Search

Autosomal dominant gain of function mutations in the gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, CMV and/or EBV viremia and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. Here we describe three patients in two families who were diagnosed with HIGM at a young age and were recently found to carry heterozygous mutations in PIK3CD. These patients had an abnormal circulating B cell distribution featuring a preponderance of early transitional (T1) B cells and plasmablasts. When stimulated in vitro, PIK3CD mutated B cells were able to secrete class-switched immunoglobulins. This finding implies that the patients' elevated serum IgM levels were unlikely a product of an intrinsic B cell functional inability to class switch. All three patients developed malignant lymphoproliferative syndromes that were not associated with EBV. Thus, we identified a novel subset of patients with PIK3CD mutations associated with HIGM, despite indications of preserved in vitro B cell class switch recombination, as well as susceptibility to non-EBV-associated malignancies.

Epstein-Barr virus (EBV) infection is usually asymptomatic and latency occurs in B cells. We studied three patients with NK cell chronic active EBV (CAEBV) disease and EBV hepatitis. All patients had an increased number of NK cells, and high levels of EBV in the blood, with EBV predominantly in NK cells; two patients who were tested had an NK cell receptor repertoire consistent with immature cells. All three patients had increased phosphorylation of Akt and ribosomal protein S6 in NK cells and a marked increase in STAT1; two of the patients had increased phosphorylation of STAT1 in their T cells, NK cells, and monocytes. Treatment of one of these patients with sirolimus, an mTOR inhibitor, reduced phosphorylation of S6 in the patient’s T and B cells, but not in her NK cells and did not reduce the level of NK cells or EBV DNA in the peripheral blood. The increased STAT1 activity observed in two of the patients was reduced to normal when the cells were treated with JAK inhibitors in vitro. Unlike other patients with STAT1 gain-of-function (GOF) mutations, no mutations were identified in STAT1 or STAT1 regulatory proteins. Patients with T or B cell CAEBV also had increased phosphorylation of Akt and S6, but not STAT1. In summary, the increase in Akt and S6 phosphorylation, and the increase in STAT1 protein, as well as immature NK cells in our NK cell patients describe a novel phenotype in NK cell CAEBV disease.

Abstract 1033 Chronic, nonmalignant, noninfectious lymphadenopathy associated with splenomegaly and elevated circulating TCRα β+, CD4-/CD8- Double Negative T (DNT) cells are the hallmarks of the apoptosis disorder called autoimmune lymphpoproliferative syndrome (ALPS). ALPS is most frequently associated with defects in the FAS gene affecting the extrinsic pathway of apoptosis. Patients with ALPS-like syndromes caused by somatic mutations in NRAS and KRAS affecting the intrinsic (mitochondrial) pathway of apoptosis are recently recognized and currently classified separately as ALPS-related apoptosis disorders. Reduced peripheral blood lymphocyte apoptosis after withdrawal of IL-2 is a key feature of their diagnosis. They present with autoimmune phenomena, massive splenomegaly, modest lymphadenopathy, and normal or only marginally elevated DNT cells. Concomitant abnormalities of the myeloid compartment, with an absolute monocytosis with or without concurrent leukocytosis and neutrophilia, mimic juvenile or chronic myelomonocytic leukemia in otherwise asymptomatic younger patients. These patients are classified as RAS-associated autoimmune leukoproliferative disorder (RALD) (Oliveira et al, Blood 2010 and Niemela et al, Blood 2011). Here we summarize the clinical features in our cohort of 10 patients with RALD. They are currently 4–54 years old (M:F ratio 5:5) and all are alive except one patient who died at age 13. Their median age of disease onset was 2 years; while 50% presented with symptoms at or before age of 1year; one patient (380.1) presented with symptoms as an adult (age 36) with hypersplenism and leukocytosis. All presented with significantly enlarged spleens and 3/10 have undergone splenectomy. All 5 patients with NRAS mutation are male in our cohort, whereas all 5 patients with KRAS mutation are female and three of them presented with ecchymotic periarthritis. As a group these patients have an absolute monocytosis (median 1.7 K/uL, range 0.8–49.7 K/uL), increased circulating B lymphocytes (median 743, range 497–5970) and elevated serum IgG (median 1960 mg/dL, range 1310–2450 mg/dL). Monocytes appear to express unique activation markers in the two patients studied to date. Monocytes from patients 380.1 and 381.1 showed atypical expression of CD56 and 40% of them from patient 381.1 were atypical with an immunophenotype consistent with dendritic cells (non-classical monocytes) that expressed CD16, bright CD45, bright HLA-DR, dim CD14, and dim CD36. Bone marrow biopsies performed on a subset of patients show hypercellular marrow with monocytosis, granulocytic hyperplasia, and no increase in blasts. Atypical megakaryocytes and myeloid maturation asynchrony have been noted in some, without overt dysplasia. Autoantibodies noted in these patients include DAT, lupus anticoagulant, ACA IgG, ACA IgM, and ANA. Importantly cytokine (IL-2) withdrawal assay showed diminished apoptosis in each of these patients while GM-CSF hypersensitivity assay was positive in the 3/5 patients tested. Only one patient in our cohort (Patient 58.1) with an NRAS mutation has developed B cell lymphoma at age 44 and is alive 10 years after receiving chemotherapy. RALD should be considered in patients with unexplained splenomegaly, autoimmune phenomena and persistent monocytosis. Diligent clinical and laboratory assessments and long-term follow up will help us understand the natural history of this rare leukoproliferative disorder and distinguish it from myelodysplastic conditions like JMML and CMML. Disclosures: No relevant conflicts of interest to declare.

Regulators of G-protein Signaling (RGS) proteins negatively regulate GPCRs by accelerating the GTPase activity of Gα. In recent years, non-GPCR-related functions have also been described for these proteins. We found previously that RGS13 was enriched in mast cells and inhibited anaphylaxis in mice by suppressing IgE-mediated mast cell degranulation. We therefore examined the transcriptional regulation of RGS13 in mast cells. We confirmed the transcriptional start site (TSS) by 5’ RACE in cDNA from the human mast cell line LAD2 and demonstrated promoter activity in a ~ 1 kb element upstream of exon 1 using luciferase reporter assays. We also identified 2 potential p53 response elements (RE) in the region 2 kb upstream of the TSS by bioinformatic analysis. Overexpression and shRNA-mediated knockdown of endogenous p53 in LAD2 cells revealed an inverse relationship between p53 levels and RGS13 expression. Endogenous p53 in these cells bound exclusively to oligonucleotides containing the p53 RE most upstream of the TSS. Cells expressing double-stranded oligonucleotides containing this site or a p53 consensus site had enhanced promoter activity. These studies indicate that p53 suppresses RGS13 transcription in mast cells. Thus, upregulation of p53 activity in mast cells exposed to stress could reduce the threshold for degranulation as a result of decreased RGS13 expression.