Molecular pixelation: spatial proteomics of single cells by sequencing.

The spatial distribution of cell surface proteins governs vital processes of the immune system such as intercellular communication and mobility. However, fluorescence microscopy has limited scalability in the multiplexing and throughput needed to drive spatial proteomics discoveries at subcellular level. We present Molecular Pixelation (MPX), an optics-free, DNA sequence-based method for spatial proteomics of single cells using antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. The relative locations of AOCs are inferred by sequentially associating them into local neighborhoods using the sequence-unique DNA pixels, forming >1,000 spatially connected zones per cell in 3D. For each single cell, DNA-sequencing reads are computationally arranged into spatial proteomics networks for 76 proteins. By studying immune cell dynamics using spatial statistics on graph representations of the data, we identify known and new patterns of spatial organization of proteins on chemokine-stimulated T cells, highlighting the potential of MPX in defining cell states by the spatial arrangement of proteins.

Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C).

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease.

No link between type I interferon autoantibody positivity and adverse reactions to COVID-19 vaccines.

Type I interferons act as gatekeepers against viral infection, and autoantibodies that neutralize these signaling molecules have been associated with COVID-19 severity and adverse reactions to the live-attenuated yellow fever vaccine. On this background, we sought to examine whether autoantibodies against type I interferons were associated with adverse events following COVID-19 vaccination. Our nationwide analysis suggests that type I interferon autoantibodies were not associated with adverse events after mRNA or viral-vector COVID-19 vaccines.

Persistent complement dysregulation with signs of thromboinflammation in active Long Covid.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

Immune system perturbations in patients with long COVID.

Klein et al. report multimodal analyses of immune cells, proteins, and physiological parameters in patients with long COVID (LC). At the group level, LC subjects exhibited elevated antibody responses to SARS-CoV-2, but also to herpes viruses, pointing to a general suppression of viral control mechanisms in LC.

Antisecretory factor in breastmilk is associated with reduced incidence of sepsis in preterm infants.

BACKGROUND: Antisecretory Factor (AF) is a protein present in breastmilk that regulates inflammatory processes. We aimed to investigate the level of AF in mothers' own milk (MOM) in relation to sepsis and other neonatal morbidities in preterm infants. METHODS: Samples of breastmilk and infant plasma were collected at 1, 4, and 12 weeks after birth from 38 mothers and their 49 infants born before 30 weeks gestation. AF-compleasome in MOM was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) and inflammatory markers in infant plasma by a panel of 92 inflammatory proteins. Neonatal treatments and outcomes were recorded. RESULTS: The level of AF in MOM week 1 was lower for infants with later sepsis compared to no sepsis (p = 0.005). Corrected for nutritional intake of MOM, higher levels of AF decreased the risk for sepsis, OR 0.24. AF in MOM week 1 was negatively correlated to inflammatory proteins in infant plasma week 4, markedly IL-8, which was also associated with infant sepsis. Overall, higher AF levels in MOM was associated with fewer major morbidities of prematurity. CONCLUSION: Mother's milk containing high levels of antisecretory factor is associated with reduced risk for sepsis and inflammation in preterm infants. IMPACT: High level of antisecretory factor (AF) in mothers' own milk is associated with less risk for later sepsis in preterm infants. Receiving mothers' milk with low AF levels during the first week after birth is correlated with more inflammatory proteins in infant's plasma 2-4 weeks later. Human breastmilk has anti-inflammatory properties, and antisecretory factor in mothers' own milk is a component of potential importance for infants born preterm. The findings suggest that food supplementation with AF to mothers of preterm infants to increase AF-levels in breastmilk may be a means to decrease the risk of inflammatory morbidities of prematurity.

The maternal gut microbiome in pregnancy: implications for the developing immune system.

The gut microbiome has important roles in host metabolism and immunity, and microbial dysbiosis affects human physiology and health. Maternal immunity and microbial metabolites during pregnancy, microbial transfer during birth, and transfer of immune factors, microorganisms and metabolites via breastfeeding provide critical sources of early-life microbial and immune training, with important consequences for human health. Only a few studies have directly examined the interactions between the gut microbiome and the immune system during pregnancy, and the subsequent effect on offspring development. In this Review, we aim to describe how the maternal microbiome shapes overall pregnancy-associated maternal, fetal and early neonatal immune systems, focusing on the existing evidence and highlighting current gaps to promote further research.

Immune responses to SARS-CoV-2 infection and vaccination in children.

During the three years since SARS-CoV-2 infections were first described a wealth of information has been gathered about viral variants and their changing properties, the disease presentations they elicit and how the many vaccines developed in record time protect from COVID-19 severe disease in different populations. A general theme throughout the pandemic has been the observation that children and young people in general fare well, with mild symptoms during acute infection and full recovery thereafter. It has also become clear that this is not universally true, as some children develop severe COVID-19 hypoxic pneumonia and even succumb to the infection, while another group of children develop a rare but serious multisystem inflammatory syndrome (MIS-C) and some other children experience prolonged illness following acute infection, post-COVID. Here I will discuss some of the findings made to explain these diverse disease manifestations in children and young people infected by SARS-CoV-2. I will also discuss the vaccines developed at record speed and their efficacy in protecting children from disease.

Making human immune systems more interpretable through systems immunology.

The human immune system is a distributed system of specialized cell populations with unique functions that collectively give rise to immune responses to infections and during immune-mediated diseases. Cell composition, plasma proteins, and functional responses vary among individuals, making the system difficult to interpret, but this variation is nonrandom. With careful analyses using novel experimental and computational tools, human immune system composition and function carry interpretable information. Here, we propose that systems-level analyses offer an opportunity to make human immune responses more interpretable in the future, and we discuss herein important considerations and lessons learned to this end. Predictable human immunology holds implications for better diagnostic and curative precision in patients with infectious and immune-associated diseases.

Achieving symptom relief in patients with myalgic encephalomyelitis by targeting the neuro-immune interface and optimizing disease tolerance.

Myalgic encephalomyelitis (ME) previously also known as chronic fatigue syndrome is a heterogeneous, debilitating syndrome of unknown etiology responsible for long-lasting disability in millions of patients worldwide. The most well-known symptom of ME is post-exertional malaise, but many patients also experience autonomic dysregulation, cranial nerve dysfunction and signs of immune system activation. Many patients also report a sudden onset of disease following an infection. The brainstem is a suspected focal point in ME pathogenesis and patients with structural impairment to the brainstem often show ME-like symptoms. The brainstem is also where the vagus nerve originates, a critical neuro-immune interface and mediator of the inflammatory reflex which regulate systemic inflammation. Here, we report the results of a randomized, placebo-controlled trial using intranasal mechanical stimulation targeting nerve endings in the nasal cavity, likely from the trigeminal nerve, possibly activating additional centers in the brainstem of ME patients and correlating with a ∼30% reduction in overall symptom scores after 8 weeks of treatment. By performing longitudinal, systems-level monitoring of the blood immune system in these patients, we uncover signs of chronic immune activation in ME, as well as immunological correlates of improvement that center around gut-homing immune cells and reduced inflammation. The mechanisms of symptom relief remain to be determined, but transcriptional analyses suggest an upregulation of disease tolerance mechanisms. We believe that these results are suggestive of ME as a condition explained by a maladaptive disease tolerance response following infection.

Age-dependent differences in efferocytosis determine the outcome of opsonophagocytic protection from invasive pathogens.

In early life, susceptibility to invasive infection skews toward a small subset of microbes, whereas other pathogens associated with diseases later in life, including Streptococcus pneumoniae (Spn), are uncommon among neonates. To delineate mechanisms behind age-dependent susceptibility, we compared age-specific mouse models of invasive Spn infection. We show enhanced CD11b-dependent opsonophagocytosis by neonatal neutrophils improved protection against Spn during early life. The augmented function of neonatal neutrophils was mediated by higher CD11b surface expression at the population level due to dampened efferocytosis, which also resulted in more CD11bhi "aged" neutrophils in peripheral blood. Dampened efferocytosis during early life could be attributed to the lack of CD169+ macrophages in neonates and reduced systemic expressions of multiple efferocytic mediators, including MerTK. On experimentally impairing efferocytosis later in life, CD11bhi neutrophils increased and protection against Spn improved. Our findings reveal how age-dependent differences in efferocytosis determine infection outcome through the modulation of CD11b-driven opsonophagocytosis and immunity.

Exaggerated responses to a virus long gone.

Multisystem inflammatory syndrome in children is caused by abnormal cell activation.

Three Adult Cases of STAT1 Gain-of-Function with Chronic Mucocutaneous Candidiasis Treated with JAK Inhibitors.

PURPOSE: The aim of this study was to characterize clinical effects and biomarkers in three patients with chronic mucocutaneous candidiasis (CMC) caused by gain-of-function (GOF) mutations in the STAT1 gene during treatment with Janus kinase (JAK) inhibitors. METHODS: Mass cytometry (CyTOF) was used to characterize mononuclear leukocyte populations and Olink assay to quantify 265 plasma proteins. Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) was used to quantify the reactivity against Candida albicans. RESULTS: Overall, JAK inhibitors improved clinical symptoms of CMC, but caused side effects in two patients. Absolute numbers of neutrophils, T cells, B cells, and NK cells were sustained during baricitinib treatment. Detailed analysis of cellular subsets, using CyTOF, revealed increased expression of CD45, CD52, and CD99 in NK cells, reflecting a more functional phenotype. Conversely, monocytes and eosinophils downregulated CD16, consistent with reduced inflammation. Moreover, T and B cells showed increased expression of activation markers during treatment. In one patient with a remarkable clinical effect of baricitinib treatment, the immune response to C. albicans increased after 7 weeks of treatment. Alterations in plasma biomarkers involved downregulation of cellular markers CXCL10, annexin A1, granzyme B, granzyme H, and oncostatin M, whereas FGF21 was the only upregulated marker after 7 weeks. After 3 months, IFN-É£ and CXCL10 were downregulated. CONCLUSIONS: The clinical effect of JAK inhibitor treatment of CMC is promising. Several biological variables were altered during baricitinib treatment demonstrating that lymphocytes, NK cells, monocytes, and eosinophils were affected. In parallel, cellular reactivity against C. albicans was enhanced.

Making inroads to precision medicine for the treatment of autoimmune diseases: Harnessing genomic studies to better diagnose and treat complex disorders.

Precision Medicine is an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle. Autoimmune diseases are those in which the body's natural defense system loses discriminating power between its own cells and foreign cells, causing the body to mistakenly attack healthy tissues. These conditions are very heterogeneous in their presentation and therefore difficult to diagnose and treat. Achieving precision medicine in autoimmune diseases has been challenging due to the complex etiologies of these conditions, involving an interplay between genetic, epigenetic, and environmental factors. However, recent technological and computational advances in molecular profiling have helped identify patient subtypes and molecular pathways which can be used to improve diagnostics and therapeutics. This review discusses the current understanding of the disease mechanisms, heterogeneity, and pathogenic autoantigens in autoimmune diseases gained from genomic and transcriptomic studies and highlights how these findings can be applied to better understand disease heterogeneity in the context of disease diagnostics and therapeutics.

Enhanced TLR3 responsiveness in hepatitis C virus resistant women from the Irish anti-D cohort.

Natural resistance to infection is an overlooked outcome after hepatitis C virus (HCV) exposure. Between 1977 and 1979, 1,200 Rhesus D-negative Irish women were exposed to HCV-contaminated anti-D immunoglobulin. Here, we investigate why some individuals appear to resist infection despite exposure (exposed seronegative [ESN]). We screen HCV-resistant and -susceptible donors for anti-HCV adaptive immune responses using ELISpots and VirScan to profile antibodies against all know human viruses. We perform standardized ex vivo whole blood stimulation (TruCulture) assays with antiviral ligands and assess antiviral responses using NanoString transcriptomics and Luminex proteomics. We describe an enhanced TLR3-type I interferon response in ESNs compared with seropositive women. We also identify increased inflammatory cytokine production in response to polyIC in ESNs compared with seropositive women. These enhanced responses may have contributed to innate immune protection against HCV infection in our cohort.

Selective transfer of maternal antibodies in preterm and fullterm children.

Preterm newborns are more likely to suffer from infectious diseases at birth compared to children delivered at term. Whether this is due to compromised cellular, humoral, or organ-specific development remains unclear. To begin to define whether maternal-fetal antibody transfer profiles differ across preterm (PT) and fullterm (FT) infants, the overall quantity and functional quality of an array of 24 vaccine-, endemic pathogen-, and common antigen-specific antibodies were assessed across a cohort of 11 PT and 12 term-delivered maternal:infant pairs from birth through week 12. While total IgG levels to influenza, pneumo, measles, rubella, EBV, and RSV were higher in FT newborns, selective Fc-receptor binding antibodies was noted in PT newborns. In fact, near equivalent antibody-effector functions were observed across PT and FT infants, despite significant quantitative differences in transferred antibody levels. Moreover, temporal transfer analysis revealed the selective early transfer of FcRn, FcγR2, and FcγR3 binding antibodies, pointing to differential placental sieving mechanisms across gestation. These data point to selectivity in placental transfer at distinct gestational ages, to ensure that children are endowed with the most robust humoral immunity even if born preterm.

Transplantation of maternal intestinal flora to the newborn after elective cesarean section (SECFLOR): study protocol for a double blinded randomized controlled trial.

BACKGROUND: A complication of elective cesarean section (CS) delivery is its interference with the normal intestinal colonization of the infant, affecting the immune and metabolic signaling in early life- a process that has been associated with long-term morbidity, such as allergy and diabetes. We evaluate, in CS-delivered infants, whether the normal intestinal microbiome and its early life development can be restored by immediate postnatal transfer of maternal fecal microbiota (FMT) to the newborn, and how this procedure influences the maturation of the immune system. METHODS: Sixty healthy mothers with planned elective CS are recruited and screened thoroughly for infections. A maternal fecal sample is taken prior to delivery and processed according to a transplantation protocol. After double blinded randomization, half of the newborns will receive a diluted aliquot of their own mother's stool orally administered in breast milk during the first feeding while the other half will be similarly treated with a placebo. The infants are clinically followed, and fecal samples are gathered weekly until the age of 4 weeks, then at the ages of 8 weeks, 3, 6, 12 and 24 months. The parents fill in questionnaires until the age of 24 months. Blood samples are taken at the age of 2-3 days and 3, 6, 12 and 24 months to assess development of major immune cell populations and plasma proteins throughout the first years of life. DISCUSSION: This is the first study to assess long-time effects on the intestinal microbiome and the development of immune system of a maternal fecal transplant given to term infants born by CS. TRIAL REGISTRATION: ClinicalTrials.gov NCT04173208 , registration date 21.11.2019.

Prostate cancer disease recurrence after radical prostatectomy is associated with HLA type and local cytomegalovirus immunity.

Prostate cancer is a heterogeneous disease with a need for new prognostic biomarkers. Human leukocyte antigen (HLA) genes are highly polymorphic genes central to antigen presentation to T-cells. Two alleles, HLA-A*02:01 and HLA-A*24:02, have been associated with prognosis in patients diagnosed with de novo metastatic prostate cancer. We leveraged the next-generation sequenced cohorts CPC-GENE and TCGA-PRAD to examine HLA alleles, antiviral T-cell receptors and prostate cancer disease recurrence after prostatectomy. Carrying HLA-A*02:01 (111/229; 48% of patients) was independently associated with disease recurrence in patients with low-intermediate risk prostate cancer. HLA-A*11 (carried by 42/441; 10% of patients) was independently associated with rapid disease recurrence in patients with high-risk prostate cancer. Moreover, HLA-A*02:01 carriers in which anti-cytomegalovirus T-cell receptors (CMV-TCR) were identified in tumors (13/144; 10% of all patients in the cohort) had a higher risk of disease recurrence than CMV-TCR-negative patients. These findings suggest that HLA-type and CMV immunity may be valuable biomarkers for prostate cancer progression.