Treatment with Elapegademase Restores Immunity in Infants with Adenosine Deaminase Deficient Severe Combined Immunodeficiency.

PURPOSE: Patients with adenosine deaminase 1 deficient severe combined immunodeficiency (ADA-SCID) are initially treated with enzyme replacement therapy (ERT) with polyethylene glycol-modified (PEGylated) ADA while awaiting definitive treatment with hematopoietic stem cell transplant (HSCT) or gene therapy. Beginning in 1990, ERT was performed with PEGylated bovine intestinal ADA (ADAGEN®). In 2019, a PEGylated recombinant bovine ADA (Revcovi®) replaced ADAGEN following studies in older patients previously treated with ADAGEN for many years. There are limited longitudinal data on ERT-naïve newborns treated with Revcovi. METHODS: We report our clinical experience with Revcovi as initial bridge therapy in three newly diagnosed infants with ADA-SCID, along with comprehensive biochemical and immunologic data. RESULTS: Revcovi was initiated at twice weekly dosing (0.2 mg/kg intramuscularly), and monitored by following plasma ADA activity and the concentration of total deoxyadenosine nucleotides (dAXP) in erythrocytes. All patients rapidly achieved a biochemically effective level of plasma ADA activity, and red cell dAXP were eliminated within 2-3 months. Two patients reconstituted B-cells and NK-cells within the first month of ERT, followed by naive T-cells one month later. The third patient reconstituted all lymphocyte subsets within the first month of ERT. One patient experienced declining lymphocyte counts with improvement following Revcovi dose escalation. Two patients developed early, self-resolving thrombocytosis, but no thromboembolic events occurred. CONCLUSION: Revcovi was safe and effective as initial therapy to restore immune function in these newly diagnosed infants with ADA-SCID, however, time course and degree of reconstitution varied. Revcovi dose may need to be optimized based on immune reconstitution, clinical status, and biochemical data.

Case Report: Profound newborn leukopenia related to a novel RAC2 variant.

We report the case of a 1-week-old male born full-term, who had two inconclusive severe combined immunodeficiency (SCID) newborn screens and developed scalp cellulitis and Escherichia coli bacteremia. He did not pass early confirmatory hearing screens. Initial blood counts and lymphocyte flow cytometry revealed profound neutropenia and lymphopenia with a T-/B-/NK- phenotype. Red blood cell adenosine deaminase 1 activity was within normal limits. A presumptive diagnosis of reticular dysgenesis was considered. Granulocyte colony-stimulating factor was started, but there was no improvement in neutrophil counts. Subsequent lymphocyte flow cytometry at around 4 weeks of age demonstrated an increase in T-, B- and NK-cell numbers, eliminating suspicion for SCID and raising concern for congenital neutropenia and bone marrow failure syndromes. Genetic testing revealed a novel variant in RAC2 [c.181C>A (p.Gln61Lys)] (Q61K). RAC2, a Ras-related GTPase, is the dominant RAC protein expressed in hematopoietic cells and is involved with various downstream immune-mediated responses. Pathogenic RAC2 variants show significant phenotypic heterogeneity (spanning from neutrophil defects to combined immunodeficiency) across dominant, constitutively activating, dominant activating, dominant negative, and autosomal recessive subtypes. Given the identification of a novel variant, functional testing was pursued to evaluate aberrant pathways described in other RAC2 pathogenic variants. In comparison to wild-type RAC2, the Q61K variant supported elevated superoxide production under both basal and PMA-stimulated conditions, increased PAK1 binding, and enhanced plasma membrane ruffling, consistent with other dominant, constitutively active mutations. This case highlights the diagnostic challenge associated with genetic variants identified via next-generation sequencing panels and the importance of functional assays to confirm variant pathogenicity.

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.

Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. OBJECTIVE: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. METHODS: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. RESULTS: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. CONCLUSION: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.

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.

Bone marrow transplantation for CVID-like humoral immune deficiency associated with red cell aplasia.

Patients with common variable immunodeficiency (CVID) have a higher incidence of autoimmune disease, which may mark the disease onset; however, anemia secondary to pure red cell aplasia is an uncommon presenting feature. Here, we describe a case of CVID-like humoral immune deficiency in a child who initially presented with red cell aplasia and ultimately developed progressive bone marrow failure. Although bone marrow transplantation (BMT) has been associated with high mortality in CVID, our patient was successfully treated with a matched sibling BMT and engrafted with >98% donor chimerism and the development of normal antibody titers to diphtheria and tetanus toxoids.

Dominant Splice Site Mutations in PIK3R1 Cause Hyper IgM Syndrome, Lymphadenopathy and Short Stature.

The purpose of this research was to use next generation sequencing to identify mutations in patients with primary immunodeficiency diseases whose pathogenic gene mutations had not been identified. Remarkably, four unrelated patients were found by next generation sequencing to have the same heterozygous mutation in an essential donor splice site of PIK3R1 (NM_181523.2:c.1425 + 1G > A) found in three prior reports. All four had the Hyper IgM syndrome, lymphadenopathy and short stature, and one also had SHORT syndrome. They were investigated with in vitro immune studies, RT-PCR, and immunoblotting studies of the mutation's effect on mTOR pathway signaling. All patients had very low percentages of memory B cells and class-switched memory B cells and reduced numbers of naïve CD4+ and CD8+ T cells. RT-PCR confirmed the presence of both an abnormal 273 base-pair (bp) size and a normal 399 bp size band in the patient and only the normal band was present in the parents. Following anti-CD40 stimulation, patient's EBV-B cells displayed higher levels of S6 phosphorylation (mTOR complex 1 dependent event), Akt phosphorylation at serine 473 (mTOR complex 2 dependent event), and Akt phosphorylation at threonine 308 (PI3K/PDK1 dependent event) than controls, suggesting elevated mTOR signaling downstream of CD40. These observations suggest that amino acids 435-474 in PIK3R1 are important for its stability and also its ability to restrain PI3K activity. Deletion of Exon 11 leads to constitutive activation of PI3K signaling. This is the first report of this mutation and immunologic abnormalities in SHORT syndrome.

Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta.

Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK alpha and zeta synergistically promote T cell maturation in the thymus. Absence of both DGKalpha and zeta (DGKalpha(-/-)zeta(-/-)) results in a severe decrease in the number of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKalpha(-/-)zeta(-/-) mice. The developmental blockage in DGKalpha(-/-)zeta(-/-) mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.

Ceramide regulates SR protein phosphorylation during adenoviral infection.

In this study, we show that adenoviral infection induced accumulation of the sphingolipid ceramide in a dose- and time-dependent manner. This accumulation preceded cell lysis, occurred in the absence of biochemical evidence of apoptosis, and was derived from de novo synthesis of ceramide. An adenovirus mutant that lacks the adenovirus death protein (ADP) produced ceramide accumulation in the absence of cell lysis. This suggested that ceramide accumulation was either driven by adenovirus or was a cellular stress response but was unlikely a result of cell death. The use of inhibitors of ceramide synthesis resulted in a significant delay in cell lysis, suggesting that ceramide was necessary for the lytic phase of the infection. Serine/arginine-rich (SR) proteins were dephosphorylated during the late phase of the viral cycle, and inhibitors of ceramide synthesis reversed this. These findings suggest that adenovirus utilizes the ceramide pathway to regulate SR proteins during infection.