Characterization of a new WHIM syndrome mutant reveals mechanistic differences in regulation of the chemokine receptor CXCR4.

WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild-type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild-type CXCR4 including agonist-promoted calcium flux and extracellular-signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, ß-arrestin binding, and endocytosis of S339fs5 and R334X compared with wild-type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared with that of R334X and wild-type CXCR4. In contrast to wild-type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild-type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, which promotes enhanced signaling, reduced phosphorylation, ß-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment.

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.

Long-term outcome of LRBA deficiency in 76 patients after various treatment modalities as evaluated by the immune deficiency and dysregulation activity (IDDA) score.

BACKGROUND: Recent findings strongly support hematopoietic stem cell transplantation (HSCT) in patients with severe presentation of LPS-responsive beige-like anchor protein (LRBA) deficiency, but long-term follow-up and survival data beyond previous patient reports or meta-reviews are scarce for those patients who do not receive a transplant. OBJECTIVE: This international retrospective study was conducted to elucidate the longitudinal clinical course of patients with LRBA deficiency who do and do not receive a transplant. METHOD: We assessed disease burden and treatment responses with a specially developed immune deficiency and dysregulation activity score, reflecting the sum and severity of organ involvement and infections, days of hospitalization, supportive care requirements, and performance indices. RESULTS: Of 76 patients with LRBA deficiency from 29 centers (median follow-up, 10 years; range, 1-52), 24 underwent HSCT from 2005 to 2019. The overall survival rate after HSCT (median follow-up, 20 months) was 70.8% (17 of 24 patients); all deaths were due to nonspecific, early, transplant-related mortality. Currently, 82.7% of patients who did not receive a transplant (43 of 52; age range, 3-69 years) are alive. Of 17 HSCT survivors, 7 are in complete remission and 5 are in good partial remission without treatment (together, 12 of 17 [70.6%]). In contrast, only 5 of 43 patients who did not receive a transplant (11.6%) are without immunosuppression. Immune deficiency and dysregulation activity scores were significantly lower in patients who survived HSCT than in those receiving conventional treatment (P = .005) or in patients who received abatacept or sirolimus as compared with other therapies, and in patients with residual LRBA expression. Higher disease burden, longer duration before HSCT, and lung involvement were associated with poor outcome. CONCLUSION: The lifelong disease activity, implying a need for immunosuppression and risk of malignancy, must be weighed against the risks of HSCT.

Juvenile Dermatomyositis: New Clues to Diagnosis and Therapy.

PURPOSE OF REVIEW: To identify clues to disease activity and discuss therapy options. RECENT FINDINGS: The diagnostic evaluation includes documenting symmetrical proximal muscle damage by exam and MRI, as well as elevated muscle enzymes-aldolase, creatine phosphokinase, LDH, and SGOT-which often normalize with a longer duration of untreated disease. Ultrasound identifies persistent, occult muscle inflammation. The myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) are associated with specific disease course variations. Anti-NXP-2 is found in younger children and is associated with calcinosis; anti-TIF-1γ+ juvenile dermatomyositis has a longer disease course. The diagnostic rash-involving the eyelids, hands, knees, face, and upper chest-is the most persistent symptom and is associated with microvascular compromise, reflected by loss of nailfold (periungual) end row capillaries. This loss is associated with decreased bioavailability of oral prednisone; the bioavailability of other orally administered medications should also be considered. At diagnosis, at least 3 days of intravenous methyl prednisolone may help control the HLA-restricted and type 1/2 interferon-driven inflammatory process. The requirement for avoidance of ultraviolet light exposure mandates vitamin D supplementation. SUMMARY: This often chronic illness targets the cardiovascular system; mortality has decreased from 30 to 1-2% with corticosteroids. New serological biomarkers indicate occult inflammation: ↑CXCL-10 predicts a longer disease course. Some biologic therapies appear promising.

Neutrophil Extracellular Traps in Tissue and Periphery in Juvenile Dermatomyositis.

OBJECTIVE: Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). This study was undertaken to investigate the role of neutrophils in juvenile dermatomyositis (JDM). METHODS: Electron microscopy was used to identify neutrophils in tissue. NETs were also imaged using fluorescence microscopy and quantified using a myeloperoxidase-DNA enzyme-linked immunosorbent assay (ELISA) in plasma obtained from healthy children (n = 20), disease controls (n = 29), JDM patients (n = 66), and JDM patients with history of calcifications (n = 20). Clinical data included disease activity scores and complement C4 levels. Levels of immune complexes (ICs) and calprotectin were analyzed using ELISA. RESULTS: Using electron microscopy, neutrophils were found to infiltrate affected muscle tissue, engulfing deposited calcium crystals. Uptake of the crystals led to neutrophil activation (P < 0.01) and subsequent phosphatidylinositol 3-kinase- and NADPH oxidase-dependent but peptidylarginine deiminase 4-independent formation of NETs, which contained mitochondrial DNA (P < 0.05), as confirmed in vivo (P < 0.001) and in vitro (P < 0.01). Peripheral NET levels were associated with calcinosis (P = 0.01), ICs (P = 0.008), and interleukin-8 levels (P = 0.004). Children with JDM had impaired NET clearance (P = 0.01), associated with autoantibody profiles including melanoma differentiation-associated protein 5 (P = 0.005), and depressed complement C4 levels (r = -0.72, P = 0.002). Furthermore, children with JDM showed evidence of neutrophil activation, with elevated levels of peroxidase activity (P = 0.02) and calprotectin (P < 0.01), which were associated with disease activity (P = 0.007), and dyslipidemia (odds ratio 4.7, P < 0.05). CONCLUSION: We found novel mechanisms of both calcium crystal-mediated neutrophil activation and cell death in JDM pathophysiology. Targeting this pathway may reduce the frequency and extent of calcinosis, as well as prevent long-term development of comorbidities, including atherosclerosis.

Rituximab-associated Hypogammaglobulinemia in pediatric patients with autoimmune diseases.

BACKGROUND: Despite the increased use of rituximab in treating pediatric patients with autoimmune diseases in the last decade, there are limited data on rituximab safety in those subjects who have a developing immune system. The objective of this study is to determine the prevalence of hypogammaglobulinemia in children with autoimmune disease receiving rituximab within the first three years of treatment in the pediatric rheumatology clinic at a tertiary care center. METHODS: We conducted a retrospective chart review of 63 pediatric subjects who received rituximab for the treatment of their autoimmune disease. Immunoglobulin gamma (IgG) levels, immunosuppressive medication and the need for immunoglobulin replacement therapy were evaluated. Hypogammaglobulinemia was defined as a serum IgG level less than two standard deviations below the mean for age-matched healthy controls. RESULTS: Twenty-eight patients (44%) were found to have hypogammaglobulinemia. Hypogammaglobulinemia occurred within the first six months of rituximab treatment in the majority of patients (22 out of 28). The occurrence of hypogammaglobulinemia varied based on the rituximab indication: 46% pediatric Systemic Lupus Erythematosus (SLE), 71% autoimmune CNS disease, 60% ANCA vasculitis, and 12% in the miscellaneous group. Autoimmune CNS disease had more severe hypogammaglobulinemia, more persistent and was associated with more frequent or severe infections. Three patients with autoimmune CNS disease and one with SLE were given IgG replacement therapy to prevent recurrent or severe infections. CONCLUSIONS: The prevalence of hypogammaglobulinemia in rituximab treated children with autoimmune disease seems to be higher than published data for adults, especially for children with autoimmune CNS disease. The onset of hypogammaglobulinemia is usually within six months of initiation of rituximab therapy. We recommend: 1) obtaining an IgG level prior to starting rituximab; 2) close monitoring for hypogammaglobulinemia after the use of rituximab in pediatric patients and 3) early institution of immunoglobulin replacement therapy if patients develop recurrent infections.