Measurement of circulating viral antigens post-SARS-CoV-2 infection in a multicohort study.

OBJECTIVES: To determine the proportion of individuals with detectable antigen in plasma or serum after SARS-CoV-2 infection and the association of antigen detection with postacute sequelae of COVID-19 (PASC) symptoms. METHODS: Plasma and serum samples were collected from adults participating in four independent studies at different time points, ranging from several days up to 14 months post-SARS-CoV-2 infection. The primary outcome measure was to quantify SARS-CoV-2 antigens, including the S1 subunit of spike, full-length spike, and nucleocapsid, in participant samples. The presence of 34 commonly reported PASC symptoms during the postacute period was determined from participant surveys or chart reviews of electronic health records. RESULTS: Of the 1569 samples analysed from 706 individuals infected with SARS-CoV-2, 21% (95% CI, 18-24%) were positive for either S1, spike, or nucleocapsid. Spike was predominantly detected, and the highest proportion of samples was spike positive (20%; 95% CI, 18-22%) between 4 and 7 months postinfection. In total, 578 participants (82%) reported at least one of the 34 PASC symptoms included in our analysis ≥1 month postinfection. Cardiopulmonary, musculoskeletal, and neurologic symptoms had the highest reported prevalence in over half of all participants, and among those participants, 43% (95% CI, 40-45%) on average were antigen-positive. Among the participants who reported no ongoing symptoms (128, 18%), antigen was detected in 28 participants (21%). The presence of antigen was associated with the presence of one or more PASC symptoms, adjusting for sex, age, time postinfection, and cohort (OR, 1.8; 95% CI, 1.4-2.2). DISCUSSION: The findings of this multicohort study indicate that SARS-CoV-2 antigens can be detected in the blood of a substantial proportion of individuals up to 14 months after infection. While approximately one in five asymptomatic individuals was antigen-positive, roughly half of all individuals reporting ongoing cardiopulmonary, musculoskeletal, and neurologic symptoms were antigen-positive.

Longitudinal Multi-omic Immune Profiling Reveals Age-Related Immune Cell Dynamics in Healthy Adults.

The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.

MOCHA's advanced statistical modeling of scATAC-seq data enables functional genomic inference in large human cohorts.

Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is being increasingly used to study gene regulation. However, major analytical gaps limit its utility in studying gene regulatory programs in complex diseases. In response, MOCHA (Model-based single cell Open CHromatin Analysis) presents major advances over existing analysis tools, including: 1) improving identification of sample-specific open chromatin, 2) statistical modeling of technical drop-out with zero-inflated methods, 3) mitigation of false positives in single cell analysis, 4) identification of alternative transcription-starting-site regulation, and 5) modules for inferring temporal gene regulatory networks from longitudinal data. These advances, in addition to open chromatin analyses, provide a robust framework after quality control and cell labeling to study gene regulatory programs in human disease. We benchmark MOCHA with four state-of-the-art tools to demonstrate its advances. We also construct cross-sectional and longitudinal gene regulatory networks, identifying potential mechanisms of COVID-19 response. MOCHA provides researchers with a robust analytical tool for functional genomic inference from scATAC-seq data.

Hallmarks of tumor-experienced T cells are absent in multiple myeloma patients from diagnosis through maintenance therapy.

Dysregulation of the bone marrow (BM) niche in multiple myeloma (MM) alters the composition and state of resident immune cells, potentially impeding anti-tumor immunity. One common mechanism of immune inhibition in solid tumors is the induction of exhaustion in tumor-specific T cells. However, the extent of T cell tumor recognition and exhaustion is not well-characterized in MM. As the specific mechanisms of immune evasion are critical for devising effective therapeutic strategies, we deeply profiled the CD8+ T cell compartment of newly-diagnosed MM (NDMM) patients for evidence of tumor reactivity and T cell exhaustion. We applied single-cell multi-omic sequencing and antigen-specific mass cytometry to longitudinal BM and peripheral blood (PB) samples taken from timepoints spanning from diagnosis through induction therapy, autologous stem cell transplant (ASCT), and maintenance therapy. We identified an exhausted-like population that lacked several canonical exhaustion markers, was not significantly enriched in NDMM patients, and consisted of small, nonpersistent clones. We also observed an activated population with increased frequency in the PB of NDMM patients exhibiting phenotypic and clonal features consistent with homeostatic, antigen-nonspecific activation. However, there was no evidence of "tumor-experienced" T cells displaying hallmarks of terminal exhaustion and/or tumor-specific activation/expansion in NDMM patients at any timepoint.

Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia.

Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/µL, there was a higher proliferative response with the PHA flow assay compared to the 3H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID.

Allogeneic hematopoietic cell transplantation is effective for p47phox chronic granulomatous disease: A Primary Immune Deficiency Treatment Consortium study.

BACKGROUND: P47phox (neutrophil cytosolic factor-1) deficiency is the most common cause of autosomal recessive chronic granulomatous disease (CGD) and is considered to be associated with a milder clinical phenotype. Allogeneic hematopoietic cell transplantation (HCT) for p47phox CGD is not well-described. OBJECTIVES: We sought to study HCT for p47phox CGD in North America. METHODS: Thirty patients with p47phox CGD who received allogeneic HCT at Primary Immune Deficiency Treatment Consortium centers since 1995 were included. RESULTS: Residual oxidative activity was present in 66.7% of patients. In the year before HCT, there were 0.38 CGD-related infections per person-years. Inflammatory diseases, predominantly of the lungs and bowel, occurred in 36.7% of the patients. The median age at HCT was 9.1 years (range 1.5-23.6 years). Most HCTs (90%) were performed after using reduced intensity/toxicity conditioning. HCT sources were HLA-matched (40%) and -mismatched (10%) related donors or HLA-matched (36.7%) and -mismatched (13.3%) unrelated donors. CGD-related infections after HCT decreased significantly to 0.06 per person-years (P = .038). The frequency of inflammatory bowel disease and the use of steroids also decreased. The cumulative incidence of graft failure and second HCT was 17.9%. The 2-year overall and event-free survival were 92.3% and 82.1%, respectively, while at 5 years they were 85.7% and 77.0%, respectively. In the surviving patients evaluated, ≥95% donor myeloid chimerism at 1 and 2 years after HCT was 93.8% and 87.5%, respectively. CONCLUSIONS: Patients with p47phox CGD suffer from a significant disease burden that can be effectively alleviated by HCT. Similar to other forms of CGD, HCT should be considered for patients with p47phox CGD.

Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment.

Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.

Dysregulated IFN-γ signals promote autoimmunity in STAT1 gain-of-function syndrome.

Heterozygous signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations promote a clinical syndrome of immune dysregulation characterized by recurrent infections and predisposition to humoral autoimmunity. To gain insights into immune characteristics of STAT1-driven inflammation, we performed deep immunophenotyping of pediatric patients with STAT1 GOF syndrome and age-matched controls. Affected individuals exhibited dysregulated CD4+ T cell and B cell activation, including expansion of TH1-skewed CXCR3+ populations that correlated with serum autoantibody titers. To dissect underlying immune mechanisms, we generated Stat1 GOF transgenic mice (Stat1GOF mice) and confirmed the development of spontaneous humoral autoimmunity that recapitulated the human phenotype. Despite clinical resemblance to human regulatory T cell (Treg) deficiency, Stat1GOF mice and humans with STAT1 GOF syndrome exhibited normal Treg development and function. In contrast, STAT1 GOF autoimmunity was characterized by adaptive immune activation driven by dysregulated STAT1-dependent signals downstream of the type 1 and type 2 interferon (IFN) receptors. However, in contrast to the prevailing type 1 IFN-centric model for STAT1 GOF autoimmunity, Stat1GOF mice lacking the type 1 IFN receptor were only partially protected from STAT1-driven systemic inflammation, whereas loss of type 2 IFN (IFN-γ) signals abrogated autoimmunity. Last, germline STAT1 GOF alleles are thought to enhance transcriptional activity by increasing total STAT1 protein, but the underlying biochemical mechanisms have not been defined. We showed that IFN-γ receptor deletion normalized total STAT1 expression across immune lineages, highlighting IFN-γ as the critical driver of feedforward STAT1 elevation in STAT1 GOF syndrome.

Persistent serum protein signatures define an inflammatory subcategory of long COVID.

Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood. Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals. Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.

Evaluation and Management of Deficiency of Adenosine Deaminase 2: An International Consensus Statement.

Importance: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited disease characterized by systemic vasculitis, early-onset stroke, bone marrow failure, and/or immunodeficiency affecting both children and adults. DADA2 is among the more common monogenic autoinflammatory diseases, with an estimate of more than 35 000 cases worldwide, but currently, there are no guidelines for diagnostic evaluation or management. Objective: To review the available evidence and develop multidisciplinary consensus statements for the evaluation and management of DADA2. Evidence Review: The DADA2 Consensus Committee developed research questions based on data collected from the International Meetings on DADA2 organized by the DADA2 Foundation in 2016, 2018, and 2020. A comprehensive literature review was performed for articles published prior to 2022. Thirty-two consensus statements were generated using a modified Delphi process, and evidence was graded using the Oxford Center for Evidence-Based Medicine Levels of Evidence. Findings: The DADA2 Consensus Committee, comprising 3 patient representatives and 35 international experts from 18 countries, developed consensus statements for (1) diagnostic testing, (2) screening, (3) clinical and laboratory evaluation, and (4) management of DADA2 based on disease phenotype. Additional consensus statements related to the evaluation and treatment of individuals with DADA2 who are presymptomatic and carriers were generated. Areas with insufficient evidence were identified, and questions for future research were outlined. Conclusions and Relevance: DADA2 is a potentially fatal disease that requires early diagnosis and treatment. By summarizing key evidence and expert opinions, these consensus statements provide a framework to facilitate diagnostic evaluation and management of DADA2.

A comprehensive platform for analyzing longitudinal multi-omics data.

Longitudinal bulk and single-cell omics data is increasingly generated for biological and clinical research but is challenging to analyze due to its many intrinsic types of variations. We present PALMO ( https://github.com/aifimmunology/PALMO ), a platform that contains five analytical modules to examine longitudinal bulk and single-cell multi-omics data from multiple perspectives, including decomposition of sources of variations within the data, collection of stable or variable features across timepoints and participants, identification of up- or down-regulated markers across timepoints of individual participants, and investigation on samples of same participants for possible outlier events. We have tested PALMO performance on a complex longitudinal multi-omics dataset of five data modalities on the same samples and six external datasets of diverse background. Both PALMO and our longitudinal multi-omics dataset can be valuable resources to the scientific community.

Clinically Complex LRBA Deficiency Due to a Founder Allele in the Georgian Jewish Population.

Pathogenic variants in LRBA, encoding the LPS Responsive Beige-Like Anchor (LRBA) protein, are responsible for recessive, early-onset hypogammaglobulinemia, severe multi-organ autoimmunity, and lymphoproliferation, with increased risk for malignancy. LRBA deficiency has a wide clinical spectrum with variable age of onset and disease severity. Three apparently unrelated patients with LRBA deficiency, of Georgian Jewish descent, were homozygous for LRBA c.6640C > T, p.R2214*, leading to a stop upstream of the LRBA BEACH domain. Despite carrying the same LRBA genotype, the three patients differed in clinical course: the first patient was asymptomatic until age 25 years; the second presented with failure to thrive at age 3 months; and the third presented at age 7 years with immune cytopenias and severe infections. Two of the patients developed malignancies: the first patient was diagnosed with recurrent Hodgkin's disease at age 36 years, and the second patient developed aggressive gastric cancer at age 15 years. Among Georgian Jews, the carrier frequency of the LRBA p.R2214* allele was 1.6% (4 of 236 Georgian Jewish controls). The allele was absent from other populations. Haplotype analysis showed a shared origin of the mutation. These three patients revealed a pathogenic LRBA founder allele in the Georgian Jewish population, support the diverse and complex clinical spectrum of LRBA deficiency, and support the possibility that LRBA deficiency predisposes to malignancy.

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.

The 2022 Update of IUIS Phenotypical Classification for Human Inborn Errors of Immunity.

The International Union of Immunological Societies (IUIS) expert committee (EC) on Inborn Errors of Immunity (IEI) reports here the 2022 updated phenotypic classification, which accompanies and complements the most-recent genotypic classification. This phenotypic classification is aimed for clinicians at the bedside and focuses on clinical features and laboratory phenotypes of specific IEI. In this classification, 485 IEI underlying phenotypes as diverse as infection, malignancy, allergy, auto-immunity and auto-inflammation are described, including 55 novel monogenic defects and 1 autoimmune phenocopy. Therefore, all 485 diseases of the genetic classification are presented in this paper in the form of colored tables with essential clinical or immunological phenotype entries.