Serum Cytokine Panels in Pediatric Clinical Practice.

BACKGROUND: Cytokines are soluble signaling proteins that regulate inflammation and coordinate immune responses. Serum cytokine assays are increasingly used in medical practice, yet our understanding of cytokines as biomarkers for disease remains limited. OBJECTIVES: We aimed to analyze real-world single center usage of a multiplexed cytokine panel, correlate its results with diagnosis and severity, and explore its utility in pediatric practice. METHODS: A multiplexed cytokine panel, able to return same-day results, was implemented in April 2020 at our institution and its performance was validated for clinical use. Coded patient data were collected using a REDCap database, and correlations between cytokine levels and outcomes of interest were analyzed retrospectively. RESULTS: Cytokine levels correlate with acuity of care, with patients admitted to the pediatric intensive care unit (PICU) having the highest cytokine values. Patients with familial HLH (fHLH) showed prominent peaks in IFNγ, IL-10, and TNF, while patients with sepsis exhibited high IL-6 and IL-8 with relatively modest IFNγ, and cytokine release syndrome (CRS) post-CAR T cell therapy often demonstrated pan-panel positivity at peak levels, with a similar pattern as that of fHLH. A ratio of [IFNγ]+[IL-10]/[IL-6]+[IL-8] levels was able to distinguish fHLH and CRS from severe sepsis. CONCLUSIONS: Cytokine levels correlate with severity of illness and can help differentiate between syndromes that present similarly, including fHLH and CRS compared to sepsis. Cytokine panels can be used as biomarkers to inform diagnosis and management decisions, but significant work remains to dissect complex clinical patterns of disease.

3D chromatin-based variant-to-gene maps across 57 human cell types reveal the cellular and genetic architecture of autoimmune disease susceptibility.

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS), but GWAS do not reveal causal variants, effector genes, or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C, Hi-C, ATAC-seq, and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features, and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific, but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types, suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However, trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this, we pharmacologically validated squalene synthase, a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE, as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms, and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression, function, and pathology across multiple, distinct tissues and cell types.

Inborn Errors of Immunity and Cytokine Storm Syndromes.

Inborn errors of immunity (IEI) are a diverse and growing category of more than 430 chronic disorders that share susceptibilities to infections. Whether the result of a genetic lesion that causes defective granule-dependent cytotoxicity, excessive lymphoproliferation, or an overwhelming infection represents a unique antigenic challenge, IEIs can display a proclivity for cytokine storm syndrome (CSS) development. This chapter provides an overview of CSS pathophysiology as it relates to IEIs. For each IEI, the immunologic defect and how it promotes or discourages CSS phenomena are reviewed. The IEI-associated molecular defects in pathways that are postulated to be critical to CSS physiology (i.e., toll-like receptors, T regulatory cells, the IL-12/IFNγ axis, IL-6) and, whenever possible, review strategies for treating CSS in IEI patients with molecularly directed therapies are highlighted.

PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells.

The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta (PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not. In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice coincides with upregulation of PIK3CG expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals instructing commitment to the ASC fate.

IL-12 drives the differentiation of human T follicular regulatory cells.

T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.

The common variable immunodeficiency IgM repertoire narrowly recognizes erythrocyte and platelet glycans.

BACKGROUND: Autoimmune cytopenias (AICs) regularly occur in profoundly IgG-deficient patients with common variable immunodeficiency (CVID). The isotypes, antigenic targets, and origin(s) of their disease-causing autoantibodies are unclear. OBJECTIVE: We sought to determine reactivity, clonality, and provenance of AIC-associated IgM autoantibodies in patients with CVID. METHODS: We used glycan arrays, patient erythrocytes, and platelets to determine targets of CVID IgM autoantibodies. Glycan-binding profiles were used to identify autoreactive clones across B-cell subsets, specifically circulating marginal zone (MZ) B cells, for sorting and IGH sequencing. The locations, transcriptomes, and responses of tonsillar MZ B cells to different TH- cell subsets were determined by confocal microscopy, RNA-sequencing, and cocultures, respectively. RESULTS: Autoreactive IgM coated erythrocytes and platelets from many CVID patients with AICs (CVID+AIC). On glycan arrays, CVID+AIC plasma IgM narrowly recognized erythrocytic i antigens and platelet i-related antigens and failed to bind hundreds of pathogen- and tumor-associated carbohydrates. Polyclonal i antigen-recognizing B-cell receptors were highly enriched among CVID+AIC circulating MZ B cells. Within tonsillar tissues, MZ B cells secreted copious IgM when activated by the combination of IL-10 and IL-21 or when cultured with IL-10/IL-21-secreting FOXP3-CD25hi T follicular helper (Tfh) cells. In lymph nodes from immunocompetent controls, MZ B cells, plentiful FOXP3+ regulatory T cells, and rare FOXP3-CD25+ cells that represented likely CD25hi Tfh cells all localized outside of germinal centers. In CVID+AIC lymph nodes, cellular positions were similar but CD25hi Tfh cells greatly outnumbered regulatory cells. CONCLUSIONS: Our findings indicate that glycan-reactive IgM autoantibodies produced outside of germinal centers may contribute to the autoimmune pathogenesis of CVID.

22q11.2 Deletion-Associated Blood-Brain Barrier Permeability Potentiates Systemic Capillary Leak Syndrome Neurologic Features.

We present a case study of a young male with a history of 22q11.2 deletion syndrome (22qDS), diagnosed with systemic capillary leak syndrome (SCLS) who presented with acute onset of diffuse anasarca and sub-comatose obtundation. We hypothesized that his co-presentation of neurological sequelae might be due to blood-brain barrier (BBB) susceptibility conferred by the 22q11.2 deletion, a phenotype that we have previously identified in 22qDS. Using pre- and post-intravenous immunoglobulins (IVIG) patient serum, we studied circulating biomarkers of inflammation and assessed the potential susceptibility of the 22qDS BBB. We employed in vitro cultures of differentiated BBB-like endothelial cells derived from a 22qDS patient and a healthy control. We found evidence of peripheral inflammation and increased serum lipopolysaccharide (LPS) alongside endothelial cells in circulation. We report that the patient's serum significantly impairs barrier function of the 22qDS BBB compared to control. Only two other cases of pediatric SCLS with neurologic symptoms have been reported, and genetic risk factors have been suggested in both instances. As the third case to be reported, our findings are consistent with the hypothesis that genetic susceptibility of the BBB conferred by genes such as claudin-5 deleted in the 22q11.2 region promoted neurologic involvement during SCLS in this patient.

NF-κB Signaling is Required for X-Chromosome Inactivation Maintenance Following T cell Activation.

X Chromosome Inactivation (XCI) is a female-specific process which balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of Xist RNA and heterochromatic modifications on the inactive X chromosome (Xi), and these modifications become enriched at the Xi after cell stimulation. Here, we examined allele-specific gene expression and the epigenomic profiles of the Xi following T cell stimulation. We found that the Xi in unstimulated T cells is largely dosage compensated and is enriched with the repressive H3K27me3 modification, but not the H2AK119-ubiquitin (Ub) mark, even at promoters of XCI escape genes. Upon CD3/CD28-mediated T cell stimulation, the Xi accumulates H2AK119-Ub and H3K27me3 across the Xi. Next, we examined the T cell signaling pathways responsible for Xist RNA localization to the Xi and found that T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of TCR, is required. Disruption of NF-κB signaling, using inhibitors or genetic deletions, in mice and patients with immunodeficiencies prevents Xist/XIST RNA accumulation at the Xi and alters expression of some X-linked genes. Our findings reveal a novel connection between NF-κB signaling pathways which impact XCI maintenance in female T cells.

Common variable immunodeficiency, cross currents, and prevailing winds.

Common variable immunodeficiency (CVID) is a heterogenous disease category created to distinguish late-onset antibody deficiencies from early-onset diseases like agammaglobulinemia or more expansively dysfunctional combined immunodeficiencies. Opinions vary on which affected patients should receive a CVID diagnosis which confuses clinicians and erects reproducibility barriers for researchers. Most experts agree that CVID's most indeliable feature is defective germinal center (GC) production of isotype-switched, affinity-maturated antibodies. Here, we review the biological factors contributing to CVID-associated GC dysfunction including genetic, epigenetic, tolerogenic, microbiome, and regulatory abnormalities. We also discuss the consequences of these biological phenomena to the development of non-infectious disease complications. Finally, we opine on topics and lines of investigation we think hold promise for expanding our mechanistic understanding of this protean condition and for improving the lives of affected patients.

Human T follicular helper clones seed the germinal center-resident regulatory pool.

The mechanisms by which FOXP3+ T follicular regulatory (Tfr) cells simultaneously steer antibody formation toward microbe or vaccine recognition and away from self-reactivity remain incompletely understood. To explore underappreciated heterogeneity in human Tfr cell development, function, and localization, we used paired TCRVA/TCRVB sequencing to distinguish tonsillar Tfr cells that are clonally related to natural regulatory T cells (nTfr) from those likely induced from T follicular helper (Tfh) cells (iTfr). The proteins iTfr and nTfr cells differentially expressed were used to pinpoint their in situ locations via multiplex microscopy and establish their divergent functional roles. In silico analyses and in vitro tonsil organoid tracking models corroborated the existence of separate Treg-to-nTfr and Tfh-to-iTfr developmental trajectories. Our results identify human iTfr cells as a distinct CD38+, germinal center-resident, Tfh-descended subset that gains suppressive function while retaining the capacity to help B cells, whereas CD38- nTfr cells are elite suppressors primarily localized in follicular mantles. Interventions differentially targeting specific Tfr cell subsets may provide therapeutic opportunities to boost immunity or more precisely treat autoimmune diseases.

Distinct stage-specific transcriptional states of B cells derived from human tonsillar tissue.

B cells within secondary lymphoid tissues encompass a diversity of activation states and multiple maturation processes that reflect antigen recognition and transition through the germinal center (GC) reaction, in which mature B cells differentiate into memory and antibody-secreting cells (ASCs). Here, utilizing single-cell RNA-seq, we identify a range of distinct activation and maturation states of tonsil-derived B cells. In particular, we identify what we believe is a previously uncharacterized CCL4/CCL3 chemokine-expressing B cell population with an expression pattern consistent with B cell receptor/CD40 activation. Furthermore, we present a computational method that leverages regulatory network inference and pseudotemporal modeling to identify upstream transcription factor modulation along a GC-to-ASC axis of transcriptional maturation. Our data set provides valuable insight into diverse B cell functional profiles and will be a useful resource for further studies into the B cell immune compartment.

Monogenic early-onset lymphoproliferation and autoimmunity: Natural history of STAT3 gain-of-function syndrome.

BACKGROUND: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset lymphoproliferation and autoimmunity. OBJECTIVE: This pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants. METHODS: We identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3. RESULTS: Overall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD4-CD8-) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate. CONCLUSION: STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome.

Implicating effector genes at COVID-19 GWAS loci using promoter-focused Capture-C in disease-relevant immune cell types.

BACKGROUND: SARS-CoV-2 infection results in a broad spectrum of COVID-19 disease, from mild or no symptoms to hospitalization and death. COVID-19 disease severity has been associated with some pre-existing conditions and the magnitude of the adaptive immune response to SARS-CoV-2, and a recent genome-wide association study (GWAS) of the risk of critical illness revealed a significant genetic component. To gain insight into how human genetic variation attenuates or exacerbates disease following SARS-CoV-2 infection, we implicated putatively functional COVID risk variants in the cis-regulatory landscapes of human immune cell types with established roles in disease severity and used high-resolution chromatin conformation capture to map these disease-associated elements to their effector genes. RESULTS: This functional genomic approach implicates 16 genes involved in viral replication, the interferon response, and inflammation. Several of these genes (PAXBP1, IFNAR2, OAS1, OAS3, TNFAIP8L1, GART) were differentially expressed in immune cells from patients with severe versus moderate COVID-19 disease, and we demonstrate a previously unappreciated role for GART in T cell-dependent antibody-producing B cell differentiation in a human tonsillar organoid model. CONCLUSIONS: This study offers immunogenetic insight into the basis of COVID-19 disease severity and implicates new targets for therapeutics that limit SARS-CoV-2 infection and its resultant life-threatening inflammation.

Genetic obstacles to developing and tolerizing human B cells.

Early in development, B cells explosively diversify B-cell receptors (BCRs) to recognize a wide variety of microbial antigens. A variety of developmental and tolerance checkpoints are subsequently deployed at later developmental stages to purge useless or potentially dangerous autoreactive B-cell clones. Once B cells recognize cognate antigens within secondary lymphoid tissues, their BCRs are genetically modified to increase the specificity and strength of antigen binding. Identification and investigation of monogenic inborn errors of immunity (IEI) diseases demonstrate which specific molecules and pathways are essential for developing well-tolerized human B cells. Although rare, IEI patients have provided important mechanistic insights into, and therapeutic clues for, patients afflicted with more common autoantibody associated autoimmune diseases like lupus, rheumatoid arthritis, and type 1 diabetes. This article is categorized under: Immune System Diseases > Stem Cells and Development > Genetics/Genomics/Epigenetics.

BTK inhibition limits B-cell-T-cell interaction through modulation of B-cell metabolism: implications for multiple sclerosis therapy.

Inhibition of Bruton's Tyrosine Kinase (BTKi) is now viewed as a promising next-generation B-cell-targeting therapy for autoimmune diseases including multiple sclerosis (MS). Surprisingly little is known; however, about how BTKi influences MS disease-implicated functions of B cells. Here, we demonstrate that in addition to its expected impact on B-cell activation, BTKi attenuates B-cell:T-cell interactions via a novel mechanism involving modulation of B-cell metabolic pathways which, in turn, mediates an anti-inflammatory modulation of the B cells. In vitro, BTKi, as well as direct inhibition of B-cell mitochondrial respiration (but not glycolysis), limit the B-cell capacity to serve as APC to T cells. The role of metabolism in the regulation of human B-cell responses is confirmed when examining B cells of rare patients with mitochondrial respiratory chain mutations. We further demonstrate that both BTKi and metabolic modulation ex vivo can abrogate the aberrant activation and costimulatory molecule expression of B cells of untreated MS patients. Finally, as proof-of-principle in a Phase 1 study of healthy volunteers, we confirm that in vivo BTKi treatment reduces circulating B-cell mitochondrial respiration, diminishes their activation-induced expression of costimulatory molecules, and mediates an anti-inflammatory shift in the B-cell responses which is associated with an attenuation of T-cell pro-inflammatory responses. These data collectively elucidate a novel non-depleting mechanism by which BTKi mediates its effects on disease-implicated B-cell responses and reveals that modulating B-cell metabolism may be a viable therapeutic approach to target pro-inflammatory B cells.

Lymphocytes Utilize Somatic Mutations, Epigenetic Silencing, and the Proteasome to Escape Truncated WASP Expression.

Wiskott-Aldrich Syndrome Protein (WASP) deficiency causes Wiskott-Aldrich Syndrome (WAS), a sex-linked disorder characterized by combined immunodeficiency, microthrombocytopenia, and eczema. Like WASP-deficient humans, WASP-deficient mice produce normal numbers of functionally defective T cells. Here, we report a WAS patient with a novel germline frameshifting WAS mutation encoding a truncated form of WASP lacking the C-terminal cofilin homology (C) and the acidic region (A) domains (WASPΔCA). Although stably overexpressed in embryonic kidney cell lines, WASPΔCA was undetectable in circulating patient leukocytes. Deep sequencing, transcript profiling, and protein degradation analyses demonstrated patient lymphocytes employ an array of genetic, epigenetic, and proteasomal strategies to avoid expressing WASPΔCA.

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. Herein, through a fine-needle aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant recipients (KTXs). We found that, unlike healthy subjects, KTXs presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cell, SARS-CoV-2 receptor binding domain-specific memory B cell, and neutralizing antibody responses. KTXs also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals and suggest a GC origin for certain humoral and memory B cell responses following mRNA vaccination.

Identification of germline monoallelic mutations in IKZF2 in patients with immune dysregulation.

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3 encoding Ikaros and Aiolos have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of proinflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation.