JAK/STAT defects and immune dysregulation, and guiding therapeutic choices.

Inborn errors of immunity (IEIs) encompass a diverse spectrum of genetic disorders that disrupt the intricate mechanisms of the immune system, leading to a variety of clinical manifestations. Traditionally associated with an increased susceptibility to recurrent infections, IEIs have unveiled a broader clinical landscape, encompassing immune dysregulation disorders characterized by autoimmunity, severe allergy, lymphoproliferation, and even malignancy. This review delves into the intricate interplay between IEIs and the JAK-STAT signaling pathway, a critical regulator of immune homeostasis. Mutations within this pathway can lead to a wide array of clinical presentations, even within the same gene. This heterogeneity poses a significant challenge, necessitating individually tailored therapeutic approaches to effectively manage the diverse manifestations of these disorders. Additionally, JAK-STAT pathway defects can lead to simultaneous susceptibility to both infection and immune dysregulation. JAK inhibitors, with their ability to suppress JAK-STAT signaling, have emerged as powerful tools in controlling immune dysregulation. However, questions remain regarding the optimal selection and dosing regimens for each specific condition. Hematopoietic stem cell transplantation (HSCT) holds promise as a curative therapy for many JAK-STAT pathway disorders, but this procedure carries significant risks. The use of JAK inhibitors as a bridge to HSCT has been proposed as a potential strategy to mitigate these risks.

Interplay between epigenetic and genetic alterations in inborn errors of immunity.

Inborn errors of immunity (IEIs) comprise a variety of immune conditions leading to infections, autoimmunity, allergy, and cancer. Some IEIs have no identified mutation(s), while others with identical mutations can display heterogeneous presentations. These observations suggest the involvement of epigenetic mechanisms. Epigenetic alterations can arise from downstream activation of cellular pathways through both extracellular stimulation and genetic-associated changes, impacting epigenetic enzymes or their interactors. Therefore, we posit that epigenetic alterations and genetic defects do not exclude each other as a disease-causing etiology. In this opinion, encompassing both basic and clinical viewpoints, we focus on selected IEIs with mutations in transcription factors that interact with epigenetic enzymes. The intricate interplay between these factors offers insights into genetic and epigenetic mechanisms in IEIs.

Genotype, oxidase status, and preceding infection or autoinflammation do not affect allogeneic HCT outcomes for CGD.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections and inflammatory conditions. Hematopoietic cell transplantation (HCT) is the definitive treatment for CGD, but questions remain regarding patient selection and impact of active disease on transplant outcomes. We performed a multi-institutional retrospective and prospective study of 391 patients with CGD treated either conventionally (non-HCT) enrolled from 2004 to 2018 or with HCT from 1996 to 2018. Median follow-up after HCT was 3.7 years with a 3-year overall survival of 82% and event-free survival of 69%. In a multivariate analysis, a Lansky/Karnofsky score <90 and use of HLA-mismatched donors negatively affected survival. Age, genotype, and oxidase status did not affect outcomes. Before HCT, patients had higher infection density, higher frequency of noninfectious lung and liver diseases, and more steroid use than conventionally treated patients; however, these issues did not adversely affect HCT survival. Presence of pre-HCT inflammatory conditions was associated with chronic graft-versus-host disease. Graft failure or receipt of a second HCT occurred in 17.6% of the patients and was associated with melphalan-based conditioning and/or early mixed chimerism. At 3 to 5 years after HCT, patients had improved growth and nutrition, resolved infections and inflammatory disease, and lower rates of antimicrobial prophylaxis or corticosteroid use compared with both their baseline and those of conventionally treated patients. HCT leads to durable resolution of CGD symptoms and lowers the burden of the disease. Patients with active infection or inflammation are candidates for transplants; HCT should be considered before the development of comorbidities that could affect performance status. This trial was registered at www.clinicaltrials.gov as #NCT02082353.

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Posttransplantation late complications increase over time for patients with SCID: A Primary Immune Deficiency Treatment Consortium (PIDTC) landmark study.

BACKGROUND: The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children in the United States and Canada onto a retrospective multicenter natural history study of hematopoietic cell transplantation (HCT). OBJECTIVE: We investigated outcomes of HCT for severe combined immunodeficiency (SCID). METHODS: We evaluated the chronic and late effects (CLE) after HCT for SCID in 399 patients transplanted from 1982 to 2012 at 32 PIDTC centers. Eligibility criteria included survival to at least 2 years after HCT without need for subsequent cellular therapy. CLE were defined as either conditions present at any time before 2 years from HCT that remained unresolved (chronic), or new conditions that developed beyond 2 years after HCT (late). RESULTS: The cumulative incidence of CLE was 25% in those alive at 2 years, increasing to 41% at 15 years after HCT. CLE were most prevalent in the neurologic (9%), neurodevelopmental (8%), and dental (8%) categories. Chemotherapy-based conditioning was associated with decreased-height z score at 2 to 5 years after HCT (P < .001), and with endocrine (P < .001) and dental (P = .05) CLE. CD4 count of ≤500 cells/µL and/or continued need for immunoglobulin replacement therapy >2 years after transplantation were associated with lower-height z scores. Continued survival from 2 to 15 years after HCT was 90%. The presence of any CLE was associated with increased risk of late death (hazard ratio, 7.21; 95% confidence interval, 2.71-19.18; P < .001). CONCLUSION: Late morbidity after HCT for SCID was substantial, with an adverse impact on overall survival. This study provides evidence for development of survivorship guidelines based on disease characteristics and treatment exposure for patients after HCT for SCID.

Outcomes following treatment for ADA-deficient severe combined immunodeficiency: a report from the PIDTC.

Adenosine deaminase (ADA) deficiency causes ∼13% of cases of severe combined immune deficiency (SCID). Treatments include enzyme replacement therapy (ERT), hematopoietic cell transplant (HCT), and gene therapy (GT). We evaluated 131 patients with ADA-SCID diagnosed between 1982 and 2017 who were enrolled in the Primary Immune Deficiency Treatment Consortium SCID studies. Baseline clinical, immunologic, genetic characteristics, and treatment outcomes were analyzed. First definitive cellular therapy (FDCT) included 56 receiving HCT without preceding ERT (HCT); 31 HCT preceded by ERT (ERT-HCT); and 33 GT preceded by ERT (ERT-GT). Five-year event-free survival (EFS, alive, no need for further ERT or cellular therapy) was 49.5% (HCT), 73% (ERT-HCT), and 75.3% (ERT-GT; P < .01). Overall survival (OS) at 5 years after FDCT was 72.5% (HCT), 79.6% (ERT-HCT), and 100% (ERT-GT; P = .01). Five-year OS was superior for patients undergoing HCT at <3.5 months of age (91.6% vs 68% if ≥3.5 months, P = .02). Active infection at the time of HCT (regardless of ERT) decreased 5-year EFS (33.1% vs 68.2%, P < .01) and OS (64.7% vs 82.3%, P = .02). Five-year EFS (90.5%) and OS (100%) were best for matched sibling and matched family donors (MSD/MFD). For patients treated after the year 2000 and without active infection at the time of FDCT, no difference in 5-year EFS or OS was found between HCT using a variety of transplant approaches and ERT-GT. This suggests alternative donor HCT may be considered when MSD/MFD HCT and GT are not available, particularly when newborn screening identifies patients with ADA-SCID soon after birth and before the onset of infections. This trial was registered at www.clinicaltrials.gov as #NCT01186913 and #NCT01346150.

NOD2 Polymorphisms May Direct a Crohn Disease Phenotype in Patients With Very Early-Onset Inflammatory Bowel Disease.

NOD2/CARD15 was the first susceptibility gene recognized for adult-onset Crohn's (or Crohn) disease (CD). Recessive inheritance of NOD2 polymorphisms has been implicated as a mechanistic driver of pediatric-onset CD. In patients with very early-onset inflammatory bowel disease (VEO-IBD), however, the clinical relevance of NOD2 polymorphisms has not been fully established. Ten VEO-IBD patients with NOD2 polymorphisms ( NOD2 +) were compared to 16 VEO-IBD patients without genetic variants in NOD2 or any other VEO-IBD susceptibility genes ( NOD2 -). The majority of NOD2 + patients exhibited a CD-like phenotype (90%), linear growth impairment (90%), and arthropathy (60%), all of which were significantly more common than in the NOD2 - group ( P = 0.037, P = 0.004, P = 0.026, respectively). We propose that the presence of NOD2 polymorphisms in patients with VEO-IBD might confer a CD-like phenotype, linear growth impairment, and arthropathy. These findings should be validated in larger cohorts and may guide precision medicine for patients with VEO-IBD in the future.

STAT5B restrains human B-cell differentiation to maintain humoral immune homeostasis.

BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

Diagnostic Modalities in Primary Immunodeficiency.

As the field of inborn errors of immunity expands, providers continually update and fine-tune their diagnostic approach and selection of testing modalities to increase diagnostic accuracy. Here, we first describe a mechanistic consideration of laboratory testing, highlighting both benefits and drawbacks of currently clinically available testing modalities. Next, we provide methods in evaluation of patients presenting with concern for inborn errors of immunity as defined by the International Union of Immunological Societies 2019 phenotypic categories: primary antibody deficiencies, cellular and humoral immune deficiency, disorders of the innate immune system, and syndrome-associated and primary immune regulation disorders (PIRDs). Using the suggested approach in this paper as a roadmap highlights the importance of thorough history taking and physical examination as the foundation to guide further diagnostic tests. This is followed by enumeration and functional testing. Finally, to determine the underlying molecular etiology-specific genetic panels, chromosomal microarrays, and broad genetic testing (whole exome sequencing or whole genome sequencing) are available.

Plasma Metagenomic Sequencing Expedites Diagnosis of Disseminated BCG in an Infant With IKBKB Mutation.

BACKGROUND: Infants with inborn errors of immunity (IEI), born in countries where Bacillus-Calmette-Guerin (BCG) vaccination is recommended at birth, are at risk of developing infectious complications following vaccination. A prompt diagnosis of disseminated BCG infection in these infants is essential, as many will require stem cell transplantation (SCT) for the immunologic cure. In patients with IEI, the mortality risk from disseminated mycobacterial infection is high, both before and following SCT. METHODS: A 7-month-old Qatari infant with an IEI, homozygous IKBKB gene mutation, was evaluated at our institution for SCT. He had a history of recurrent pneumonias, but pretransplant evaluation revealed negative cultures from bronchoalveolar fluid, blood and urine. At 8 months of age, the infant developed skin nodules of unclear etiology, prompting additional evaluation. RESULTS: Given his profound immunosuppression and receipt of broad-spectrum antimicrobials, plasma metagenomic next-generation sequencing (mNGS) was obtained and identified Mycobacterium tuberculosis complex within 72 hours. A skin biopsy was performed, and antimycobacterial therapy was initiated. Mycobacterium bovis-BCG was confirmed from cultures 3 weeks later. Treatment was complicated by elevated serum liver transaminases and aminoglycoside-associated high-frequency hearing loss. The infant completed 14 months of treatment from engraftment. Evaluation for active BCG infection after SCT was negative. CONCLUSION: In an infant with a unique IEI, plasma mNGS provided the first diagnosis of disseminated BCG infection. We believe that early initiation of antimycobacterial treatment improved the infant's clinical outcome. Plasma mNGS testing should be considered as a noninvasive screen for infectious pathogens in children with IEIs before SCT.

STAT3 gain-of-function syndrome.

STAT3 gain-of-function (GOF) syndrome is a multi-organ primary immune regulatory disorder characterized by early onset autoimmunity. Patients present early in life, most commonly with lymphoproliferation, autoimmune cytopenias, and growth delay. However, disease is often progressive and can encompass a wide range of clinical manifestations such as: enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, and rarely neurologic disease, vasculopathy, and malignancy. Treatment of the autoimmune and immune dysregulatory features of STAT3-GOF patients relies heavily on immunosuppression and is often challenging and fraught with complications including severe infections. Defects in the T cell compartment leading to effector T cell accumulation and decreased T regulatory cells may contribute to autoimmunity. While T cell exhaustion and apoptosis defects likely contribute to the lymphoproliferative phenotype, no conclusive correlations are yet established. Here we review the known mechanistic and clinical characteristics of this heterogenous PIRD.

An ELF4 hypomorphic variant results in NK cell deficiency.

NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.

A Toolkit and Framework for Optimal Laboratory Evaluation of Individuals with Suspected Primary Immunodeficiency.

Knowledge related to the biology of inborn errors of immunity and associated laboratory testing methods continues to expand at a tremendous rate. Despite this, many patients with inborn errors of immunity suffer for prolonged periods of time before identification of their underlying condition, thereby delaying appropriate care. Understanding that test selection and optimal evaluation for patients with recurrent infections or unusual patterns of inflammation can be unclear, we present a document that distills relevant clinical features of immunologic disease due to inborn errors of immunity and related appropriate and available test options. This document is intended to serve the practicing clinical immunologist and, in turn, patients by describing best available test options for initial and expanded immunologic evaluations across the disease spectrum. Our goal is to demystify the process of evaluating patients with suspected immune dysfunction and to enable more rapid and accurate diagnosis of such individuals.

Case Report: Secondary Hemophagocytic Lymphohistiocytosis With Disseminated Infection in Chronic Granulomatous Disease-A Serious Cause of Mortality.

Chronic granulomatous disease (CGD) is a primary immune deficiency due to defects in phagocyte respiratory burst leading to severe and life-threatening infections. Patients with CGD also suffer from disorders of inflammation and immune dysregulation including colitis and granulomatous lung disease, among others. Additionally, patients with CGD may be at increased risk of systemic inflammatory disorders such as hemophagocytic lymphohistiocytosis (HLH). The presentation of HLH often overlaps with symptoms of systemic inflammatory response syndrome (SIRS) or sepsis and therefore can be difficult to identify, especially in patients with a primary immune deficiency in which incidence of infection is increased. Thorough evaluation and empiric treatment for bacterial and fungal infections is necessary as HLH in CGD is almost always secondary to infection. Simultaneous treatment of infection with anti-microbials and inflammation with immunosuppression may be needed to blunt the hyperinflammatory response in secondary HLH. Herein, we present a series of X-linked CGD patients who developed HLH secondary to or with concurrent disseminated CGD-related infection. In two patients, CGD was a known diagnosis prior to development of HLH and in the other two CGD was diagnosed as part of the evaluation for HLH. Concurrent infection and HLH were fatal in three; one case was successfully treated, ultimately receiving hematopoietic stem cell transplantation. The current literature on presentation, diagnosis, and treatment of HLH in CGD is reviewed.