Autoimmunity in Down's syndrome via cytokines, CD4 T cells and CD11c+ B cells.

Down's syndrome (DS) presents with a constellation of cardiac, neurocognitive and growth impairments. Individuals with DS are also prone to severe infections and autoimmunity including thyroiditis, type 1 diabetes, coeliac disease and alopecia areata1,2. Here, to investigate the mechanisms underlying autoimmune susceptibility, we mapped the soluble and cellular immune landscape of individuals with DS. We found a persistent elevation of up to 22 cytokines at steady state (at levels often exceeding those in patients with acute infection) and detected basal cellular activation: chronic IL-6 signalling in CD4 T cells and a high proportion of plasmablasts and CD11c+TbethighCD21low B cells (Tbet is also known as TBX21). This subset is known to be autoimmune-prone and displayed even greater autoreactive features in DS including receptors with fewer non-reference nucleotides and higher IGHV4-34 utilization. In vitro, incubation of naive B cells in the plasma of individuals with DS or with IL-6-activated T cells resulted in increased plasmablast differentiation compared with control plasma or unstimulated T cells, respectively. Finally, we detected 365 auto-antibodies in the plasma of individuals with DS, which targeted the gastrointestinal tract, the pancreas, the thyroid, the central nervous system, and the immune system itself. Together, these data point to an autoimmunity-prone state in DS, in which a steady-state cytokinopathy, hyperactivated CD4 T cells and ongoing B cell activation all contribute to a breach in immune tolerance. Our findings also open therapeutic paths, as we demonstrate that T cell activation is resolved not only with broad immunosuppressants such as Jak inhibitors, but also with the more tailored approach of IL-6 inhibition.

Autoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq.

Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

Serological responses to human virome define clinical outcomes of Italian patients infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic respiratory infectious disease COVID-19. However, clinical manifestations and outcomes differ significantly among COVID-19 patients, ranging from asymptomatic to extremely severe, and it remains unclear what drives these disparities. Here, we studied 159 sequentially enrolled hospitalized patients with COVID-19-associated pneumonia from Brescia, Italy using the VirScan phage-display method to characterize circulating antibodies binding to 96,179 viral peptides encoded by 1,276 strains of human viruses. SARS-CoV-2 infection was associated with a marked increase in immune antibody repertoires against many known pathogenic and non-pathogenic human viruses. This antiviral antibody response was linked to longitudinal trajectories of disease severity and was further confirmed in additional 125 COVID-19 patients from the same geographical region in Northern Italy. By applying a machine-learning-based strategy, a viral exposure signature predictive of COVID-19-related disease severity linked to patient survival was developed and validated. These results provide a basis for understanding the role of memory B-cell repertoire to viral epitopes in COVID-19-related symptoms and suggest that a unique anti-viral antibody repertoire signature may be useful to define COVID-19 clinical severity.

Autoantibodies against type I IFNs in patients with critical influenza pneumonia.

Autoantibodies neutralizing type I interferons (IFNs) can underlie critical COVID-19 pneumonia and yellow fever vaccine disease. We report here on 13 patients harboring autoantibodies neutralizing IFN-α2 alone (five patients) or with IFN-ω (eight patients) from a cohort of 279 patients (4.7%) aged 6-73 yr with critical influenza pneumonia. Nine and four patients had antibodies neutralizing high and low concentrations, respectively, of IFN-α2, and six and two patients had antibodies neutralizing high and low concentrations, respectively, of IFN-ω. The patients' autoantibodies increased influenza A virus replication in both A549 cells and reconstituted human airway epithelia. The prevalence of these antibodies was significantly higher than that in the general population for patients <70 yr of age (5.7 vs. 1.1%, P = 2.2 × 10-5), but not >70 yr of age (3.1 vs. 4.4%, P = 0.68). The risk of critical influenza was highest in patients with antibodies neutralizing high concentrations of both IFN-α2 and IFN-ω (OR = 11.7, P = 1.3 × 10-5), especially those <70 yr old (OR = 139.9, P = 3.1 × 10-10). We also identified 10 patients in additional influenza patient cohorts. Autoantibodies neutralizing type I IFNs account for ∼5% of cases of life-threatening influenza pneumonia in patients <70 yr old.

COVID-19 and Inborn Errors of Immunity.

Inborn errors of immunity (IEI) are a heterogeneous group of disorders affecting immune host defense and immunoregulation. Considering the predisposition to develop severe and chronic infections, it is crucial to understand the clinical evolution of COVID-19 in IEI patients. This review analyzes clinical outcomes following SARS-CoV-2 infection, as well as response to COVID-19 vaccines in patients with IEI.

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening ‘breakthrough’ cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS-CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals;however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals (age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto-Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-α2 and IFN-ω, while two neutralized IFN-ω only. No patient neutralized IFN-β. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population. Type I IFN auto-Abs are found in 20% of hypoxemic, mRNA vaccinated COVID-19 patients despite SARS-CoV-2 neutralizing antibodies. Description

Temporal Dynamics of Anti-Type 1 Interferon Autoantibodies in Patients With Coronavirus Disease 2019.

Binding levels and neutralization activity of anti-type 1 interferon autoantibodies peaked during acute coronavirus disease 2019 and markedly decreased thereafter. Most patients maintained some ability to neutralize type 1 interferon into convalescence despite lower levels of binding immunoglobulin G. Identifying these autoantibodies in healthy individuals before the development of critical viral disease may be challenging.

Congenital and acquired defects of immunity: An ever-evolving story.

Inborn errors of immunity (IEI), also referred to as primary immunodeficiencies (PID), are disorders that, for the most part, result from mutations in genes involved in immune host defense and immune regulation. With the increased availability of high-throughput DNA sequencing and improved genomic data interpretation, the number of newly identified genes associated with IEI has exponentially increased over the last decade. Here, we focus on the newly described IEI associated with severe COVID-19 and SASH3 deficiency, the most recently reported IEI with impaired T-cell receptor (TCR) signaling.

Prognostic value of serum/plasma neurofilament light chain for COVID-19-associated mortality.

OBJECTIVE: Given the continued spread of coronavirus 2, the early predictors of coronavirus disease 19 (COVID-19) associated mortality might improve patients' outcomes. Increased levels of circulating neurofilament light chain (NfL), a biomarker of neuronal injury, have been observed in severe COVID-19 patients. We investigated whether NfL provides non-redundant clinical value to previously identified predictors of COVID-19 mortality. METHODS: We measured serum or plasma NfL concentrations in a blinded fashion in 3 cohorts totaling 338 COVID-19 patients. RESULTS: In cohort 1, we found significantly elevated NfL levels only in critically ill COVID-19 patients. Longitudinal cohort 2 data showed that NfL is elevated late in the course of the disease, following the two other prognostic markers of COVID-19: decrease in absolute lymphocyte count (ALC) and increase in lactate dehydrogenase (LDH). Significant correlations between ALC and LDH abnormalities and subsequent rise of NfL implicate that the multi-organ failure is the most likely cause of neuronal injury in severe COVID-19 patients. The addition of NfL to age and gender in cohort 1 significantly improved the accuracy of mortality prediction and these improvements were validated in cohorts 2 and 3. INTERPRETATION: A substantial increase in serum/plasma NfL reproducibly enhanced COVID-19 mortality prediction. Combined with other prognostic markers, such as ALC and LDH that are routinely measured in ICU patients, NfL measurements might be useful to identify the patients at a high risk of COVID-19-associated mortality, who might still benefit from escalated care.

Autoantibodies Against Proteins Previously Associated With Autoimmunity in Adult and Pediatric Patients With COVID-19 and Children With MIS-C.

The antibody profile against autoantigens previously associated with autoimmune diseases and other human proteins in patients with COVID-19 or multisystem inflammatory syndrome in children (MIS-C) remains poorly defined. Here we show that 30% of adults with COVID-19 had autoantibodies against the lung antigen KCNRG, and 34% had antibodies to the SLE-associated Smith-D3 protein. Children with COVID-19 rarely had autoantibodies; one of 59 children had GAD65 autoantibodies associated with acute onset of insulin-dependent diabetes. While autoantibodies associated with SLE/Sjögren's syndrome (Ro52, Ro60, and La) and/or autoimmune gastritis (gastric ATPase) were detected in 74% (40/54) of MIS-C patients, further analysis of these patients and of children with Kawasaki disease (KD), showed that the administration of intravenous immunoglobulin (IVIG) was largely responsible for detection of these autoantibodies in both groups of patients. Monitoring in vivo decay of the autoantibodies in MIS-C children showed that the IVIG-derived Ro52, Ro60, and La autoantibodies declined to undetectable levels by 45-60 days, but gastric ATPase autoantibodies declined more slowly requiring >100 days until undetectable. Further testing of IgG and/or IgA antibodies against a subset of potential targets identified by published autoantigen array studies of MIS-C failed to detect autoantibodies against most (16/18) of these proteins in patients with MIS-C who had not received IVIG. However, Troponin C2 and KLHL12 autoantibodies were detected in 2 of 20 and 1 of 20 patients with MIS-C, respectively. Overall, these results suggest that IVIG therapy may be a confounding factor in autoantibody measurements in MIS-C and that antibodies against antigens associated with autoimmune diseases or other human proteins are uncommon in MIS-C.

SARS-CoV-2 infection in dialysis and kidney transplant patients: immunological and serological response.

BACKGROUND: Dialysis and kidney transplant patients with moderate-severe COVID-19 have a high mortality rate, around 30%, that is similar in the two populations, despite differences in their baseline characteristics. In these groups, the immunology of the disease has been poorly explored. METHODS: Thirty-two patients on dialysis or with kidney transplant and SARS-CoV-2 infection requiring hospitalization (COV group) were included in our study. Lymphocyte subsets, dendritic cell (DC) counts and monocyte activation were studied. SARS-CoV-2 anti-spike/anti-nucleocapsid were monitored, and baseline cytokines and chemokines were measured in 10 patients. RESULTS: The COV group, compared to healthy subjects and uninfected dialysis/kidney transplant controls, showed lower numbers of CD4 + and CD8 + T cells, Natural-Killer (NK), B cells, plasmacytoid and myeloid DCs, while the proportion of terminally differentiated B-cells was increased. IL6, IL10, IFN-α and chemokines involved in monocyte and neutrophil recruitment were higher in the COV group, compared to uninfected dialysis/kidney transplant controls. Patients with severe disease had lower CD4 + , CD8 + and B-cell counts and lower monocyte HLA-DR expression. Of note, when comparing dialysis and kidney transplant patients with COVID-19, the latter group presented lower NK and pDC counts and monocyte HLA-DR expression. Up to 60 days after symptom onset, kidney transplant recipients showed lower levels of anti-spike antibodies compared to dialysis patients. CONCLUSIONS: During SARS-CoV-2 infection, dialysis and kidney transplant patients manifest immunophenotype abnormalities; these are similar in the two groups, however kidney transplant recipients show more profound alterations of the innate immune system and lower anti-spike antibody response.

Evidence of SARS-CoV-2-Specific T-Cell-Mediated Myocarditis in a MIS-A Case.

A 26-year-old otherwise healthy man died of fulminant myocarditis. Nasopharyngeal specimens collected premortem tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Histopathological evaluation of the heart showed myocardial necrosis surrounded by cytotoxic T-cells and tissue-repair macrophages. Myocardial T-cell receptor (TCR) sequencing revealed hyper-dominant clones with highly similar sequences to TCRs that are specific for SARS-CoV-2 epitopes. SARS-CoV-2 RNA was detected in the gut, supporting a diagnosis of multisystem inflammatory syndrome in adults (MIS-A). Molecular targets of MIS-associated inflammation are not known. Our data indicate that SARS-CoV-2 antigens selected high-frequency T-cell clones that mediated fatal myocarditis.

SARS-CoV-2 Spike Protein-Directed Monoclonal Antibodies May Ameliorate COVID-19 Complications in APECED Patients.

Patients with the monogenic immune dysregulatory syndrome autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene, uniformly carry neutralizing autoantibodies directed against type-I interferons (IFNs) and many develop autoimmune pneumonitis, both of which place them at high risk for life-threatening COVID-19 pneumonia. Bamlanivimab and etesevimab are monoclonal antibodies (mAbs) that target the SARS-CoV-2 spike protein and block entry of SARS-CoV-2 in host cells. The use of bamlanivimab and etesevimab early during infection was associated with reduced COVID-19-associated hospitalization and death in patients at high risk for progressing to severe disease, which led the US Food and Drug Administration to issue an emergency use authorization for their administration in non-hypoxemic, non-hospitalized high-risk patients. However, the safety and efficacy of these mAbs has not been evaluated in APECED patients. We enrolled two siblings with APECED on an IRB-approved protocol (NCT01386437) and admitted them prophylactically at the NIH Clinical Center for evaluation of mild-to-moderate COVID-19. We assessed the safety and clinical effects of early treatment with bamlanivimab and etesevimab. The administration of bamlanivimab and etesevimab was well tolerated and was associated with amelioration of COVID-19 symptoms and prevention of invasive ventilatory support, admission to the intensive care, and death in both patients without affecting the production of antibodies to the nucleocapsid protein of SARS-CoV-2. If given early in the course of COVID-19 infection, bamlanivimab and etesevimab may be beneficial in APECED and other high-risk patients with neutralizing autoantibodies directed against type-I IFNs.

Antibody responses to the SARS-CoV-2 vaccine in individuals with various inborn errors of immunity.

BACKGROUND: SARS-CoV-2 vaccination is recommended in patients with inborn errors of immunity (IEIs); however, little is known about immunogenicity and safety in these patients. OBJECTIVE: We sought to evaluate the impact of genetic diagnosis, age, and treatment on antibody response to COVID-19 vaccine and related adverse events in a cohort of patients with IEIs. METHODS: Plasma was collected from 22 health care worker controls, 81 patients with IEIs, and 2 patients with thymoma; the plasma was collected before immunization, 1 to 6 days before the second dose of mRNA vaccine, and at a median of 30 days after completion of the immunization schedule with either mRNA vaccine or a single dose of Johnson & Johnson's Janssen vaccine. Anti-spike (anti-S) and anti-nucleocapsid antibody titers were measured by using a luciferase immunoprecipitation systems method. Information on T- and B-cell counts and use of immunosuppressive drugs was extracted from medical records, and information on vaccine-associated adverse events was collected after each dose. RESULTS: Anti-S antibodies were detected in 27 of 46 patients (58.7%) after 1 dose of mRNA vaccine and in 63 of 74 fully immunized patients (85.1%). A lower rate of seroconversion (7 of 11 [63.6%]) was observed in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Previous use of rituximab and baseline counts of less than 1000 CD3+ T cells/mL and less than 100 CD19+ B cells/mL were associated with lower anti-S IgG levels. No significant adverse events were reported. CONCLUSION: Vaccinating patients with IEIs is safe, but immunogenicity is affected by certain therapies and gene defects. These data may guide the counseling of patients with IEIs regarding prevention of SARS-CoV-2 infection and the need for subsequent boosts.

Robust Antibody and T Cell Responses to SARS-CoV-2 in Patients with Antibody Deficiency.

Immunocompromised patients, including those with inborn errors of immunity (IEI), may be at increased risk for severe or prolonged infections with SARS-CoV-2 (Zhu et al. N Engl J Med. 382:727-33, 2020; Guan et al. 2020; Minotti et al. J Infect. 81:e61-6, 2020). While antibody and T cell responses to SARS-CoV-2 structural proteins are well described in healthy convalescent donors, adaptive humoral and cellular immunity has not yet been characterized in patients with antibody deficiency (Grifoni et al. Cell. 181:1489-1501 e1415, 2020; Burbelo et al. 2020; Long et al. Nat Med. 26:845-8, 2020; Braun et al. 2020). Herein, we describe the clinical course, antibody, and T cell responses to SARS-CoV-2 structural proteins in a cohort of adult and pediatric patients with antibody deficiencies (n = 5) and controls (related and unrelated) infected with SARS-CoV-2. Five patients within the same family (3 with antibody deficiency, 2 immunocompetent controls) showed antibody responses to nucleocapsid and spike proteins, as well as SARS-CoV-2 specific T cell immunity at days 65-84 from onset of symptoms. No significant difference was identified between immunocompromised patients and controls. Two additional unrelated, adult patients with common variable immune deficiency were assessed. One did not show antibody response, but both demonstrated SARS-CoV-2-specific T cell immunity when evaluated 33 and 76 days, respectively, following SARS-CoV-2 diagnosis. This report is the first to show robust T cell activity and humoral immunity against SARS-CoV-2 structural proteins in some patients with antibody deficiency. Given the reliance on spike protein in most candidate vaccines (Folegatti et al. Lancet. 396:467-78, 2020; Jackson et al. N Engl J Med. 383:1920-31, 2020), the responses are encouraging. Additional studies will be needed to further define the timing of onset of immunity, longevity of the immune response, and variability of response in immunocompromised patients.

Association of rare predicted loss-of-function variants of influenza-related type I IFN genes with critical COVID-19 pneumonia/Reply

[...]the power computation shown in their Figure 1 is based on an incorrect hypothesis about the odds ratio, which would be expected to be lower when using general population controls (as they did) than when using paucisymptomatic and asymptomatic infected individuals (as we did). (iv) The ethnic origin of the patients differs between the 2 studies: 58% of our 659 patients (and 8 of our 9 pLOF carriers) were European, versus only 10% of their 713 patients with severe disease (and the pLOF carrier was East Asian). (v) Age is a key factor neglected in their comparison: our sample was much younger (mean age, 51.8 years) than theirs (mean, 65.9 years), and 7 of our 9 pLOF carriers were younger than 60 years. Because the rates of pLOFs vary considerably across populations, adjustment for only 3 principal components of ancestry in rare-variant association tests of multiethnic cohorts does not provide adequate control for population structure. [...]none of the associations showed even marginal significance. [...]consistent with our study, these findings do not support substantial contributions of inborn errors in type I IFN immunity to COVID-19 severity.