudC domain-containing 3 (NUDCD3). Two infants had severe combined immunodeficiency with the complete absence of T and B cells (T -B- SCID), whereas nine showed classical features of Omenn syndrome (OS). Restricted antigen receptor gene usage by residual T lymphocytes suggested impaired V(D)J recombination. Patient cells showed reduced expression of NUDCD3 protein and diminished ability to support RAG-mediated recombination in vitro, which was associated with pathologic sequestration of RAG1 in the nucleoli. Although impaired V(D)J recombination in a mouse model bearing the homologous variant led to milder immunologic abnormalities, NUDCD3 is absolutely required for healthy T and B cell development in humans.

Recurrent group B stretococcus infection in an extremely premature infant: as a preterm neonate, infant and toddler.

We report five discrete episodes of group B streptococcus (GBS) bacteraemia in an extremely premature infant, extending into early childhood. The first four episodes occurred during infancy despite appropriate treatment. Breastmilk was positive for group B streptococcal 16S DNA by polymerase chain reaction. The fifth episode occurred at 17 months of age, shortly after stopping antimicrobial prophylaxis.Radiological investigations did not identify a focus for recurrence of GBS bacteraemia, and immunological investigations and targeted whole genome sequencing yielded only transient hypogammaglobulinaemia of infancy, which resolved.This case highlights invasive GBS infection as a cause of infant morbidity. Premature infants are at particular risk of invasive as well as recurrent disease. GBS is typically a sensitive organism and each episode of GBS in our patient was effectively treated with penicillin. The role of breastmilk in recurrent GBS is controversial; in this case infant and mother isolated identical GBS serotypes and were concurrently treated with rifampicin.

Gestational Development of the Human Immune System.

Building an immune system is a monumental task critical to the survival of the fetus and newborn. A functional fetal immune system must complement the maternal immune system in handling in utero infection; abstain from damaging non-self-reactions that would compromise the materno-fetal interface; mobilize in response to infection and equip mucosal tissues for pathogen exposure at birth. There is growing appreciation that immune cells also have noncanonical roles in development and specifically may contribute to tissue morphogenesis. In this review we detail how hematopoietic and lymphoid organs jointly establish cellular constituents of the immune system; how these constituents are organized in 2 mucosal sites-gut and lung-where early life immune function has long-term consequences for health; and how exemplar diseases of prematurity and inborn errors of immunity reveal dominant pathways in prenatal immunity.

Clinical frailty, and not features of acute infection, is associated with late mortality in COVID-19: a retrospective cohort study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with excess mortality after hospital discharge. Identification of patients at increased risk of death following hospital discharge is needed to guide clinical monitoring and early intervention. Herein, we aimed to identify predictors of early vs. late mortality in COVID-19 patients. METHODS: A total of 471 patients with polymerase chain reaction-confirmed COVID-19 were followed up for 9 months [median (inter-quartile range) of follow-up time: 271 (14) days] after hospital admission. COVID-19-related signs and symptoms, laboratory features, co-morbidities, Coronavirus Clinical Characterisation Consortium (4C) mortality and Clinical Frailty Scale (CFS) scores were analysed by logistic regression for association with early (28 day) vs. late mortality. Receiver operating characteristic (ROC) analysis was used to determine the discriminative value of 4C and CFS scores for early vs. late mortality. RESULTS: A total of 120 patients died within 28 days from hospital admission. Of the remaining 351 patients, 41 died within the next 8 months. Respiratory failure, systemic inflammation, and renal impairment were associated with early mortality, while active cancer and dementia were associated with late mortality, after adjustment for age and sex. 4C mortality score and CFS were associated with both early [odds ratio (OR) (95% confidence interval-CI): 4C: 1.34 (1.25-1.45); CFS: 1.49 (1.33-1.66)] and late [OR (95% CI): 4C: 1.23 (1.12-1.36); CFS: 2.04 (1.62-2.56)] mortality. After adjustment for CFS, the association between 4C and late mortality was lost. By ROC analysis, 4C mortality score was superior to CFS for 28 day mortality [area under the curve (AUC) (95% CI): 0.779 (0.732-0.825) vs. 0.723 (0.673-0.773), respectively; P = 0.039]. In contrast, CFS had higher predictive value for late mortality compared with 4C mortality score [AUC (95% CI): 0.830 (0.776-0.883) vs. 0.724 (0.650-0.798), respectively; P = 0.007]. CONCLUSIONS: In our cohort, late mortality in COVID-19 patients is more strongly associated with premorbid clinical frailty than with severity of the acute infection phase.

National Early Warning Score 2 (NEWS2) to identify inpatient COVID-19 deterioration: a retrospective analysis.

INTRODUCTION: We sought to provide the first report of the use of NEWS2 monitoring to pre-emptively identify clinical deterioration within hospitalised COVID-19 patients. METHODS: Consecutive adult admissions with PCR-confirmed COVID-19 were included in this single-centre retrospective UK cohort study. We analysed all electronic clinical observations recorded within 28 days of admission until discharge or occurrence of a serious event, defined as any of the following: initiation of respiratory support, admission to intensive care, initiation of end of life care, or in-hospital death. RESULTS: 133/296 (44.9%) patients experienced at least one serious event. NEWS2 ≥ 5 heralded the first occurrence of a serious event with sensitivity 0.98 (95% CI 0.96-1.00), specificity 0.28 (0.21-0.35), positive predictive value (PPV) 0.53 (0.47-0.59), and negative predictive value (NPV) 0.96 (0.90-1.00). The NPV (but not PPV) of NEWS2 monitoring exceeded that of other early warning scores including the Modified Early Warning Score (MEWS) (0.59 [0.52-0.66], p<0.001) and quick Sepsis Related Organ Failure Assessment (qSOFA) score (0.58 [0.51-0.65], p<0.001). CONCLUSION: Our results support the use of NEWS2 monitoring as a sensitive method to identify deterioration of hospitalised COVID-19 patients, albeit at the expense of a relatively high false-trigger rate.

Evaluation of procalcitonin-guided antimicrobial stewardship in patients admitted to hospital with COVID-19 pneumonia.

BACKGROUND: Procalcitonin is a biomarker that may be able to identify patients with COVID-19 pneumonia who do not require antimicrobials for bacterial respiratory tract co-infections. OBJECTIVES: To evaluate the safety and effectiveness of a procalcitonin-guided algorithm in rationalizing empirical antimicrobial prescriptions in non-critically ill patients with COVID-19 pneumonia. METHODS: Retrospective, single-site, cohort study in adults hospitalized with confirmed or suspected COVID-19 pneumonia and receiving empirical antimicrobials for potential bacterial respiratory tract co-infection. Regression models were used to compare the following outcomes in patients with and without procalcitonin testing within 72 h of starting antimicrobials: antimicrobial consumption (DDD); antimicrobial duration; a composite safety outcome of death, admission to HDU/ICU or readmission to hospital within 30 days; and length of admission. Procalcitonin levels of ≤0.25 ng/L were interpreted as negatively predictive of bacterial co-infection. Effects were expressed as ratios of means (ROM) or prevalence ratios (PR) accordingly. RESULTS: 259 patients were included in the final analysis. Antimicrobial use was lower in patients who had procalcitonin measured within 72 h of starting antimicrobials: mean antimicrobial duration 4.4 versus 5.4 days, adjusted ROM 0.7 (95% CI 0.6-0.9); mean antimicrobial consumption 6.8 versus 8.4 DDD, adjusted ROM 0.7 (95% CI 0.6-0.8). Both groups had similar composite safety outcomes (adjusted PR 0.9; 95% CI 0.6-1.3) and lengths of admission (adjusted ROM 1.3; 95% CI 0.9-1.6). CONCLUSIONS: A procalcitonin-guided algorithm may allow for the safe reduction of antimicrobial usage in hospitalized non-critically ill patients with COVID-19 pneumonia.

Estimated pulse wave velocity improves risk stratification for all-cause mortality in patients with COVID-19.

Accurate risk stratification in COVID-19 patients consists a major clinical need to guide therapeutic strategies. We sought to evaluate the prognostic role of estimated pulse wave velocity (ePWV), a marker of arterial stiffness which reflects overall arterial integrity and aging, in risk stratification of hospitalized patients with COVID-19. This retrospective, longitudinal cohort study, analyzed a total population of 1671 subjects consisting of 737 hospitalized COVID-19 patients consecutively recruited from two tertiary centers (Newcastle cohort: n = 471 and Pisa cohort: n = 266) and a non-COVID control cohort (n = 934). Arterial stiffness was calculated using validated formulae for ePWV. ePWV progressively increased across the control group, COVID-19 survivors and deceased patients (adjusted mean increase per group 1.89 m/s, P < 0.001). Using a machine learning approach, ePWV provided incremental prognostic value and improved reclassification for mortality over the core model including age, sex and comorbidities [AUC (core model + ePWV vs. core model) = 0.864 vs. 0.755]. ePWV provided similar prognostic value when pulse pressure or hs-Troponin were added to the core model or over its components including age and mean blood pressure (p < 0.05 for all). The optimal prognostic ePWV value was 13.0 m/s. ePWV conferred additive discrimination (AUC: 0.817 versus 0.779, P < 0.001) and reclassification value (NRI = 0.381, P < 0.001) over the 4C Mortality score, a validated score for predicting mortality in COVID-19 and the Charlson comorbidity index. We suggest that calculation of ePWV, a readily applicable estimation of arterial stiffness, may serve as an additional clinical tool to refine risk stratification of hospitalized patients with COVID-19 beyond established risk factors and scores.

SARS-CoV-2 Testing of 11,884 Healthcare Workers at an Acute NHS Hospital Trust in England: A Retrospective Analysis.

Healthcare workers (HCWs) are known to be at increased risk of infection with SARS-CoV-2, although whether these risks are equal across all roles is uncertain. Here we report a retrospective analysis of a large real-world dataset obtained from 10 March to 6 July 2020 in an NHS Foundation Trust in England with 17,126 employees. 3,338 HCWs underwent symptomatic PCR testing (14.4% positive, 2.8% of all staff) and 11,103 HCWs underwent serological testing for SARS-CoV-2 IgG (8.4% positive, 5.5% of all staff). Seropositivity was lower than other hospital settings in England but higher than community estimates. Increased test positivity rates were observed in HCWs from BAME backgrounds and residents in areas of higher social deprivation. A multiple logistic regression model adjusting for ethnicity and social deprivation confirmed statistically significant increases in the odds of testing positive in certain occupational groups, most notably domestic services staff, nurses, and health-care assistants. PCR testing of symptomatic HCWs appeared to underestimate overall infection levels, probably due to asymptomatic seroconversion. Clinical outcomes were reassuring, with only a small minority of HCWs with COVID-19 requiring hospitalization (2.3%) or ICU management (0.7%) and with no deaths. Despite a relatively low level of HCW infection compared to other UK cohorts, there were nevertheless important differences in test positivity rates between occupational groups, robust to adjustment for demographic factors such as ethnic background and social deprivation. Quantitative and qualitative studies are needed to better understand the factors contributing to this risk. Robust informatics solutions for HCW exposure data are essential to inform occupational monitoring.

COVID-19 Management in a UK NHS Foundation Trust with a High Consequence Infectious Diseases Centre: A Retrospective Analysis.

Recent large national and international cohorts describe the baseline characteristics and outcome of hospitalised patients with COVID-19, however there is limited granularity to these reports. We aimed to provide a detailed description of a UK COVID-19 cohort, focusing on management and outcome. We performed a retrospective single-centre analysis of clinical management and 28-day outcomes of consecutive adult inpatients with SARS-CoV-2 PCR-confirmed COVID-19 from 31 January to 16 April 2020 inclusive. In total, 316 cases were identified. Most patients were elderly (median age 75) with multiple comorbidities. One quarter were admitted from residential or nursing care. Mortality was 84 out of 316 (26.6%). Most deaths occurred in patients in whom a ceiling of inpatient treatment had been determined and for whom end of life care and specialist palliative care input was provided where appropriate. No deaths occurred in patients aged under 56 years. Decisions to initiate respiratory support were individualised after consideration of patient wishes, premorbid frailty and comorbidities. In total, 59 (18%) patients were admitted to intensive care, of which 31 (10% overall cohort) required intubation. Multiple logistic regression identified associations between death and age, frailty, and disease severity, with age as the most significant factor (odds ratio 1.07 [95% CI 1.03-1.10] per year increase, p < 0.001). These findings provide important clinical context to outcome data. Mortality was associated with increasing age. Most deaths were anticipated and occurred in patients with advance decisions on ceilings of treatment.

Single-cell multi-omics analysis of the immune response in COVID-19.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

COVID-19-associated hyperinflammation and escalation of patient care: a retrospective longitudinal cohort study.

BACKGROUND: A subset of patients with severe COVID-19 develop a hyperinflammatory syndrome, which might contribute to morbidity and mortality. This study explores a specific phenotype of COVID-19-associated hyperinflammation (COV-HI), and its associations with escalation of respiratory support and survival. METHODS: In this retrospective cohort study, we enrolled consecutive inpatients (aged ≥18 years) admitted to University College London Hospitals and Newcastle upon Tyne Hospitals in the UK with PCR-confirmed COVID-19 during the first wave of community-acquired infection. Demographic data, laboratory tests, and clinical status were recorded from the day of admission until death or discharge, with a minimum follow-up time of 28 days. We defined COV-HI as a C-reactive protein concentration greater than 150 mg/L or doubling within 24 h from greater than 50 mg/L, or a ferritin concentration greater than 1500 µg/L. Respiratory support was categorised as oxygen only, non-invasive ventilation, and intubation. Initial and repeated measures of hyperinflammation were evaluated in relation to the next-day risk of death or need for escalation of respiratory support (as a combined endpoint), using a multi-level logistic regression model. FINDINGS: We included 269 patients admitted to one of the study hospitals between March 1 and March 31, 2020, among whom 178 (66%) were eligible for escalation of respiratory support and 91 (34%) patients were not eligible. Of the whole cohort, 90 (33%) patients met the COV-HI criteria at admission. Despite having a younger median age and lower median Charlson Comorbidity Index scores, a higher proportion of patients with COV-HI on admission died during follow-up (36 [40%] of 90 patients) compared with the patients without COV-HI on admission (46 [26%] of 179). Among the 178 patients who were eligible for full respiratory support, 65 (37%) met the definition for COV-HI at admission, and 67 (74%) of the 90 patients whose respiratory care was escalated met the criteria by the day of escalation. Meeting the COV-HI criteria was significantly associated with the risk of next-day escalation of respiratory support or death (hazard ratio 2·24 [95% CI 1·62-2·87]) after adjustment for age, sex, and comorbidity. INTERPRETATION: Associations between elevated inflammatory markers, escalation of respiratory support, and survival in people with COVID-19 indicate the existence of a high-risk inflammatory phenotype. COV-HI might be useful to stratify patient groups in trial design. FUNDING: None.

The long-term survival potential of mature T lymphocytes is programmed during development in the thymus.

The homeostatic maintenance of normal numbers of mature T lymphocytes in the immune system depends on signaling from the T cell antigen receptor (TCR) and the interleukin-7 receptor (IL-7R); however, it is unclear whether there is crosstalk between these two receptors. Here, we have identified a central role for TCR signaling during the development of T lymphocytes in the thymus in the determination of IL-7 function in mature T lymphocytes. We showed that Il7r expression in mature T cells was modulated by developmental TCR-dependent signals elicited during the process of positive selection in the thymus and that this mechanism was common to both CD4(+) and CD8(+) T cells. Control of Il7r expression by the TCR was limited to thymocytes because neither the abundance nor the function of IL-7Rα was affected by TCR signaling in peripheral T cells. Finally, we showed that thymocytes without optimal IL-7Rα abundance failed to form part of the pool of mature T lymphocytes that patrol the periphery of normal hosts, highlighting the importance of this mechanism in shaping the repertoire of lymphocytes that make up this population.