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.

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.

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.

Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Mapping effector genes at lupus GWAS loci using promoter Capture-C in follicular helper T cells.

Systemic lupus erythematosus (SLE) is mediated by autoreactive antibodies that damage multiple tissues. Genome-wide association studies (GWAS) link >60 loci with SLE risk, but the causal variants and effector genes are largely unknown. We generated high-resolution spatial maps of SLE variant accessibility and gene connectivity in human follicular helper T cells (TFH), a cell type required for anti-nuclear antibodies characteristic of SLE. Of the ~400 potential regulatory variants identified, 90% exhibit spatial proximity to genes distant in the 1D genome sequence, including variants that loop to regulate the canonical TFH genes BCL6 and CXCR5 as confirmed by genome editing. SLE 'variant-to-gene' maps also implicate genes with no known role in TFH/SLE disease biology, including the kinases HIPK1 and MINK1. Targeting these kinases in TFH inhibits production of IL-21, a cytokine crucial for class-switched B cell antibodies. These studies offer mechanistic insight into the SLE-associated regulatory architecture of the human genome.

Common variable immunodeficiency-associated endotoxemia promotes early commitment to the T follicular lineage.

BACKGROUND: Although chiefly a B-lymphocyte disorder, several research groups have identified common variable immunodeficiency (CVID) subjects with numeric and/or functional TH cell alterations. The causes, interrelationships, and consequences of CVID-associated CD4+ T-cell derangements to hypogammaglobulinemia, autoantibody production, or both remain unclear. OBJECTIVE: We sought to determine how circulating CD4+ T cells are altered in CVID subjects with autoimmune cytopenias (AICs; CVID+AIC) and the causes of these derangements. METHODS: Using hypothesis-generating, high-dimensional single-cell analyses, we created comprehensive phenotypic maps of circulating CD4+ T cells. Differences between subject groups were confirmed in a large and genetically diverse cohort of CVID subjects (n = 69) by using flow cytometry, transcriptional profiling, multiplex cytokine/chemokine detection, and a suite of in vitro functional assays measuring naive T-cell differentiation, B-cell/T-cell cocultures, and regulatory T-cell suppression. RESULTS: Although CD4+ TH cell profiles from healthy donors and CVID subjects without AICs were virtually indistinguishable, T cells from CVID+AIC subjects exhibited follicular features as early as thymic egress. Follicular skewing correlated with IgA deficiency-associated endotoxemia and endotoxin-induced expression of activin A and inducible T-cell costimulator ligand. The resulting enlarged circulating follicular helper T-cell population from CVID+AIC subjects provided efficient help to receptive healthy donor B cells but not unresponsive CVID B cells. Despite this, circulating follicular helper T cells from CVID+AIC subjects exhibited aberrant transcriptional profiles and altered chemokine/cytokine receptor expression patterns that interfered with regulatory T-cell suppression assays and were associated with autoantibody production. CONCLUSIONS: Endotoxemia is associated with early commitment to the follicular T-cell lineage in IgA-deficient CVID subjects, particularly those with AICs.

Patients with common variable immunodeficiency with autoimmune cytopenias exhibit hyperplastic yet inefficient germinal center responses.

BACKGROUND: The lack of pathogen-protective, isotype-switched antibodies in patients with common variable immunodeficiency (CVID) suggests germinal center (GC) hypoplasia, yet a subset of patients with CVID is paradoxically affected by autoantibody-mediated autoimmune cytopenias (AICs) and lymphadenopathy. OBJECTIVE: We sought to compare the physical characteristics and immunologic output of GC responses in patients with CVID with AIC (CVID+AIC) and without AIC (CVID-AIC). METHODS: We analyzed GC size and shape in excisional lymph node biopsy specimens from 14 patients with CVID+AIC and 4 patients with CVID-AIC. Using paired peripheral blood samples, we determined how AICs specifically affected B-and T-cell compartments and antibody responses in patients with CVID. RESULTS: We found that patients with CVID+AIC displayed irregularly shaped hyperplastic GCs, whereas GCs were scarce and small in patients with CVID-AIC. GC hyperplasia was also evidenced by an increase in numbers of circulating follicular helper T cells, which correlated with decreased regulatory T-cell frequencies and function. In addition, patients with CVID+AIC had serum endotoxemia associated with a dearth of isotype-switched memory B cells that displayed significantly lower somatic hypermutation frequencies than their counterparts with CVID-AIC. Moreover, IgG+ B cells from patients with CVID+AIC expressed VH4-34-encoded antibodies with unmutated Ala-Val-Tyr and Asn-His-Ser motifs, which recognize both erythrocyte I/i self-antigens and commensal bacteria. CONCLUSIONS: Patients with CVID+AIC do not contain mucosal microbiota and exhibit hyperplastic yet inefficient GC responses that favor the production of untolerized IgG+ B-cell clones that recognize both commensal bacteria and hematopoietic I/i self-antigens.

BCL6 Inhibitor-Mediated Downregulation of Phosphorylated SAMHD1 and T Cell Activation Are Associated with Decreased HIV Infection and Reactivation.

Clearance of HIV-infected germinal center (GC) CD4+ follicular helper T cells (Tfh) after combination antiretroviral therapy (ART) is essential to an HIV cure. Blocking B cell lymphoma 6 (BCL6; the master transcription factor for Tfh cells) represses HIV infection of tonsillar CD4+ Tfh ex vivo, reduces GC formation, and limits immune activation in vivo We assessed the anti-HIV activity of a novel BCL6 inhibitor, FX1, in Tfh/non-Tfh CD4+ T cells and its impact on T cell activation and SAMHD1 phosphorylation (Thr592). FX1 repressed HIV-1 infection of peripheral CD4+ T cells and tonsillar Tfh/non-Tfh CD4+ T cells (P < 0.05) and total elongated and multispliced HIV-1 RNA production during the first round of viral life cycle (P < 0.01). Using purified circulating CD4+ T cells from uninfected donors, we demonstrate that FX1 treatment resulted in downregulation pSAMHD1 expression (P < 0.05) and T cell activation (HLA-DR, CD25, and Ki67; P < 0.01) ex vivo corresponding with inhibition of HIV-1 and HIV-2 replication. Ex vivo HIV-1 reactivation using purified peripheral CD4+ T cells from HIV-infected ART-suppressed donors was also blocked by FX1 treatment (P < 0.01). Our results indicate that BCL6 function contributes to Tfh/non-Tfh CD4+ T cell activation and cellular susceptibility to HIV infection. BCL6 inhibition represents a novel therapeutic strategy to potentiate HIV suppression in Tfh/non-Tfh CD4+ T cells without reactivation of latent virus.IMPORTANCE The expansion and accumulation of HIV-infected BCL6+ Tfh CD4+ T cells are thought to contribute to the persistence of viral reservoirs in infected subjects undergoing ART. Two mechanisms have been raised for the preferential retention of HIV within Tfh CD4+ T cells: (i) antiretroviral drugs have limited tissue distribution, resulting in insufficient tissue concentration and lower efficacy in controlling HIV replication in lymphoid tissues, and (ii) cytotoxic CD8+ T cells within lymphoid tissues express low levels of chemokine receptor (CXCR5), thus limiting their ability to enter the GCs to control/eliminate HIV-infected Tfh cells. Our results indicate that the BCL6 inhibitor FX1 can not only repress HIV infection of tonsillar Tfh ex vivo but also suppress HIV infection and reactivation in primary, non-Tfh CD4+ T cells. Our study provides a rationale for targeting BCL6 protein to extend ART-mediated reduction of persistent HIV and/or support strategies toward HIV remission beyond ART cessation.

Cytotoxic T-Lymphocyte-Associated Protein 4 Haploinsufficiency-Associated Inflammation Can Occur Independently of T-Cell Hyperproliferation.

Located contiguously on the long arm of the second chromosome are gene paralogs encoding the immunoglobulin-family co-activation receptors CD28 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). CD28 and CTLA4 share the same B7 ligands yet each provides opposing proliferative signals to T cells. Herein, we describe for the first time two unrelated subjects with coexisting CD28 and CTLA4 haploinsufficiency due to heterozygous microdeletions of chromosome 2q. Although their clinical phenotype, multi-organ inflammatory disease, is superficially similar to that of CTLA4 haploinsufficient autoimmune lymphoproliferative syndrome type V (ALPS5) patients, we demonstrate our subjects' underlying immunopathology to be distinct. Unlike ALPS5 T cells which hyperproliferate to T-cell receptor-mediated activation and infiltrate organs, T cells from our subjects are hypoproliferative and do not. Instead of T cell infiltrates, biopsies of affected subject tissues demonstrated infiltrates of lineage negative lymphoid cells. This histologic feature correlated with significant increases in circulating type 3 innate lymphoid cells (ILC3s) and ILC3 cytokines, interleukin 22, and interleukin-17A. CTLA4-Ig monotherapy, which we trialed in one subject, was remarkably effective in controlling inflammatory diseases, normalizing ILC3 frequencies, and reducing ILC3 cytokine concentrations.

Smith-Magenis Syndrome Patients Often Display Antibody Deficiency but Not Other Immune Pathologies.

BACKGROUND: Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder associated with recurrent otitis. Most SMS cases result from heterozygous interstitial chromosome 17p11.2 deletions that encompass not only the intellectual disability gene retinoic acid-induced 1 but also other genes associated with immunodeficiency, autoimmunity, and/or malignancy. OBJECTIVES: The goals of this study were to describe the immunological consequence of 17p11.2 deletions by determining the prevalence of immunological diseases in subjects with SMS and by assessing their immune systems via laboratory methods. METHODS: We assessed clinical histories of 76 subjects with SMS with heterozygous 17p11.2 deletions and performed in-depth immunological testing on 25 representative cohort members. Laboratory testing included determination of serum antibody concentrations, vaccine titers, and lymphocyte subset frequencies. Detailed reactivity profiles of SMS serum antibodies were performed using custom-made antigen microarrays. RESULTS: Of 76 subjects with SMS, 74 reported recurrent infections including otitis (88%), pneumonia (47%), sinusitis (42%), and gastroenteritis (34%). Infections were associated with worsening SMS-related neurobehavioral symptoms. The prevalence of autoimmune and atopic diseases was not increased. Malignancy was not reported. Laboratory evaluation revealed most subjects with SMS to be deficient of isotype-switched memory B cells and many to lack protective antipneumococcal antibodies. SMS antibodies were not more reactive than control antibodies to self-antigens. CONCLUSIONS: Patients with SMS with heterozygous 17p.11.2 deletions display an increased susceptibility to sinopulmonary infections, but not to autoimmune, allergic, or malignant diseases. SMS sera display an antibody reactivity profile favoring neither recognition of pathogen-associated antigens nor self-antigens. Prophylactic strategies to prevent infections may also provide neurobehavioral benefits to selected patients with SMS.

Circulating TFH subset distribution is strongly affected in lupus patients with an active disease.

Follicular helper T cells (TFH) represent a distinct subset of CD4(+) T cells specialized in providing help to B lymphocytes, which may play a central role in autoimmune diseases having a major B cell component such as systemic lupus erythematosus. Recently, TFH subsets that share common phenotypic and functional characteristics with TFH cells from germinal centers, have been described in the peripheral blood from healthy individuals. The aim of this study was to analyze the distribution of such populations in lupus patients. Circulating TFH cell subsets were defined by multicolor flow cytometry as TFH17 (CXCR3(-)CCR6(+)), TFH1 (CXCR3 (+) CCR6(-)) or TFH2 (CXCR3(-)CCR6(-)) cells among CXCR5 (+) CD45RA(-)CD4(+) T cells in the peripheral blood of 23 SLE patients and 23 sex and age-matched healthy controls. IL-21 receptor expression by B cells was analyzed by flow cytometry and the serum levels of IL-21 and Igs were determined by ELISA tests. We found that the TFH2 cell subset frequency is strongly and significantly increased in lupus patients with an active disease (SLEDAI score>8), while the TFH1 cell subset percentage is greatly decreased. The TFH2 and TFH1 cell subset frequency alteration is associated with the presence of high Ig levels and autoantibodies in patient's sera. Moreover, the TFH2 cell subset enhancement correlates with an increased frequency of double negative memory B cells (CD27(-)IgD(-)CD19(+) cells) expressing the IL-21R. Finally, we found that IgE levels in lupus patients' sera correlate with disease activity and seem to be associated with high TFH2 cell subset frequency. In conclusion, our study describes for the first time the distribution of circulating TFH cell subsets in lupus patients. Interestingly, we found an increased frequency of TFH2 cells, which correlates with disease activity. Our results suggest that this subset might play a key role in lupus pathogenesis.