Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

Clinical and Treatment History of Patients with Partial DiGeorge Syndrome and Autoimmune Cytopenia at Multiple Centers.

BACKGROUND: Patients with partial DiGeorge syndrome (pDGS) can present with immune dysregulation, the most common being autoimmune cytopenia (AIC). There is a lack of consensus on the approach to type, combination, and timing of therapies for AIC in pDGS. Recognition of immune dysregulation early in pDGS clinical course may help individualize treatment and prevent adverse outcomes from chronic immune dysregulation. OBJECTIVES: Objectives of this study were to characterize the natural history, immune phenotype, and biomarkers in pDGS with AIC. METHODS: Data on clinical presentation, disease severity, immunological phenotype, treatment selection, and response for patients with pDGS with AIC were collected via retrospective chart review. Flow cytometric analysis was done to assess T and B cell subsets, including biomarkers of immune dysregulation. RESULTS: Twenty-nine patients with the diagnosis of pDGS and AIC were identified from 5 international institutions. Nineteen (62%) patients developed Evan's syndrome (ES) during their clinical course and twenty (69%) had antibody deficiency syndrome. These patients demonstrated expansion in T follicular helper cells, CD19hiCD21lo B cells, and double negative cells and reduction in CD4 naïve T cells and regulatory T cells. First-line treatment for 17/29 (59%) included corticosteroids and/or high-dose immunoglobulin replacement therapy. Other overlapping therapies included eltrombopag, rituximab, and T cell immunomodulators. CONCLUSIONS: AIC in pDGS is often refractory to conventional AIC treatment paradigms. Biomarkers may have utility for correlation with disease state and potentially even response to therapy. Immunomodulating therapies could be initiated early based on early immune phenotyping and biomarkers before the disease develops or significantly worsens.

Disease Progression of WHIM Syndrome in an International Cohort of 66 Pediatric and Adult Patients.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

Lineage Reconstruction of In Vitro Identified Antigen-Specific Autoreactive B Cells from Adaptive Immune Receptor Repertoires.

The emergence, survival, growth and maintenance of autoreactive (AR) B-cell clones, the hallmark of humoral autoimmunity, leave their footprints in B-cell receptor repertoires. Collecting IgH sequences related to polyreactive (PR) ones from adaptive immune receptor repertoire (AIRR) datasets make the reconstruction and analysis of PR/AR B-cell lineages possible. We developed a computational approach, named ImmChainTracer, to extract members and to visualize clonal relationships of such B-cell lineages. Our approach was successfully applied on the IgH repertoires of patients suffering from monogenic hypomorphic RAG1 and 2 deficiency (pRD) or polygenic systemic lupus erythematosus (SLE) autoimmune diseases to identify relatives of AR IgH sequences and to track their fate in AIRRs. Signs of clonal expansion, affinity maturation and class-switching events in PR/AR and non-PR/AR B-cell lineages were revealed. An extension of our method towards B-cell expansion caused by any trigger (e.g., infection, vaccination or antibody development) may provide deeper insight into antigen specific B-lymphogenesis.

Evolving Approach to Clinical Cytometry for Immunodeficiencies and Other Immune Disorders.

Primary immunodeficiencies were initially identified on the basis of recurrent, severe or unusual infections. Subsequently, it was noted that these diseases can also manifest with autoimmunity, autoinflammation, allergy, lymphoproliferation and malignancy, hence a conceptual change and their renaming as inborn errors of immunity. Ongoing advances in flow cytometry provide the opportunity to expand or modify the utility and scope of existing laboratory tests in this field to mirror this conceptual change. Here we have used the B cell subset, variably known as CD21low B cells, age-associated B cells and T-bet+ B cells, as an example to demonstrate this possibility.

Case Report: Initial Treatment Adjustments and Complications in Ovarian Cancer Patient With Inborn Error of Immunity.

Background: Patients with inborn errors of immunity (IEI) have increased risk of developing cancers secondary to impaired anti-tumor immunity. Treatment of patients with IEI and cancer is challenging as chemotherapy can exacerbate infectious susceptibility. However, the literature on optimal cancer treatment in the setting of IEI is sparse. Objectives: We present a patient with specific antibody deficiency with normal immunoglobins (SADNI), immune dysregulation (ID), and stage III ovarian carcinoma as an example of the need to modify conventional treatment in the context of malignancy, IEI, and ongoing infections. Methods: This is a retrospective chart review of the patient's clinical manifestations, laboratory evaluation and treatment course. Results: Our patient is a female with SADNI and ID diagnosed with stage III ovarian carcinoma at 60 years of age. Her ID accounted for antinuclear antibody positive (ANA+) mixed connective tissue diseases, polyarthralgia, autoimmune neutropenia, asthma, autoimmune thyroiditis, and Celiac disease. Due to the lack of precedent in the literature, her treatment was modified with continuous input from infectious disease, allergy/immunology and oncology specialist using a multidisciplinary approach.The patient completed debulking surgery and 6 cycles of chemotherapy. The dosing for immunoglobulin replacement therapy was increased for prophylaxis. Chemotherapy doses were lowered for all cycles preemptively for IEI. The therapy included carboplatin, paclitaxel, bevacizumab, and pegfilgrastim. The patient completed six-months of maintenance medication involving bevacizumab.Her treatment course was complicated by Mycobacterium avium-complex (MAC) infection, elevated bilirubin and liver enzymes attributed to excessive immunoglobulin replacement therapy, and urinary tract infection (UTI) and incontinence.Cancer genetic analysis revealed no targetable markers and primary immunodeficiency gene panel of 407 genes by Invitae was unrevealing. Lab tests revealed no evidence of Epstein-Barr Virus (EBV) infection. Post-chemotherapy imaging revealed no evidence of cancer for 1 year and 4 months, but the disease relapsed subsequently. The patient's lung scarring requires vigilance. Conclusions: Our patient with ovarian cancer and IEI required modified treatment and prevention of complications. In cases of IEI, optimal chemotherapy should be titrated to minimize immunosuppression yet treat cancer aggressively while decreasing the risk of infection with prophylactic antibiotics and prolonged post-treatment surveillance, including pulmonary evaluation.

Corrigendum: Case report: Initial treatment adjustments and complications in ovarian cancer patient with inborn error of immunity.

[This corrects the article DOI: 10.3389/fonc.2022.843741.].

Cellular Mechanisms Underlying B Cell Abnormalities in Patients With Gain-of-Function Mutations in the PIK3CD Gene.

Background: Activated phosphoinositide 3 kinase (PI3K) -delta syndrome (APDS) is an inborn error of immunity with variable clinical phenotype of immunodeficiency and immune dysregulation and caused by gain-of-function mutations in PIK3CD. The hallmark of immune phenotype is increased proportions of transitional B cells and plasmablasts (PB), progressive B cell loss, and elevated levels of serum IgM. Objective: To explore unique B cell subsets and the pathomechanisms driving B cell dysregulation beyond the transitional B cell stage in APDS. Methods: Clinical and immunological data was collected from 24 patients with APDS. In five cases, we performed an in-depth analysis of B cell phenotypes and cultured purified naïve B cells to evaluate their survival, activation, Ig gene class switch recombination (CSR), PB differentiation and antibody secretion. We also analyzed PB differentiation capacity of sorted CD27-IgD- double-negative B (DNB) cells. Results: The patients had increased B cell sizes and higher proportions of IgM+ DNB cells than healthy controls (HC). Their naïve B cells exhibited increased death, impaired CSR but relatively normal PB differentiation. Upon stimulation, patient's DNB cells secreted a similar level of IgG but a higher level of IgM than DNB cells from HC. Targeted therapy of PI3K inhibition partially restored B cell phenotypes. Conclusions: The present study suggests additional mechanistic insight into B cell pathology of APDS: (1) decreased peripheral B cell numbers may be due to the increased death of naïve B cells; (2) larger B cell sizes and expanded DNB population suggest enhanced activation and differentiation of naïve B cells into DNB cells; (3) the impaired CSR yet normal PB differentiation can predominantly generate IgM-secreting cells, resulting in elevated IgM levels.

Asymptomatic Infant With Atypical SCID and Novel Hypomorphic RAG Variant Identified by Newborn Screening: A Diagnostic and Treatment Dilemma.

The T-cell receptor excision circle (TREC) assay detects T-cell lymphopenia (TCL) in newborns and is especially important to identify severe combined immunodeficiency (SCID). A spectrum of SCID variants and non-SCID conditions that present with TCL are being discovered with increasing frequency by newborn screening (NBS). Recombination-activating gene (RAG) deficiency is one the most common causes of classical and atypical SCID and other conditions with immune dysregulation. We present the case of an asymptomatic male with undetectable TRECs on NBS at 1 week of age. The asymptomatic newborn was found to have severe TCL, but normal B cell quantities and lymphocyte proliferation upon mitogen stimulation. Next generation sequencing revealed compound heterozygous hypomorphic RAG variants, one of which was novel. The moderately decreased recombinase activity of the RAG variants (16 and 40%) resulted in abnormal T and B-cell receptor repertoires, decreased fraction of CD3+ TCRVα7.2+ T cells and an immune phenotype consistent with the RAG hypomorphic variants. The patient underwent successful treatment with hematopoietic stem cell transplantation (HSCT) at 5 months of age. This case illustrates how after identification of a novel RAG variant, in vitro studies are important to confirm the pathogenicity of the variant. This confirmation allows the clinician to expedite definitive treatment with HSCT in an asymptomatic phase, mitigating the risk of serious infectious and non-infectious complications.

Vasculitis as a Major Morbidity Factor in Patients With Partial RAG Deficiency.

Vasculitis can be a life-threatening complication associated with high mortality and morbidity among patients with primary immunodeficiencies (PIDs), including variants of severe and combined immunodeficiencies ((S)CID). Our understanding of vasculitis in partial defects in recombination activating gene (RAG) deficiency, a prototype of (S)CIDs, is limited with no published systematic evaluation of diagnostic and therapeutic modalities. In this report, we sought to establish the clinical, laboratory features, and treatment outcome of patients with vasculitis due to partial RAG deficiency. Vasculitis was a major complication in eight (13%) of 62 patients in our cohort with partial RAG deficiency with features of infections and immune dysregulation. Vasculitis occurred early in life, often as first sign of disease (50%) and was complicated by significant end organ damage. Viral infections often preceded the onset of predominately non-granulomatous-small vessel vasculitis. Autoantibodies against cytokines (IFN-α, -ω, and IL-12) were detected in a large fraction of the cases tested (80%), whereas the majority of patients were anti-neutrophil cytoplasmic antibodies (ANCA) negative (>80%). Genetic diagnosis of RAG deficiency was delayed up to 2 years from the onset of vasculitis. Clinical cases with sole skin manifestation responded well to first-line steroid treatment, whereas systemic vasculitis with severe end-organ complications required second-line immunosuppression and/or hematopoietic stem cell transplantation (HSCT) for definitive management. In conclusion, our data suggest that vasculitis in partial RAG deficiency is prevalent among patients with partial RAG deficiency and is associated with high morbidity. Therefore, partial RAG deficiency should be included in the differential diagnosis of patients with early-onset systemic vasculitis. Diagnostic serology may be misleading with ANCA negative findings, and search for conventional autoantibodies should be extended to include those targeting cytokines.

Combined Immunodeficiency With Late-Onset Progressive Hypogammaglobulinemia and Normal B Cell Count in a Patient With RAG2 Deficiency.

Proteins expressed by recombination activating genes 1 and 2 (RAG1/2) are essential in the process of V(D)J recombination that leads to generation of the T and B cell repertoires. Clinical and immunological phenotypes of patients with RAG deficiencies correlate well to the degree of impaired RAG activity and this has been expanding to variants of combined immunodeficiency (CID) or even milder antibody deficiency syndromes. Pathogenic variants that severely impair recombinase activity of RAG1/2 determine a severe combined immunodeficiency (SCID) phenotype, whereas hypomorphic variants result in leaky (partial) SCID and other immunodeficiencies. We report a patient with novel pathogenic compound heterozygous RAG2 variants that result in a CID phenotype with two distinctive characteristics: late-onset progressive hypogammaglobulinemia and highly elevated B cell count. In addition, the patient had early onset of infections, T cell lymphopenia and expansion of lymphocytes after exposure to herpes family viruses. This case highlights the importance of considering pathogenic RAG variants among patients with preserved B cell count and CID phenotype.

Familial Immune Thrombocytopenia Associated With a Novel Variant in IKZF1.

We report a novel variant in IKZF1 associated with IKAROS haploinsufficiency in a patient with familial immune thrombocytopenia (ITP). IKAROS, encoded by the IKZF1 gene, is a hematopoietic zinc-finger transcription factor that can directly bind to DNA. We show that the identified IKZF1 variant (p.His195Arg) alters a completely conserved histidine residue required for the folding of the third zinc-finger of IKAROS protein, leading to a loss of characteristic immunofluorescence nuclear staining pattern. In our case, genetic testing was essential for the diagnosis of IKAROS haploinsufficiency, of which known presentations include infections, aberrant hematopoiesis, leukemia, and age-related decrease in humoral immunity. Our family study underscores that, after infections, ITP is the second most common clinical manifestation of IKAROS haploinsufficiency.

Arthritis in Two Patients With Partial Recombination Activating Gene Deficiency.

Autoimmunity is becoming an increasingly recognized complication in patients with primary immunodeficiencies (PIDs), including a variety of combined immune deficiencies such as Recombination Activating Gene (RAG) defects. The approach to treating autoimmunity in PID patients is complex, requiring a balance between immunosuppression and susceptibility to infection. Inflammatory arthritis is a feature of immune dysregulation in many PIDs, and the optimal treatment may differ from first line therapies that usually consist of disease-modifying anti rheumatic drugs (DMARDs). An example of mechanism-based therapy of arthritis in PID uses blockade of IL-6 signaling with tocilizumab for patients with STAT 3 gain-of-function (GOF) mutation and augmented IL-6 pathway. Herein, we describe two PID cases with arthritis who were found to have defects in RAG. One patient with refractory inflammatory arthritis experienced remarkable improvement in symptoms with tocilizumab therapy. Arthritis can be a clinical feature of immune dysregulation in RAG deficiency, and tocilizumab therapy has been suggested to have utility in treatment of arthritis in RAG deficiency.

Two Unique Cases of X-linked SCID: A Diagnostic Challenge in the Era of Newborn Screening.

In the era of newborn screening (NBS) for severe combined immunodeficiency (SCID) and the possibility of gene therapy (GT), it is important to link SCID phenotype to the underlying genetic disease. In western countries, X-linked interleukin 2 receptor gamma chain (IL2RG) and adenosine deaminase (ADA) deficiency SCID are two of the most common types of SCID and can be treated by GT. As a challenge, both IL2RG and ADA genes are highly polymorphic and a gene-based diagnosis may be difficult if the variant is of unknown significance or if it is located in non-coding areas of the genes that are not routinely evaluated with exon-based genetic testing (e.g., introns, promoters, and the 5'and 3' untranslated regions). Therefore, it is important to extend evaluation to non-coding areas of a SCID gene if the exon-based sequencing is inconclusive and there is strong suspicion that a variant in that gene is the cause for disease. Functional studies are often required in these cases to confirm a pathogenic variant. We present here two unique examples of X-linked SCID with variable immune phenotypes, where IL2R gamma chain expression was detected and no pathogenic variant was identified on initial genetic testing. Pathogenic IL2RG variants were subsequently confirmed by functional assay of gamma chain signaling and maternal X-inactivation studies. We propose that such tests can facilitate confirmation of suspected cases of X-linked SCID in newborns when initial genetic testing is inconclusive. Early identification of pathogenic IL2RG variants is especially important to ensure eligibility for gene therapy.