The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons.

The severity of COVID-19 is linked to excessive inflammation. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation, but their role in COVID-19 pathophysiology remains poorly understood. We conducted transcriptomic profiling of neutrophils obtained from patients with mild and severe COVID-19, as well as from SARS-CoV-2 infected mice, in comparison to non-infected healthy controls. In addition, we investigated the inflammasome formation potential in neutrophils from patients and mice upon SARS-CoV-2 infection. Transcriptomic analysis of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, revealed a striking type I interferon (IFN-I) gene signature in severe COVID-19 patients, contrasting with mild COVID-19 and healthy controls. Notably, low-density granulocytes (LDGs) from severe COVID-19 patients exhibited an immature neutrophil phenotype and lacked this IFN-I signature. Moreover, PMNs from severe COVID-19 patients showed heightened nigericin-induced caspase1 activation, but reduced responsiveness to exogenous inflammasome priming. Furthermore, IFN-I emerged as a priming stimulus for neutrophil inflammasomes. These findings suggest a potential role for neutrophil inflammasomes in driving inflammation during severe COVID-19. Altogether, these findings open promising avenues for targeted therapeutic interventions to mitigate the pathological processes associated with the disease.

Reduction in mucosal-associated invariant T cells (MAIT) in APECED patients is associated with elevated serum IFN-γ concentration.

Mucosal-associated invariant T cells (MAIT) are innate-like lymphocytes enriched in mucosal organs where they contribute to antimicrobial defense. APECED is an inborn error of immunity characterized by immune dysregulation and chronic mucocutaneous candidiasis. Reduction in the frequency of circulating MAITs has been reported in many inborn errors of immunity, but only in a few of them, the functional competence of MAITs has been assessed. Here, we show in a cohort of 24 patients with APECED, that the proportion of circulating MAITs was reduced compared with healthy age and sex-matched controls (1.1% vs. 2.6% of CD3+ T cells; p < 0.001) and the MAIT cell immunophenotype was more activated. Functionally the IFN-γ secretion of patient MAITs after stimulation was comparable to healthy controls. We observed in the patients elevated serum IFN-γ (46.0 vs. 21.1 pg/mL; p = 0.01) and IL-18 (42.6 vs. 13.7 pg/mL; p < 0.001) concentrations. Lower MAIT proportion did not associate with the levels of neutralizing anti-IL-22 or anti-IL-12/23 antibodies but had a clear negative correlation with serum concentrations of IFN-γ, IL-18, and protein C-reactive protein. Our data suggest that reduction of circulating MAITs in patients with APECED correlates with chronic type 1 inflammation but the remaining MAITs are functionally competent.

Dysregulated germinal center reaction with expanded T follicular helper cells in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy lymph nodes.

BACKGROUND: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, also called APS-1) is an inborn error of immunity with clear signs of B-cell autoimmunity such as neutralizing anti-IFN antibodies. In APECED, mutations in the AIRE gene impair thymic negative selection of T cells. The resulting T-cell alterations may then cause dysregulation of B-cell responses. However, no analysis of interactions of T and B cells in the germinal centers (GCs) in patients' secondary lymphatic tissues has been reported. OBJECTIVE: This study examined the relationship between B cells and follicular T helper cells (TfH) in peripheral blood and lymph node (LN) GCs in patients with APECED. METHODS: Immunophenotyping of peripheral blood B cells and TfH was performed for 24 patients with APECED. Highly multiplexed fluorescent immunohistochemical staining was performed on 7 LN biopsy samples from the patients to study spatial interactions of lymphocytes in the GCs at the single-cell level. RESULTS: The patients' peripheral B-cell phenotype revealed skewing toward a mature B-cell phenotype with marked loss of transitional and naive B cells. The frequency of circulating TfH cells was diminished in the patients, while in the LNs the TfH population was expanded. In LNs the overall frequency of Treg cells and interactions of Treg cells with nonfollicular T cells were reduced, suggesting that aberrant Treg cell function might fail to restrain TfH differentiation. CONCLUSIONS: GC reactions are disrupted in APECED as a result of defective T-cell control.

Increased type 1 inflammation in gynecologic cervicovaginal samples in patients with APS-1.

BACKGROUND: Inborn errors of immunity offer important insights into mucosal immunity. In autoimmune polyendocrine syndrome type-1 (APS-1), chronic mucocutaneous candidiasis has been ascribed to neutralizing IL-17 autoantibodies. Recent evidence implicates excessive T-cell IFN-γ secretion and ensuing epithelial barrier disruption in predisposition to candidiasis, but these results remain to be replicated. Whether IL-17 paucity, increased type I inflammation, or their combination underlies susceptibility to chronic mucocutaneus candidiasis in APS-1 is debated. OBJECTIVE: Our aim was to characterize the immunologic features in the cervicovaginal mucosa of females with APS-1. METHODS: Vaginal fluid was collected with a flocked swab from 17 females with APS-1 and 18 controls, and cytokine composition was analyzed using Luminex (Luminex Corporation, Austin, Tex). Cervical cell samples were obtained with a cervix brush from 6 patients and 6 healthy controls and subjected to transcriptome analysis. RESULTS: The vaginal fluid samples from patients with APS-1 had IFN-γ concentrations comparable to those of the controls (2.6 vs 2.4 pg/mL) but high concentrations of the TH1 chemokines CXCL9 and CXCL10 (1094 vs 110 pg/mL [P < .001] and 4033 vs 273 pg/mL [P = .001], respectively), whereas the IL-17 levels in the samples from the 2 groups were comparable (28 vs 8.8 pg/mL). RNA sequencing of the cervical cells revealed upregulation of pathways related to mucosal inflammation and cell death in the patients with APS-1. CONCLUSION: Excessive TH1 cell response appears to underlie disruption of the mucosal immune responses in the genital tract of patients with APS-1 and may contribute to susceptibility to candidiasis in the genital tract as well.

Helios-Illuminating the way for lymphocyte self-control.

Transcription factor Helios, encoded by the IKZF2 gene, has an important role in regulatory T cells by stabilizing their suppressive phenotype. While Helios is prominently expressed in regulatory T cells, its expression extends beyond to include effector T cells, follicular regulatory T cells, B cells, and innate-like lymphocyte populations. Recent characterizations of patients with inborn error of immunity due to damaging IKZF2 variants coupled with translational research on lymphocytes from healthy individuals, have increased our understanding on Helios' multifaceted role in controlling the human adaptive immune system. A less studied role for Helios beyond the stabilizing of regulatory T cells has emerged in directing effector T cell maturation. In the absence of functional Helios, effector T cells acquire more inflammatory phenotype and are prone to senescence. Loss of Helios expression disrupts the regulation of the germinal centre reaction, often resulting in either hypogammaglobulinemia or B cell autoimmunity. This review summarizes findings from studies in both mice and men offering a comprehensive understanding of the impact of the transcription factor Helios on the adaptive immune system.

Infective SARS-CoV-2 in Skull Sawdust at Autopsy, Finland.

We assessed the distribution of SARS-CoV-2 at autopsy in 22 deceased persons with confirmed COVID-19. SARS-CoV-2 was found by PCR (2/22, 9.1%) and by culture (1/22, 4.5%) in skull sawdust, suggesting that live virus is present in tissues postmortem, including bone. Occupational exposure risk is low with appropriate personal protective equipment.

Proteomics screening after pediatric allogenic hematopoietic stem cell transplantation reveals an association between increased expression of inhibitory receptor FCRL6 on γδ T cells and cytomegalovirus reactivation.

We studied the associations between inflammation-related proteins in circulation and complications after pediatric allogenic hematopoietic stem cell transplantation (HSCT), to reveal proteomic signatures or individual soluble proteins associated with specific complications after HSCT. We used a proteomics method called Proximity Extension Assay to repeatedly measure 180 different proteins together with clinical variables, cellular immune reconstitution and blood viral copy numbers in 27 children (1-18 years of age) during a 2-year follow-up after allogenic HSCT. Protein profile analysis was performed using unsupervised hierarchical clustering and a regression-based method, while the Bonferroni-corrected Mann-Whitney U-test was used for time point-specific comparison of individual proteins against outcome. At 6 months after allogenic HSCT, we could identify a protein profile pattern associated with occurrence of the complications such as chronic graft-versus-host disease, viral infections, relapse and death. When protein markers were analyzed separately, the plasma concentration of the inhibitory and cytotoxic T-cell surface protein FCRL6 (Fc receptor-like 6) was higher in patients with cytomegalovirus (CMV) viremia [log2-fold change 1.5 (P = 0.00099), 2.5 (P = 0.00035) and 2.2 (P = 0.045) at time points 6, 12 and 24 months]. Flow cytometry confirmed that FCRL6 expression was higher in innate-like γδ T cells, indicating that these cells are involved in controlling CMV reactivation in HSCT recipients. In conclusion, the potentially druggable FCRL6 receptor on cytotoxic T cells appears to have a role in controlling CMV viremia after HSCT. Furthermore, our results suggest that system-level analysis is a useful addition to the studying of single biomarkers in allogenic HSCT.

Working group summary of the 2023 full update of the Finnish national guidelines for paediatric lower respiratory tract infections.

AIM: The first evidence-based Finnish guidelines for paediatric lower respiratory tract infections (LRTIs) were published in 2014 and completely updated in 2023. This paper, by the interdisciplinary working group that developed the 2023 guidelines, summarises the main recommendations. METHODS: The 2023 guidelines were produced after a systematic review. Strong evidence was at least two separate, high-quality studies, moderate evidence was at least one high-quality study and weak evidence was at least one satisfactory study. The authors have now summarised the key points. RESULTS: There was strong evidence that antitussives and beta-sympathomimetics were not effective for bronchitis-related cough and that laryngitis should be treated with oral corticosteroids, with adrenaline inhalations added in severe cases. Also, that amoxicillin for 5 days provided sufficient treatment for paediatric community-acquired pneumonia and that children with apparent viral pneumonia could be observed without antimicrobial therapy. There was moderate evidence that corticosteroids or inhaled agents were not effective for bronchiolitis and that administering salbutamol with a holding chamber could relieve symptoms of wheezing bronchitis. Also, pertussis should be considered for unvaccinated infants with coughs. CONCLUSION: The 2023 guidelines aim to improve acute evidence-based treatment of LRTIs, through appropriate antibiotics, inhaled drugs, corticosteroids, radiology and laboratory testing.

Pneumonia, wheezing and asthma were more common in children after thymectomy due to open-heart surgery.

AIM: This nationwide study evaluated the clinical impact that an early thymectomy, during congenital heart defect (CHD) surgery, had on the health of children and adolescents. METHODS: The subjects were patients aged 1-15 years who had undergone CHD surgery at the University Children's Hospital, Helsinki, where all CHD surgery in Finland is carried out, from 2006 to 2018. The parents or the cases and population-based controls, matched for sex, age and hospital district, completed electronic questionnaires. We excluded those with low birth weights or a known immunodeficiency. Adjusted odds ratios (aOR) and 95% confidence intervals (CI) were calculated for prespecified outcomes. RESULTS: We received responses relating to 260/450 (58%) cases and 1403/4500 (31%) controls and excluded 73 cases with persistent cardiac or respiratory complaints after surgery. The CHD group reported more recurrent hospitalisations due to infections (aOR 6.3, 95% CI 3.0-13) than the controls and more pneumonia episodes (aOR 3.5, 95% CI 2.1-5.6), asthma (aOR 2.5, 95% CI 1.5-4.1) and wheezing (aOR 2.1, 95% CI 1.5-2.9). CONCLUSION: Hospitalisation due to infections, pneumonia, wheezing and asthma was more common in children after a thymectomy due to open-heart surgery than population-based controls, underlining the importance of immunological follow-ups.

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood.

Human adaptive-like "memory" CD56dimCD16+ natural killer (NK) cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue residency and tumor homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56brightCD16- NK cells with hallmarks of tissue residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident NK (trNK) cells were found to be present independently of adaptive-like CD56dimCD16+ NK cells and to be hyperresponsive toward target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.

FASCIA Method in the Assessment of Lymphocyte Mitogen Responses in the Laboratory Diagnostics of Primary Immunodeficiencies.

Lymphocyte responses to mitogens constitute a key part of the diagnostics of combined immunodeficiency (CID). Currently, mostly radioactive thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution methods are used. Flow-cytometric assay for specific cell-mediated immune-response in activated whole blood (FASCIA) has been put forth as an easy-to-perform option for the measurement of lymphocyte responses with the advantage of recognizing different lymphocyte subtypes and avoiding the use of radioactive reagents. Our aim was to analyze retrospectively the usefulness of FASCIA in the diagnostics of CID. We included all lymphocyte stimulation tests done with FASCIA in HUSLAB (Helsinki, Finland) between February 2015 and September 2018 in our analysis. The cohort was divided into two groups according to the patients' final diagnoses: CID (n = 30) or non-CID (n = 159). We evaluated the stimulation responses with a combined FASCIA score (the average of all mitogen responses). The FASCIA score was significantly lower among the CID group compared to the other patients (p = 0.002), and in the ROC analysis, the AUC was 0.75 (p < 0.001) for the FASCIA score. When the three mitogens were analyzed separately, phytohemagglutinin (PHA) was best in separating patients with CID from non-CID (in the ROC analysis AUC 0.71, p = 0.001). Immunosuppressive medication affected the FASCIA result significantly and needs to be considered when evaluating the results. In conclusion, FASCIA can reliably detect the CID patients in the absence of immunosuppressive medication. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

Peripheral differentiation patterns of human T cells.

Long-term T-cell memory is dependent on the maintenance of memory T cells in the lymphoid tissues, and at the surface interfaces that provide entry routes for pathogens. However, much of the current information on human T-cell memory is based on analyzing circulating T cells. Here, we have studied the distribution and age-related changes of memory T-cell subsets in samples from blood, mesenteric LNs, spleen, and ileum, obtained from donors ranging in age from 5 days to 67 years of age. Our data show that the main reservoir of polyclonal naive cells is found in the LNs, and the resting memory subsets capable of self-renewal are also prominent there. In contrast, nondividing but functionally active memory subsets dominate the spleen, and especially the ileum. In general, the replacement of naive cells with memory subsets continues throughout our period of observation, with no apparent plateau. In conclusion, the analysis of lymphoid and nonlymphoid tissues reveals a dynamic pattern of changes distinct to each tissue, and with substantial differences between CD4+ and CD8+ compartments.

Characterization of low-density granulocytes in COVID-19.

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.

A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations.

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.

Ectopic germinal centers in the thymus accurately predict prognosis of myasthenia gravis after thymectomy.

The ability of thymic histopathology to predict the long-term impact of thymectomy in non-thymomatous myasthenia gravis (NTMG) is mainly uncharted. We applied digital pathology to quantitatively characterize differences of thymic histology between early-onset (EOMG) and late-onset MG (LOMG) and to investigate the role of thymic changes for thymectomy outcomes in MG. We analyzed 83 thymic H&E slides from thymectomized NTMG patients, of which 69 had EOMG and 14 LOMG, using digital pathology open-access software QuPath. We compared the results to the retrospectively assessed clinical outcome at two years after thymectomy and at the last follow-up visit where complete stable remission and minimal use of medication were primary outcomes. The automated annotation pipeline was an effective and reliable way to analyze thymic H&E samples compared to manual annotation with mean intraclass correlation of 0.80. The ratio of thymic tissue to stroma and fat was increased in EOMG compared to LOMG (p = 8.7e-07), whereas no difference was observed in the ratio of medulla to cortex between these subtypes. AChRAb seropositivity correlated with the number of ectopic germinal centers (eGC; p = 0.00067) but not with other histological areas. Patients with an increased number of eGCs had better post-thymectomy outcomes at two years after thymectomy (p = 0.0035) and at the last follow-up (p = 0.0267). ROC analysis showed that eGC area predicts thymectomy outcome in EOMG with an AUC of 0.79. Digital pathology can thus help in providing a predictive tool to the clinician, the eGC number, to guide the post-thymectomy treatment decisions in EOMG patients.

Patients with autoimmune polyendocrine syndrome type 1 have an increased susceptibility to severe herpesvirus infections.

Almost all patients with autoimmune polyendocrine syndrome type 1 (APS-1) have neutralizing antibodies against type 1 interferons (IFN), important mediators of antiviral defense. Recently, neutralizing anti-IFN antibodies were shown to be a risk factor of severe COVID-19. Here we show in a cohort of 44 patients with APS-1 that higher titers of neutralizing anti-IFNα4 antibodies are associated with a higher and earlier incidence of VZV reactivation (herpes zoster). The patients also present with uncommonly severe clinical sequelae of herpetic infections. APS-1 patients had decreased humoral immune responses to varicella zoster virus, but cellular responses were comparable to healthy controls. These results suggest that blocking the type I interferon pathway in patients with APS-1 patients leads to a clinically significant immune deficiency, and susceptibility to herpesviruses should be taken into account when treating patients with APS-1.

Evaluation of commercial and automated SARS-CoV-2 IgG and IgA ELISAs using coronavirus disease (COVID-19) patient samples.

Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. We evaluated SARS-CoV-2 IgG and IgA ELISAs in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset.

Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.

Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69-CD56dim cells.

BACKGROUND: In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer. OBJECTIVE: We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single-cell level. METHODS: NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16-color flow cytometry and confocal microscopy. RESULTS: CD56dimCD16+ NK cells made up the vast majority of NK cells in human lungs, had a more differentiated phenotype, and more frequently expressed educating killer cell immunoglobulin-like receptors compared with NK cells in peripheral blood. Despite this, human lung NK cells were hyporesponsive toward target cell stimulation, even after priming with IFN-α. Furthermore, we detected a small subset of NK cells expressing CD69, a marker of tissue residency. These CD69+ NK cells in the lung consisted predominantly of immature CD56brightCD16- NK cells and less differentiated CD56dimCD16+ NK cells. CONCLUSION: Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue-resident CD69+ NK cells.