Nature Immunology

The immune response to SARS-CoV2 is under intense investigation, but not fully understood att this moment. Severe disease is characterized by vigorous inflammatory responses in the lung, often with a sudden onset after 5-7 days of stable disease. Efforts to modulate this hyperinflammation and the associated acute respiratory distress syndrome, rely on the unraveling of the immune cell interactions and cytokines that drive such responses. Systems-level analyses are required to simultaneously capture all immune cell populations and the many protein mediators by which cells communicate. Since every patient analyzed will be captured at different stages of his or her infection, longitudinal monitoring of the immune response is critical. Here we report on a systems-level blood immunomonitoring study of 39 adult patients, hospitalized with severe COVID-19 and followed with up to 14 blood samples from acute to recovery phases of the disease. We describe an IFN{gamma} - Eosinophil axis activated prior to lung hyperinflammation and changes in cell-cell coregulation during different stages of the disease. We also map an immune trajectory during recovery that is shared among patients with severe COVID-19. HIGHLIGHTSSystems-level immunomonitoring from acute to recovery in severe COVID-19 An IFN{gamma} - Eosinophil axis involved in lung hyperinflammation Cell-cell coregulation differ during four disease stages Basophils and hyperinflammation modulate humoral responses A shared trajectory of immunological recovery in severe COVID-19

Coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health emergency. Although SARS-CoV-2 is primarily a respiratory pathogen, extra-respiratory organs, including the central nervous system (CNS), can also be affected. Neurologic symptoms have been observed not only during acute SARS-CoV-2 infection, but also at distance from respiratory disease, also known as long-COVID or neurological post-acute sequelae of COVID-19 (neuroPASC). The pathogenesis of neuroPASC is not well understood, but hypotheses include SARS-CoV-2-induced immune dysfunctions, hormonal dysregulations, and persistence of SARS-CoV-2 reservoirs. In this study, we used a high throughput systems serology approach to dissect the humoral response to SARS-CoV-2 (and other common Coronaviruses - 229E, HKU1, NL63, OC43) in the serum and cerebrospinal fluid (CSF) from 112 infected individuals who developed or did not develop neuroPASC. Unique SARS-CoV-2 humoral profiles were observed in the CSF of neuroPASC. All antibody isotypes (IgA, IgM, IgA) and subclasses (IgA1-2; IgG1-4) were detected in serum, whereas CSF was characterized by focused IgG1 (and absence of IgM). These data argue in favor of compartmentalized brain-specific responses against SARS-CoV-2 through selective transfer of antibodies from the serum to the CSF across the blood-brain-barrier, rather than intrathecal synthesis, where more diversity in antibody classes/subclasses would be expected. Moreover, compared to individuals who did not develop post-acute neurological complications following infection (n=94), those with neuroPASC (n=18) exhibited attenuated systemic antibody responses against SARS-CoV-2, characterized by decreased capacity to activate antibody-dependent complement deposition (ADCD), NK cell activation (ADNKA) and to bind Fc{gamma} receptors. However, surprisingly, neuroPASC showed significantly expanded antibody responses to other common Coronaviruses, including 229E, HKU1, NL63, and OC43. This biased humoral activation across coronaviruses was particularly enriched in neuroPASC individuals with poor outcome, suggesting an original antigenic sin (or immunologic imprinting), where pre-existing immune responses against related viruses shape the response to current infection, as a key prognostic marker of neuroPASC disease. Overall, these findings point to a pathogenic role for compromised anti-SARS-CoV-2 responses in the CSF, likely resulting in incomplete virus clearance from the brain and persistent neuroinflammation, in the development of post-acute neurologic complications of SARS-CoV-2 infection.

Autoimmunity is a key clinical feature in both post-infectious Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) and Post-Acute Sequelae of COVID (PASC). Passive transfer of immunoglobulins from patients sera into mice induces some clinical features of PASC. IgG-induced transfer of disease phenotypes has long been appreciated, yet the exact mechanism of disease development remains largely elusive. Here, we demonstrate that IgG isolated from post-infectious ME/CFS patients selectively induces mitochondrial fragmentation in human endothelial cells, thereby altering mitochondrial energetics. This effect is lost upon cleavage of IgG into its Fab and Fc fragments. The digested Fab fragment from ME/CFS alone was able to alter the mitochondrial energetics, resembling the effect of intact IgG. In contrast, the Fc fragment alone induced a hypometabolic phenotype characterized by a trend towards reduced overall ATP content. IgG from ME/CFS and PASC patients induced distinct but separate cytokine secretion profiles in healthy PBMCs. Proteomics analysis of IgG-bound immune complexes revealed significant changes within the immune complexes of ME/CFS patients, affecting extracellular matrix organization, while the same from PASC patients pointed towards alterations in hemostasis and blood clot regulation. We demonstrate that IgGs from ME/CFS patients carry a chronic protective stress response that promotes mitochondrial adaptation via fragmentation, without altering mitochondrial ATP generation capacity in endothelial cells. Together, these results highlight a potential pathogenic role of IgG in post-infectious ME/CFS and point to novel therapeutic strategies targeting antibody-mediated metabolic dysregulation. One Sentence SummaryIgG immune complexes from ME/CFS and PASC patients differ from those of healthy individuals and affect mitochondrial structure and function.

BackgroundCytokine storm syndromes (CSS), including hemophagocytic lymphohistiocytosis (HLH), are increasingly recognized as hyper-inflammatory states leading to multi-organ failure and death. Familial HLH (FHL) in infancy results from homozygous genetic defects in perforin-mediated cytolysis by CD8 T-lymphocytes and natural killer (NK) cells. Later onset CSS are frequently associated with heterozygous defects in FHL genes, but genetic etiologies for most are unknown. We identified rare DOCK8 variants in CSS patients. ObjectiveWe explore the role of CSS patient derived DOCK8 mutations on cytolytic activity in NK cells. We further study effects of Dock8-/- in murine models of CSS. MethodsDOCK8 cDNA from 2 unrelated CSS patients with different missense mutations were introduced into human NK-92 NK cells by foamy virus transduction. NK cell degranulation (CD107a), cytolytic activity against K562 target cells, and interferon-gamma (IFN{gamma}) production were explored by flow cytometry (FCM). A third CSS patient DOCK8 mRNA splice acceptor site variant was explored by exon trapping. Dock8-/- mice were assessed for features of CSS (weight loss, splenomegaly, hepatic inflammation, cytopenias, and IFN{gamma} levels) upon challenge with lymphochoriomeningitic virus (LCMV) and excess IL-18. ResultsBoth patient DOCK8 missense mutations decreased cytolytic function in NK cells in a partial dominant-negative fashion in vitro. The patient DOCK8 splice variant disrupted mRNA splicing in vitro. Dock8-/- mice tolerated excess IL-18 but developed features of CSS upon LCMV infection. ConclusionMutations in DOCK8 may contribute to CSS-like hyper-inflammatory states by altering cytolytic function in a threshold model of disease. Key MessagesO_LIHeterozygous missense mutations in DOCK8 may contribute to decreased NK cell function via partial dominant-negative effects on perforin-mediated cytolysis. C_LIO_LIHeterozygous mutations in DOCK8 may contribute to hyper-inflammatory syndromes in a threshold model of disease. C_LIO_LILCMV infection of Dock8-/- mice recapitulates features of murine FHL. C_LI Capsule SummaryHeterozygous missense and splice site mutations in DOCK8 may contribute to hyper-inflammation in patients with CSS. DOCK8 is important for optimal NK cell cytolytic function, and LCMV infection of Dock8-/- mice resembles murine FHL.

Clinical manifestations of COVID-19 caused by the novel coronavirus SARS-CoV-2 are associated with age. While children are largely spared from severe respiratory disease, they can present with a SARS-CoV-2-associated multisystem inflammatory syndrome (MIS-C) similar to Kawasakis disease. Here, we show distinct antibody (Ab) responses in children with MIS-C compared to adults with severe COVID-19 causing acute respiratory distress syndrome (ARDS), and those who recovered from mild disease. There was a reduced breadth and specificity of anti-SARS-CoV-2-specific antibodies in MIS-C patients compared to the COVID patient groups; MIS-C predominantly generated IgG Abs specific for the Spike (S) protein but not for the nucleocapsid (N) protein, while the COVID-19 cohorts had anti-S IgG, IgM and IgA Abs, as well as anti-N IgG Abs. Moreover, MIS-C patients had reduced neutralizing activity compared to both COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children and adults who develop severe disease, with implications for optimizing treatments based on symptom and age.

Abstract/IntroductionA wide clinical spectrum has become a hallmark of the SARS-CoV-2 (COVID-19) pandemic, although its immunologic underpinnings remain to be defined. We have performed deep characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation as previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody secreting cell expansion and early production of high levels of SARS-CoV-2-specific antibodies. Yet, these patients fared poorly with elevated inflammatory biomarkers, multi-organ failure, and death. Combined, the findings strongly indicate a major pathogenic role for immune activation in subsets of COVID-19 patients. Our study suggests that, as in autoimmunity, targeted immunomodulatory therapy may be beneficial in specific patient subpopulations that can be identified by careful immune profiling.

BackgroundThe role of cellular immunity in pathogenesis of COVID-19 is unclear and conflicting data points to insufficient or pathogenic immunity as drivers of COVID-19 progression. Here we aimed to delineate the phenotype and function of the immune system in patients with moderate, severe, and critical COVID-19. MethodsIn this prospective study, we included 53 patients with moderate (n=21), severe (n=18), and critical (n=14) COVID-19 manifestations. Using multiparametric flow cytometry we compared quantitative, phenotypic, and functional characteristics of circulating immune cells, SARS-CoV-2 antigen-reactive T-cells, and humoral immunity. ResultsDeep phenotypic profiling revealed a depletion of circulating bulk CD8+ T-cells, CD4+ and CD8+ T-cell subsets with activated memory/effector T-cells expressing CD57+, HLA-DR+, and the key activation and migration molecule CD11a++ in critical COVID-19. Importantly, survival from acute respiratory distress syndrome was accompanied by a recovery of the depleted CD11++ T-cell subsets including T-cells expressing CD28, CD57, HLA-DR activation/effector molecules. We further observed a stronger response of S-protein specific T-cells producing inflammatory cytokines in critical COVID-19 cases. This seemingly contradictory observation is in fact confirmation of the underlying immunopathogenesis in patients with critical COVID-19. ConclusionOur findings suggest a CD11a-based immune signature as a possible prognostic marker for disease development. Our data further reveal that increased rather than decreased SARS-CoV-2 specific T cell immunity is associated with adverse outcome in COVID-19. Tissue migration of activated effectors T-cells may constitute a crucial cornerstone in the immunopathogenesis of SARS-CoV-2 associated tissue injury. Trial registrationThis is a prospective observational study without a trial registration number. FundingThis work was supported by grants from Mercator Foundation, the BMBF e:KID (01ZX1612A), and BMBF NoChro (FKZ 13GW0338B). 25 Word summaryStronger S-protein reactivity and decreased frequency of activated memory/effector T-cells expressing CD11a++ suggests immunopathogenesis in critical COVID-19 mediated by tissue migration of activated effector T-cells.

SARS-CoV-2 infection causes a spectrum of clinical outcomes and diverse memory responses. Population studies indicate that viral neutralizing antibody responses are protective, but do not always develop post-infection. Other antiviral antibody effector functions, T-cell responses, or immunity to seasonal coronaviruses (OC43, 229E) have been implicated but not defined in all ages. Here, we identify that children and adult subjects generate polyfunctional antibodies to the spike protein after asymptomatic infection or mild disease, with some subjects developing cellular responses without seroconversion. Diversity in immunity was explained by two clusters distinguished by CD4+ T-cell cytokines, age, and antibodies to seasonal coronaviruses. Post-vaccination neutralizing responses were predicted by specific post-infection immune measures, including IL-2, spike-IgA, OC43-IgG1, 229E-IgM. We confirm a key role for CD4+ T cell cytokines in functionality of anti-spike antibodies, and show that antibody diversity is impacted by age, Th/Th2 cytokine biases, and antibody isotypes to SARS-CoV-2 and seasonal coronaviruses.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating but poorly-understood disease. ME/CFS symptoms can range from mild to severe, and include immune system effects alongside incapacitating fatigue and post-exertional disease exacerbation. In this study, we examined immunological profiles of people living with ME/CFS by flow cytometry, focusing on cytotoxic cells, to determine whether people with mild/moderate (n= 43) or severe ME/CFS (n=53) expressed different immunological markers. We found that people with mild/moderate ME/CFS had increased expression of cytotoxic effector molecules alongside enhanced proportions of early-immunosenescence cells, determined by the CD28-CD57- phenotype, indicative of persistent viral infection. In contrast, people with severe ME/CFS had higher proportions of activated circulating lymphocytes, determined by CD69+ and CD38+ expression, and expressed more pro-inflammatory cytokines, including IFN{gamma}, TNF and IL-17, following stimulation in vitro, indicative of prolonged non-specific inflammation. These changes were consistent across different cell types including CD8+ T cells, mucosal associated invariant T cells and Natural Killer cells, indicating generalised altered cytotoxic responses across the innate and adaptive immune system. These immunological differences likely reflect different disease pathogenesis mechanisms occurring in the two clinical groups, opening up opportunities for the development of prognostic markers and stratified treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/24319359v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1e3f2e5org.highwire.dtl.DTLVardef@5fd9fborg.highwire.dtl.DTLVardef@e015eforg.highwire.dtl.DTLVardef@1c03ae1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

Coronavirus disease 2019 (COVID-19) displays high clinical variability but the parameters that determine disease severity are still unclear. Pre-existing T cell memory has been hypothesized as a protective mechanism but conclusive evidence is lacking. Here we demonstrate that all unexposed individuals harbor SARS-CoV-2-specific memory T cells with marginal cross-reactivity to common cold corona and other unrelated viruses. They display low functional avidity and broad protein target specificities and their frequencies correlate with the overall size of the CD4+ memory compartment reflecting the "immunological age" of an individual. COVID-19 patients have strongly increased SARS-CoV-2-specific inflammatory T cell responses that are correlated with severity. Strikingly however, patients with severe COVID-19 displayed lower TCR functional avidity and less clonal expansion. Our data suggest that a low avidity pre-existing T cell memory negatively impacts on the T cell response quality against neoantigens such as SARS-CoV-2, which may predispose to develop inappropriate immune reactions especially in the elderly. We propose the immunological age as an independent risk factor to develop severe COVID-19. Key points- Pre-existing SARS-CoV-2-reactive memory T cells are present in all humans, but have low functional avidity and broad target specificities - Pre-existing memory T cells show only marginal cross-reactivity to common cold corona viruses - Frequencies of pre-existing memory T cells increase with the size of the CD4+ memory compartment reflecting the "immunological age" of the individual - Low-avidity and polyclonal, but strongly enhanced SARS-CoV-2 specific T cell responses develop in severe COVID-19, suggesting their origin from pre-existing memory - The immunological age may represent a risk factor to develop severe COVID-19

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se, but rather with the delayed kinetics of NAb production.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic comorbidities remain ill-defined. Herein, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aero allergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed co-incident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine TNF-. These observations provide important insights into a potential mechanism underlying the development of allergic co-morbidity in early life in children with AD which involves altered NK-cell functional responses, and define an endotype of severe AD.

BackgroundRepeated mRNA vaccination against SARS-CoV-2 has been shown to induce class switching to IgG4, a non-inflammatory human antibody subclass linked to tolerance. Although poorly understood, prolonged antigenic stimulation and IL-4 signalling may be instrumental in IgG4 switching. We and others have previously shown that widely used immunosuppressive drugs such as methotrexate (MTX) and TNF inhibitors (TNFi) have a minor inhibitory impact on humoral SARS-CoV-2 mRNA vaccination responses. However, the impact of such immunosuppressive drugs on IgG4 switching is unknown. AimTo study the impact of widely used immunosuppressive drugs (TNFi, MTX, or the IL-4 receptor-blocking antibody dupilumab on IgG4 skewing upon repeated SARS-CoV-2 mRNA vaccination. MethodsAntibody responses to the receptor-binding domain (RBD) of the spike protein upon repeated SARS-CoV-2 mRNA vaccination were measured in 604 individuals including patients with immune-mediated inflammatory diseases treated with TNFi and/or MTX, or dupilumab, as well as healthy controls and untreated patients. ResultsWe observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after a third mRNA vaccination. This IgG4 skewing was absent when primary vaccination was adenoviral vector-based and was profoundly reduced in both dupilumab- and TNFi-treated patients (<1%), but only moderately in patients treated with MTX (7%). ConclusionOur results imply a major role for both IL-4/IL-13 as well as TNF in IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit future mRNA vaccine strategies, humoral tolerance induction, as well as treatment of IgG4 pathologies.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. Here, we investigated their role at nasal mucosa following challenge with house dust mite. We combined single cell proteome and transcriptome profiling on immune cells from nasal biopsy cells of allergic rhinitis and non-allergic subjects, before and after repeated nasal allergen challenge. Nasal biopsies of patients showed infiltrating inflammatory HLA-DRhi CD14+ monocytes and CD16+ monocytes, and transcriptional changes in resident CD1C+ CD1A+ conventional dendritic cells (cDC)2 following challenge. Importantly, although clinically silent, non-allergic individuals displayed a distinct innate MPS response to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (HLA-DRlow CD14+ monocytes), as well as cDC2 clusters expressing increased inhibitory/tolerogenic transcripts. Therefore, we identified not only clusters involved in airway inflammation but also a non-inflammatory, homeostatic blueprint of innate MPS responses to allergens in non-allergic individuals. Future therapies should target innate MPS for treatment of inflammatory airway diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/20189886v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@e6e970org.highwire.dtl.DTLVardef@1e7e8bdorg.highwire.dtl.DTLVardef@60d55forg.highwire.dtl.DTLVardef@1587d4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Little is known about the quality of polyclonal antibody responses in COVID-19 patients, and how it correlates with disease severity or patients prior exposure to other pathogens. The whole polyclonal antibody repertoire in a retrospective cohort of 538 individuals was mapped against SARS-CoV-2 spike (S) glycoprotein, the main target of antibody immune responses in SARS-CoV-2 infection. Bioinformatic predictions identified 15 major B cell epitopes for S of SARS-CoV-2. Several epitopes localised in RBD of S including those spanning the ACE2-binding site, the highly conserved cryptic epitope of the neutralizing antibody of SARS-CoV, and fusion/entry domains of HR1 and HR2 of S protein of SARS-CoV-2. Intriguingly, some of these epitopes have cross-reactivity to antigens of common pathogens, potentially affecting SARS-CoV-2 infection outcome. High level of anti-Spike SARS-CoV-2 seroreactivity in populations with no history of exposure to SARS-CoV-2 is of clinical relevance and could underpin better understanding of COVID-19 pathophysiology in different populations and provide a blueprint for design of effective vaccines and developing better strategies for antibody testing.

Novel mRNA vaccines have resulted in a reduced number of SARS-CoV-2 infections and hospitalizations. Yet, there is a paucity of studies regarding their effectiveness on immunocompromised autoimmune subjects. In this study, we enrolled subjects naive to SARS-CoV-2 infections from two cohorts of healthy donors (HD, n=56) and systemic lupus erythematosus (SLE, n=69). Serological assessments of their circulating antibodies revealed a significant reduction of potency and breadth of neutralization in the SLE group, only partially rescued by a 3rd booster dose. Immunological memory responses in the SLE cohort were characterized by a reduced magnitude of spike-reactive B and T cell responses that were strongly associated with poor seroconversion. Vaccinated SLE subjects were defined by a distinct expansion and persistence of a DN2 spike-reactive memory B cell pool and a contraction of spike-specific memory cTfh cells, contrasting with the sustained germinal center (GC)-driven activity mediated by mRNA vaccination in the healthy population. Among the SLE-associated factors that dampened the vaccine responses, treatment with the monoclonal antibody anti-BAFF/Belimumab (a lupus FDA-approved B cell targeting agent) profoundly affected the vaccine responsiveness by restricting the de novo B cell responses and promoting stronger extra-follicular (EF)-mediated responses that were associated with poor immunogenicity and impaired immunological memory. In summary, this study interrogates antigen-specific responses and characterized the immune cell landscape associated with mRNA vaccination in SLE. The identification of factors associated with reduced vaccine efficacy illustrates the impact of SLE B cell biology on mRNA vaccine responses and provides guidance for the management of boosters and recall vaccinations in SLE patients according to their disease endotype and modality of treatment.

Autoimmunity develops as a result of a breakdown in immune tolerance and activation of autoreactive immune cells. Most of the common autoimmune diseases are polygenic1 suggesting dysregulation in multiple signalling pathways. By contrast, in monogenic Inborn Errors of Immunity (IEI), which also can result in autoimmunity, the disease is triggered by a single genetic defect. Therefore, the discovery of causative mutations in IEI allows tracing the molecular mechanisms leading to autoimmunity in humans from a defect in the function of a specific gene to patients clinical and immunological phenotype. Here, we discovered an IEI patient with systemic autoimmunity caused by a private homozygous protein-truncating mutation in gene ZC3H12A leading to deficiency of Regnase-1, a regulatory RNase2-5. Flow cytometry, bulk T cell transcriptome analysis and single-cell RNA sequencing demonstrated expansion of {gamma}{delta} T cells expressing VCAM-1 and IFN{gamma} genes. We show that Regnase-1 directly targets 3UTR of VCAM1 and the coding sequence of IFNG mRNAs. These findings highlight a new autoimmunity mechanism in humans, where Regnase-1 deficiency causes expansion of VCAM1+IFNG+ T cells and their interaction with integrin 4{beta}1-expressing B cells, which showed upregulation of IFN-response genes and activation, leading to systemic autoimmunity. Furthermore, we show that VCAM1+ T cells are present in organs of donors and are expanded in the blood of patients with systemic lupus erythematosus, a common autoimmune disease characterised by systemic autoimmunity.

A dysregulated immune response against coronavirus-2 (SARS-CoV-2) plays a critical role in the outcome of patients with coronavirus disease 2019 (COVID-19). A significant increase in circulating plasmablasts is characteristic of COVID-19 though the underlying mechanisms and its prognostic implications are not known. Here, we demonstrate that in the acute phase of COVID-19, activated PD-1highCXCR5-CD4+ T cells, peripheral helper T cells, (Tph) are significantly increased and promote inflammatory tissue-homing plasmablasts in patients with stable but not severe COVID-19. Analysis of scRNA-seq data revealed that plasmablasts in stable patients express higher levels of tissue-homing receptors including CXCR3. The increased Tph cells exhibited "B cell help" signatures similar to that of circulating T follicular helper (cTfh) cells and promoted B cell differentiation in vitro. Compared with cTfh cells, Tph cells produced more IFN{gamma}, inducing tissue-homing chemokine receptors on plasmablasts. Finally, expansion of activated Tph cells was correlated with the frequency of CXCR3+ plasmablasts in the acute phase of patients with stable disease. Our results demonstrate a novel role for Tph cells in acute viral immunity by inducing ectopic, antibody secreting plasmablasts.

To address the need for a safe, efficacious vaccine against SARS-CoV-2 infection with the critical properties of enabling both blocking viral entry into cells and clearing virus from cells already infected, we have developed a bivalent, human adenovirus serotype 5 (hAd5) SARS-CoV- 2 S-Fusion + N-ETSD vaccine that is currently in clinical testing. This vaccine uses the next- generation hAd5 [E1-, E2b-, E3-] platform previously used successfully in cancer patients with pre-existing adenovirus immunity, engineered to express both SARS-CoV-2 spike (S) protein modified to improve the generation of neutralizing antibodies to block entry of the virus, and nucleocapsid (N) protein with an Enhanced T cell Stimulation Domain (ETSD) to activate CD4+ and CD8+ T cells to clear the virus and block replication by killing infected cells. The targeting of N to endosomes and lysosomes to enhance CD4+ and CD8+ T-cell responses distinguishes our vaccine. In our previously reported pre-clinical studies we showed that in mice, the hAd5 S-Fusion + N-ETSD vaccine elicits both humoral and T-cell responses that are robust and T helper cell 1 (Th1) dominant. Here we report that the hAd5 S-Fusion + N-ETSD vaccine is recognized by anti-sera and T cells from previously SARS-CoV-2 infected patients, and that the presence of N is vital for T-cell recall. The findings presented herein: (i) demonstrate specific recognition of hAd5 S-Fusion + N-ETSD infected cells by plasma antibodies from previously SARS-CoV-2 infected patients, but not antibodies from virus-naive subjects; (ii) show enhanced binding of plasma SARS-CoV-2 antibodies from previously infected patients to monocyte-derived dendritic cells (MoDCs) expressing the hAd5 S-Fusion + N-ETSD vaccine as compared to hAd5 S-Fusion alone; (iii) reveal N-ETSD localizes to vesicles associated with MHC class II antigen presentation, including endosomes, lysosomes and autophagosomes in MoDCs; (iv) demonstrate endosome/lysosome-targeted N-ETSD elicits higher interferon-{gamma} T-cell responses than cytoplasm-localized N; and (v) N-ETSD alone or in the hAd5 S-Fusion + N-ETSD construct induces both CD4+ and CD8+ T cell memory recall. This recognition of hAd5 S-Fusion + N-ETSD vaccine antigens by T cells from previously SARS-CoV-2 infected patients, together with the ability of this vaccine candidate to elicit de novo immune responses in naive mice suggests that it re-capitulates the natural immune response to SARS-CoV-2 to activate both B and T cells towards viral neutralization and recognition of infected cells, critical for prevention of COVID-19 disease. Intriguingly, our hAd5 S-Fusion + N-ETSD T-cell biased vaccine has the potential to not only provide protection for uninfected individuals, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.

Neutrophils are crucial to innate immune responses to microbes. The crosslinking of opsonized pathogens by Fc gamma receptors (Fc{gamma}Rs) on neutrophil surfaces mediates multiple antimicrobial functions, including phagocytosis and the production of reactive oxygen species (ROS). Fc{gamma}RIIIb (CD16b) is the most abundant receptor on human neutrophils. It is a GPI-anchored receptor that lacks an intracellular domain. The exact mechanisms by which Fc{gamma}RIIIb transduces signals remain unclear. A rare Fc{gamma}RIIIb-deficient phenotype has been reported in apparently healthy subjects, which is intriguing given the abundance of this receptor on neutrophil surfaces and its crucial role in neutrophil activation by immune complexes. Here, we identified 2 healthy brothers lacking Fc{gamma}RIIIb on neutrophils and characterized their neutrophil activation through Fc{gamma}R crosslinking by immune complexes. Sequencing of the FCGR3B gene revealed mutations in exon 2 resulting in translation loss. In the absence of stimulation, Fc{gamma}RIIIbnull neutrophils showed unaltered levels of Fc{gamma}RIIa, TLR-2, TLR-4 and TLR-6, but significantly higher Fc{gamma}RIIIa and Fc{gamma}RIa. Upon challenge with E. coli immune complexes, increased surface expression of Fc{gamma}RIa, TLR-4, and M integrin (CD11b) was observed exclusively in Fc{gamma}RIIIbnull neutrophils. Antibacterial functions stimulated by immune complexes were significantly lower in Fc{gamma}RIIIbnull neutrophils, including phagocytic capacity and ROS production compared to Fc{gamma}RIIIb-expressing neutrophils. Overall, the absence of Fc{gamma}RIIIb on human neutrophils correlated with impaired antimicrobial functions following stimulation through Fc{gamma}Rs. This study provides new insights into the functional relevance of Fc{gamma}RIIIb and emphasizes the importance of this receptor in neutrophil responses to bacteria.