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Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.

TO THE EDITOR: GATA2 encodes a zinc-finger transcription factor that is required for proliferation and survival of hematopoietic stem cells.[1][1] First described in 2011, germline heterozygous mutations in GATA2 lead to haploinsufficiency[2][2] and are associated with bone marrow failure

Deficiency of adenosine deaminase 2 (DADA2) is caused by biallelic deleterious mutations in CECR1 DADA2 results in variable autoinflammation and vasculopathy (recurrent fevers, livedo reticularis, polyarteritis nodosa, lacunar ischemic strokes and intracranial hemorrhages), immunodeficiency and bone marrow failure. TNF-α blockade is the treatment of choice for the autoinflammation and vascular manifestations. Hematopoietic stem cell transplantation (HSCT) represents a potential definitive treatment. We present a cohort of 14 patients from 6 countries who received HSCT for DADA2. Indication for HSCT was bone marrow dysfunction or immunodeficiency. Six of 14 patients had vasculitis pre-HSCT. The median age at HSCT was 7.5 years. Conditioning regimens were myeloablative (9), and reduced intensity (5). Donors were HLA-matched sibling (n=1), HLA-matched unrelated (n=9), HLA-mismatched unrelated (n=3), and HLA haploidentical sibling (n=1). All patients are alive and well with no new vascular events and resolution of hematological and immunological phenotype at a median follow-up of 18mo (range 5mo to 13yr). Plasma ADA2 enzyme activity normalized in those tested post-HSCT (7/7), as early as D+14 (myeloid engraftment). Post-HSCT hematological autoimmunity (cytopenias) was reported in 4 patients, acute graft versus host disease (GvHD) grade 1 in 2, grade 2 in 3 and grade 3-4 in 1, and moderate chronic GvHD in 1 patient. In conclusion, in 14 patients, HSCT was an effective and definitive treatment for DADA2. ispartof: Blood vol:130 issue:24 pages:2682-2688 ispartof: location:United States status: published

Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes including systemic infections, lymphedema, cytopenias, myelodysplasia, and a high risk of developing myeloid leukemias. GATA2 deficiency is recognized as a major MDS predisposition syndrome, germline GATA2 mutations are found in 7% of primary MDS cases in children and adolescents. Monosomy 7 and trisomy 8 are frequent chromosomal abnormalities in GATA2 deficiency and features of disease prognosis and malignant transformation. GATA2 mutations mostly affect two zinc finger domains and broadly classify into three major categories: missense, null, and regulatory region mutations. Profound monocytopenia, B-cell and NK-cell lymphopenias, and dendritic cell deficiency are specific for GATA2 deficiency. Absence of multi-lymphoid progenitor and decreased granulocyte -macrophage progenitors occur early in disease. There are limited studies of gene expression profiles in GATA2 deficiency and these usually in bulk populations, partly due to the paucity of cells in patients. How GATA2 deficiency negatively affects hematopoiesis, especially the preferential loss of specific lineages, is poorly understood. We performed single-cell RNA sequencing of sorted bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) from eight GATA2 deficiency patients, who had various well characterized pathogenic GATA2 mutations and clinically manifest myelodysplasia (Fig A). We characterized transcriptomes in lineages, computationally defined cells with chromosomal abnormalities, and described gene expression of these cells. We first used gene expression signatures of single HSPCs from healthy donors to impute identity of stem cells or progenitors of erythroid/megakaryocytic (MEP), myeloid and lymphoid lineages, and then proceeded to reconstruct hematopoietic differentiation by pseudotemporal ordering. Mapping patients' cells onto normal hematopoiesis, we observed marked deficiency in granulocyte/monocyte/lymphocyte differentiation and relatively preserved MEP (Fig B, top). Based on signature genes of each differentiated lineage, we computed a score of gene expression to corresponding lineage in individual patient and found a significantly lower score for granulocyte/monocyte and B cell progenitors in patients, MEP scores tended to be higher in patients without significance (Fig B, bottom). Upregulated genes in patients' HSPCs related to erythroid differentiation, and cell cycle and proliferation (Fig C), while downregulated gene sets were highly enriched in immune responses (Fig D). Differential co-expressed genes were defined by weighted correlation network analysis; they were enriched in immune response and DNA repair (genes exhibited higher correlation in MTOR and T cell receptor signaling pathway in patients [Fig E]). We observed differential effects of GATA2 mutations in distinct lineages, which could only be resolved with the single cell method in rare cell populations. In HSCs, upregulated genes were related to apoptosis and downregulated genes were involved in maintenance of quiescence and cell cycle; there was increased expression of erythroid/megakaryocytic priming programs and decreased expression of lymphoid priming programs. We also observed lower GATA2 expression levels at initial stages of hematopoiesis, likely contributing to disturbed stem cell homeostasis in GATA2 deficiency. DNA repair genes were downregulated in trisomy 8 cells, possibly rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetics had defects in multi-lineage differentiation and cell cycle. In conclusion, germline GATA2 mutations modulate gene expression and change gene coexpression patterns. Distinct lineages show different transcriptional profiles resulting from GATA2 mutations. The prominent deficiency in myeloid/lymphoid lineages in GATA2 deficiency is partly due to expression of aberrant gene programs in HSCs prior to lineage commitment. Disclosures No relevant conflicts of interest to declare.

Coding and regulatory human GATA2 mutations that deregulate protein expression and/or function cause immunodeficiency that often progresses to MDS/AML (McReynolds et al., 2018). In the mouse, decreased GATA2 expression impairs hematopoietic stem/progenitor cell (HSPC) genesis and function (de Pater et al., 2013; Gao et al., 2013; Tsai et al., 1994). While prior studies demonstrated Gata2 +9.5 and -77 enhancers are essential for HSC emergence (+9.5) and/or progenitor cell fate (+9.5 and -77) (Johnson et al., 2012; Johnson et al., 2015; Mehta et al., 2017) and hematopoietic regeneration (+9.5) (Soukup et al., 2019), the mechanisms mediating these processes are not completely established. The -77 enhancer is required for fetal liver progenitors to undergo erythroid, megakaryocytic, granulocytic and monocytic differentiation. By contrast, progenitors with a -77 homozygous deletion (-77-/-) exhibit a predominant monocytic cell fate and generate macrophages ex vivo (Johnson et al., 2015). Using multiomic and single-cell strategies, we asked how this enhancer orchestrates a balance between fate-promoting and -suppressing circuitry in cell populations and single cells. Quantitative proteomics was conducted to discover the -77-regulated protein ensemble conferring multiple fates in a myeloid progenitor population [Common Myeloid Progenitor (CMP) and Granulocyte-Monocyte Progenitor (GMP)] from E14.5 fetal liver of -77+/+ and -77-/- mouse embryos. -77-/- progenitors exhibited decreased levels of GATA2 (4.7-fold) and proteins generated from GATA2 target genes (GATA1: 51-fold; HDC: 52-fold). The 202 proteins upregulated in -77-/- progenitors highlighted immune and inflammatory mechanisms, while the 232 downregulated proteins were linked to erythroid, megakaryocyte and granulocyte biology, indicative of loss of these fate potentials. Innate immune machinery was upregulated in -77-/- vs. -77+/+ progenitors, including interferon (IFN) signaling pathway components such as the IFN-inducible transcription factor and critical monocytic differentiation determinant Interferon Regulatory Factor 8 (IRF8; 2.7 fold higher) (Kurotaki et al., 2013) and diverse pathogen sensors. Expressing GATA2 at physiological levels in -77-/- progenitors normalized the aberrant transcriptome. Since -77 deletion downregulated Gata2 and upregulated Irf8, we tested whether this opposing expression pattern occurs in distinct and/or identical cells in the population using single cell transcriptomics. -77 deletion decreased Gata2 expression, which was anti-correlative with Irf8, and detailed single cell analyses indicated that -77 loss downregulates GATA2, corrupting the transcriptome/proteome, Irf8 expression increases, and IRF8 enables or drives the predominant monocytic differentiation. To determine how GATA2-dependent mechanisms governing progenitor fate relate to those guiding HSPC expansion and differentiation during regeneration, we utilized our +9.5 human disease Ets motif mutation that abrogates myeloablation-dependent GATA2 induction and hematopoietic regeneration in bone marrow (Soukup et al., 2019). While population RNA-seq with Lin-Sca1+c-Kit+ (LSK) cells revealed little to no perturbations in the steady-state, 5-FU treatment of mutants led to altered expression of only 14% of the transcripts regulated in regenerating wild type cells (423/2974). Gene Ontology analysis of differentially expressed genes indicated that genes dysregulated by the Ets motif mutation included those linked to cell cycle regulation and cellular proliferation. As LSKs consist of LT-HSCs, ST-HSCs, and MPPs, we used single-cell transcriptomics to elucidate defective regenerative circuits in individual cells. Analysis of 17000-20000 wild type and mutant LSK cells revealed GATA2-dependent mechanisms distinct from those mediating progenitor cell fate control in the fetal liver. These studies have revealed context-dependent GATA2 mechanisms governing developmental and regenerative hematopoiesis, which will enable the development of strategies to detect, diagnose, and treat GATA2-linked blood diseases. Disclosures No relevant conflicts of interest to declare.

Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.

Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), “MonoMAC” syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or “MonoMAC” syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.

Background: CTLA4 is a homodimeric cell surface inhibitory receptor that plays a critical role in maintaining immune system homeostasis by suppressing T-lymphocyte activation and proliferation. Heterozygous germline, loss-of-function mutations in CTLA4, present with a diverse clinical phenotype of recurrent infections, lymphoproliferation, autoimmunity, and lymphocytic infiltration of target organs. Cytopenias are common in CTLA4 Haploinsufficiency and can be often multifactorial due to autoimmune destruction, splenic sequestration and bone marrow failure.(Kuehn HS …. Uzel G. Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.Science. 2014 Sep 26;345(6204):1623-1627. doi: 10.1126/science.1255904.) Patients and Methods: We have identified 95 CTLA4 mutation-positive subjects at the NIH CC, and a subset (n=74, 77%) of them exhibit symptomatic disease affecting brain, lungs, gut, spleen and bone marrow. Mean age for the affected cohort is 25 ± 2.0 years, and average age of onset of cytopenia is 16 years (range 6-43years). Of all patients with confirmed mutations, there are roughly equal numbers of females to males (1.15:1 ratio), but males dominate the affected cohort (69% males vs 31% females). Cytopenias due to autoimmune destruction, splenic sequestration and/or bone marrow failure with polyclonal lymphocytic infiltration are the most frequent initial presentation (59%; n=33): Thrombocytopenia (n=13), anemia (n=4), Evans' syndrome (n=7), and multilineage cytopenia (n=9). Splenomegaly is seen in 60% of the affected cohort. Results: We share our experience with short and long term immunomodulatory therapy in treating autoimmune cytopenias in 22 patients with CTLA4 deficiency,, including our experience with off-label use of the CTLA4 mimetic, abatacept(n=10), given at a dose of 500-750mg X2 as monthly intravenous infusions and sirolimus (n=19) with a target to maintain 24hr trough levels of 10-15ng/dL. The most frequent other concomitant immunomodulatory medication used was rituximab (n=10), azathioprine (n=5), mycophenolate mofetil (n=4), cyclosporine (n=3), hydroxychloroquine (n=1), ruxolitinib (n=1), mercaptopurine (n=1), and cyclophosphamide (n=1). Three patients received romiplostim and/or eltrombopag. All 22 patients received more than one course of pulse dose steroids. We have cumulative retrospective data of 17 patient-years in 10 CTLA4-deficient patients followed at NIH CC on abatacept. All of them showed either complete or partial response in terms stabilization of their cytopenias and/or improvement of their gastrointestinal symptoms and brain lesions. We did not observe any abatacept related adverse events: including infusion reactions, EBV reactivation, or diagnosis of malignancy. One patient died secondary to Klebsiella pneumoniae sepsis 3 months after stopping abatacept. His disease was complicated by large granular lymphocyte leukemia and treatment with alemtuzumab prior to his diagnosis with CTLA4 deficiency and abatacept treatment. Overall, we used mTOR inhibitors in 19 patients for a total of 37 patient years to treatment. Three most common recorded adverse events were: Clostridium difficile colitis (n=9, in 4 patients), lipid abnormalities (n =7, 3 patients required treatment) and bacterial pneumonia (n=6, in 4 patients). Conclusion: As abatacept replaces the missing CTLA4 protein, it should be part of the treatment regimen in patients with CTLA4 haploinsufficiency presenting with cytopenias. Both long term treatment outcomes and schedule of administration are yet to be determined in a larger cohort in the setting of a clinical trial. Disclosures Uzel: Novartis: Research Funding. Rao:novartis: Research Funding.

GATA2 deficiency is a rare, inherited or sporadic genetic disorder characterized by variable onset of a pleomorphic constellation of immune, hematologic and lymphatic abnormalities linked to heterozygous mutations in the Gata2 gene. Patients develop monocyte, B cell, NK cell and dendritic cell deficiencies resulting in vulnerabilities to unusual infections. Patients with GATA2 deficiency also frequently progress to bone marrow failure, myelodysplastic syndrome and/or acute myelogenous leukemia. GATA proteins are transcription factors with central roles in early embryonic development and lineage specification. GATA2 is a master regulator of hematopoiesis, implicated in the initial generation and maintenance of hematopoietic stem cells (HSC). Murine models recapitulate the human phenotype incompletely: GATA2 heterozygous knockout mice do not manifest loss of monocyte, B cells or NK cells; however, serial repopulation assays show decreased engraftment potential. Direct studies of primary HSC from patients with GATA2 deficiency are challenging due to the generally hypocellular marrow. We hypothesized that human pluripotent stem cells, particularly patient-specific iPSC, could be used to study potential developmental defects in GATA2 deficiency, overcoming a lack of primary HSC. In order to gain insights into the impact of human GATA2 deficiency on hematopoietic differentiation, we compared the single cell transcriptomes of HSPC differentiated from (i) iPSCs from a patient with GATA2 deficiency due to a mutation p.R337X (c.1009C>T) (ii) isogenic iPSCs created via homology-directed repair of Gata2 p.R337X, (iii) iPSCs from a healthy control and (iv) isogenic Gata2 heterozygous mutant iPSCs with monoallelic frameshift mutations in the second zinc finger domain. Mesodermal and hematopoietic differentiation was performed under feeder-free, defined media conditions. At day 0, iPSCs were plated in mesodermal induction media containing VEGF, SCF, Activin A and Y27632 in STEMdiff APEL media. Mesodermal induction was continued until day 4, when embryoid bodies were cultured in hematopoietic specification media with SCF, FLT3L, IL3, IL6, G-CSF and BMP4 until day 16, when CD34+CD45+ iPSC-derived hematopoietic stem and progenitor cells (iHSC) were enumerated and sorted by fluorescence-activated cell sorting. Single cell RNA-seq was performed using the 10XGenomics Chromium platform and primary analysis via CellRanger. Scater was used to filter outlier cells. Seurat as used to compute multiple manifold alignment and differential gene expression. Cell classification, pseudotemporal ordering and branch point analysis were performed with monocle. URD was used to calculate confirmatory diffusion maps and pseudotemporal ordering. We analyzed 7,855 iHSPC (2952 from GATA2-deficient patient, 241 isogenic iHSPCs after repair of Gata2 mutation, 2,605 from a healthy volunteer and 2,057 from isogenic heterozygous Gata2 knockout iHSPCs) after filtering of outliers. We computed multiple manifold alignment to mitigate batch effects. Differential gene expression across Gata2 mutation status found that 42 out of 102 (42%) target genes of GATA2 (c.f. TRANSFAC database of curated transcription factor targets) were differentially expressed with adjusted p-values less than 0.05. Semi-supervised classification of cell-types and pseudotemporal ordering via monocle revealed two branch points, consistent with developmental branchings at the level of CLP and CMP multipotent progenitors. The numbers of cells along each branch was found to be statistically different (χ2=30.07, p-value = 3e-7) with the biggest differences noted in the lymphoid branch (state 4). Differential gene expression in this branch revealed a differential up-regulation of Notch1, CD69 and FKBPs and differential down-regulation of CD14. In conclusion, iPSC/iHSPC differentiation models combined with single cell transcriptome analysis may be a valuable tool to identify pathways responsible for impaired hematopoietic/lymphatic development in GATA2 deficiency. Figure. Figure. Disclosures Dunbar: National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.

Leukocyte adhesion deficiency type-1 (LAD-1) is an autosomal recessive immunodeficiency caused by mutations in the β2 integrin, CD18, that impair CD11/CD18 heterodimer surface expression and/or function. Absence of functional CD11/CD18 integrins on leukocytes, particularly neutrophils, leads to their incapacity to adhere to the endothelium and migrate to sites of infection. We studied 3 LAD-1 patients with markedly diminished neutrophil CD18 expression, each of whom had a small population of lymphocytes with normal CD18 expression (CD18+). These CD18+ lymphocytes were predominantly cytotoxic T cells, with a memory/effector phenotype. Microsatellite analyses proved patient origin of these cells. Sequencing of T-cell subsets showed that in each patient one CD18 allele had undergone further mutation. Interestingly, all 3 patients were young adults with inflammatory bowel disease. Somatic reversions of inherited mutations in primary T-cell immunodeficiencies are typically associated with milder clinical phenotypes. We hypothesize that these somatic revertant CD18+ cytotoxic T lymphocytes (CTLs) may have altered immune regulation. The discovery of 3 cases of reversion mutations in LAD-1 at one center suggests that this may be a relatively common event in this rare disease.

The syndrome now known as GATA2 haploinsufficiency had been published at least since the 1970s under different clinical guises. With the identification of the gene in 2011, GATA2 deficiency became the overarching diagnosis for a complex syndrome with acoustic, cutaneous, lymphatic, pulmonary, leukemic, hematologic and infectious manifestations. Arising in both spontaneous and autosomal dominant patterns, the genetics made it recognizable and diagnosable even before symptoms arose. Over the last several years it has become clear that GATA2 is a major contributor to: pediatric and adolescent MDS, AML in younger adults, some cases of ALL, severe genital HPV infection, disseminated mycobacterial infections, pulmonary alveolar proteinosis, pulmonary hypertension and is associated with a variety of solid tumors. Mutations with more profound effects on GATA2 expression (nonsense and deletions) appear to have a more severe phenotype than do intronic mutations that alter transcript level but not transcript integrity. These latter intronic mutations may be associated with later onset of disease and more variable penetrance. Elucidating the gene targets on which GATA2 works and which partners it engages will lead to important observations in the molecular genetics of hematopoietic control. This variability of penetrance and expression is most apparent with the intronic mutations, in which whole generations may be relatively protected, presumably from genes in trans to GATA2 . An active search is on for these genes that appear to regulate GATA2 . One of the consequences of the variable penetrance of GATA2 deficiency is that it may be discordant within a family, allowing a false sense of security about use of a clinically well relative for bone marrow transplant donation. Hematopoietic stem cell transplantation is highly effective for cure of GATA2 deficiency. We have now done almost 40 transplants in GATA2 deficiency with high survival rates using related haploidentical donors, as well as with fully matched donors. The effects of transplantation are profound both in terms of hematopoietic and somatic results, including resolution of most HPV lesions, resolution of pulmonary alveolar proteinosis and resolution of pulmonary hypertension, when present. We have now encountered several cases of transplant from unexpectedly affected donors into a known GATA2, all of which have had poor outcomes. These cases reinforce the critical need for molecular confirmation of the GATA2 status of any related donor, regardless of lack of symptoms. GATA2 deficiency is recently recognized but successful treatments have emerged alongside the science. Efforts to understand and allow for manipulation of GATA2 expression are underway. Disclosures No relevant conflicts of interest to declare.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designatedMEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase–polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) γ, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor β inhibited such expression. Induction by IFN-γ occurred rapidly and was resistant to cycloheximide. IFN- also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-γ and the combination of IFN- and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-γ immediate early gene.

The p47phox−/− mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus–based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox−/− marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox−/− mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% ± 0.9% of phorbol myristate acetate–stimulated peripheral blood neutrophils at 4 weeks and 2.6% ± 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox−/− mice were significantly lower than untreated p47phox−/− mice (0.89 ± 0.30 colonies v 237.7 ± 83.6 colonies at 24 hours, P < .02; 4.0 ± 2.0 colonies v 110.2 ± 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox−/− mice (P < .001). Thus, stem-cell–directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.

Background:Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with atypical mycobacterial infection (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency. Methods: We carried out myeloablative allogeneic hematopoietic stem cell transplant (HSCT) on 24 patients (mean age 24.9 years; range 16 to 46 years) with mutations in GATA2, or the MonoMAC syndrome, during the preceding three years. Three matched related donor (MRD) recipients and 13 matched unrelated donor (URD) recipients received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on day's -6, -5, -4, and -3. Eight haploidentical related donor recipients received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. The majority of the MRD and URD recipients (n=13) received tacrolimus and short course methotrexate post-transplant as graft-versus host disease (GVHD) prophylaxis; three URD, while three received post-transplant cyclophosphamide (PT/CY) on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for GVHD. All 8 haploidentical related donor recipients received PT/CY as described followed by tacrolimus/methotrexate. Results:Twenty-two of the 24 patients are alive and disease-free at a mean follow-up of 13 months (range, 2 to 36 months). One matched URD recipient died from persistent AML 110 days post-transplant, and one matched URD recipient died from GVHD 2 years post-transplant. Despite a previous history of MAC in 11 patients, including 3 patients with active MAC at the time of HSCT, there were no recurrences of MAC during or following HSCT. Similarly, 13 patients had MDS with cytogenetic abnormalities at the time of HSCT (trisomy 8, monosomy 7, 5q-, trisomy 1q), and all 13 had resolution of their cytogenetic abnormalities with no late relapses following HSCT. Three patients had lymphedema at the time of HSCT (Emberger syndrome), which was not reversed. Twenty-three of the 24 patients had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, which were severely deficient pre-transplant. One URD who received PT/CT had poor graft reconstitution and required a CD34+ donor cell boost three months post-transplant. Four of the 13 MRD and URD recipients who received tacrolimus/methotrexate developed grade III-IV acute GVHD, and 3 of the 7 who were more than one-year post-HSCT developed moderate to severe chronic GVHD. No haploidentical related donor recipient developed grade III-IV GVHD or moderate to severe chronic GVHD. Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of clinical manifestations of GATA2 deficiency with low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We are currently using PT/CY in MRD and URD recipients with normal, favorable, or intermediate cytogenetics with isolated trisomy 8 to reduce the incidence of acute and chronic GVHD. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular MDS with or without trisomy 8. Disclosures No relevant conflicts of interest to declare.

We performed nonmyeloablative HSCT in 6 patients with a newly described genetic immunodeficiency syndrome caused by mutations in GATA2—a disease characterized by nontuberculous mycobacterial infection, monocytopenia, B- and NK-cell deficiency, and the propensity to transform to myelodysplastic syndrome/acute myelogenous leukemia. Two patients received peripheral blood stem cells (PBSCs) from matched-related donors, 2 received PBSCs from matched-unrelated donors, and 2 received stem cells from umbilical cord blood (UCB) donors. Recipients of matched-related and -unrelated donors received fludarabine and 200 cGy of total body irradiation (TBI); UCB recipients received cyclophosphamide in addition to fludarabine and TBI as conditioning. All patients received tacrolimus and sirolimus posttransplantation. Five patients were alive at a median follow-up of 17.4 months (range, 10-25). All patients achieved high levels of donor engraftment in the hematopoietic compartments that were deficient pretransplantation. Adverse events consisted of delayed engraftment in the recipient of a single UCB, GVHD in 4 patients, and immune-mediated pancytopenia and nephrotic syndrome in the recipient of a double UCB transplantation. Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B-cell, and NK-cell populations and reversal of the clinical phenotype. Registered at www.clinicaltrials.gov as NCT00923364.

We identified 18 patients with the distinct clinical phenotype of susceptibility to disseminated nontuberculous mycobacterial infections, viral infections, especially with human papillomaviruses, and fungal infections, primarily histoplasmosis, and molds. This syndrome typically had its onset in adulthood (age range, 7-60 years; mean, 31.1 years; median, 32 years) and was characterized by profound circulating monocytopenia (mean, 13.3 cells/μL; median, 14.5 cells/μL), B lymphocytopenia (mean, 9.4 cells/μL; median, 4 cells/μL), and NK lymphocytopenia (mean, 16 cells/μL; median, 5.5 cells/μL). T lymphocytes were variably affected. Despite these peripheral cytopenias, all patients had macrophages and plasma cells at sites of inflammation and normal immunoglobulin levels. Ten of these patients developed 1 or more of the following malignancies: 9 myelodysplasia/leukemia, 1 vulvar carcinoma and metastatic melanoma, 1 cervical carcinoma, 1 Bowen disease of the vulva, and 1 multiple Epstein-Barr virus+ leiomyosarcoma. Five patients developed pulmonary alveolar proteinosis without mutations in the granulocyte-macrophage colony-stimulating factor receptor or anti–granulocyte-macrophage colony-stimulating factor autoantibodies. Among these 18 patients, 5 families had 2 generations affected, suggesting autosomal dominant transmission as well as sporadic cases. This novel clinical syndrome links susceptibility to mycobacterial, viral, and fungal infections with malignancy and can be transmitted in an autosomal dominant pattern.

Background: DOCK8 deficiency- a combined immunodeficiency characterized by recurrent sinopulmonary infections, severe cutaneous DNA viral infections, eczema, food and drug allergies, virally driven malignancies and vasculopathy- results from recessive mutations in Dedicator of Cytokinesis 8 (DOCK8). We have previously reported our experience with allogeneic hematopoietic stem cell transplant (HSCT) for DOCK8 deficiency using matched donors. We now report our recent experience with haploidentical transplantation for this disease. Methods: Six patients with DOCK8 deficiency (median age 19.5 yrs, range 6-25 yrs) received a related donor, haploidentical HSCT. Co-morbitidies, in addition to active infections, included: 1 subject with prior history of Burkitt's lymphoma, cardiomyopathy and renal artery stenosis; 2 subjects with prior histories of stroke and critical basilar artery stenosis; 1 subject with a prior history of Hodgkin lymphoma and bleomycin pulmonary toxicity; and 1 subject with end-stage liver disease from Cryptosporidium who underwent living donor liver transplant from the haploidentical HSCT donor 2 months prior to HSCT. All donors underwent bone marrow harvest for collection of stem cells. Recipients of haploidentical related donor HSCT were conditioned with cyclophosphamide 14.5 mg/kg on days -6 and -5, fludarabine 30 mg/m2/day on days -6 to -2, busulfan 3.2 mg/kg/day on days -4 and -3 (pharmacokinetically targeted to attain an AUC of 4000), and 200 cGy TBI on day -1. Graft-versus host disease (GVHD) prophylaxis consisted of post-transplant cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus from day +5 to day +180, and mycophenolate mofetil from day +5 to day +35. Results: The median follow-up was 7 mo (range, 1-13 mo). Five patients engrafted with a mean time of 15.8 days (range, 13 to 18 days). One patient who had received a liver transplant 2 months before HSCT had primary graft failure and received a CD34+ selected stem cell boost. All 5 patients who engrafted had > 90% donor chimerism by day 30. All five of these patients have had reversal of the disease phenotype, which correlated with lymphocyte reconstitution. No subject developed steroid-refractory GVHD-2 subjects had no GVHD, 1 had grade 1 skin GVHD only and was treated with topical steroids; 2 developed grade 2 GVHD with skin/GI GVHD and were steroid responsive. Conclusions: There was minimal regimen-related toxicity, and no incidence of steroid-refractory GVHD despite complete donor chimerism. With genetic testing for DOCK8 deficiency becoming more widely available, we anticipate that earlier diagnosis will enable patients to be transplanted earlier in their clinical course, before significant organ damage or the development of viral-driven malignancies, and that the outcome will continue to improve. Despite extensive co-morbidities in this patient population, haploidentical HSCT was a feasible transplant strategy in patients without matched donors and resulted in reconstitution of the deficient lymphocyte compartments leading to complete reversal of the infection susceptibility phenotype in most patients. Disclosures No relevant conflicts of interest to declare.

GATA2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis as well as lymphatic angiogenesis. Human GATA2 deficiency disease is caused by a variety of mutations in GATA2 which lead to the clinical phenotypes variously known as monocytopenia and mycobacterial disease (MonoMac), familial MDS/AML, dendritic cell, myeloid and NK cell lymphopenia (DCML), Emberger syndrome, and classical NK cell deficiency. Patients with GATA2 deficiency have an immunodeficiency with monocytopenia, NK, and B cell leukopenia; are susceptible to mycobacterial, viral and fungal infections as well as MDS and progression to AML. Additionally, patients may suffer from lymphedema and pulmonary alveolar proteinosis. GATA2 mutations have also been associated with aplastic anemia and severe congenital neutropenia. In order to understand the mechanisms underlying the development of MDS/AML in GATA2 deficiency we developed and analyzed a unique mouse model. The previously characterized Gata2+9.5+/- mouse has an intronic regulatory region mutation identical to one found in some patients. However, these mice do not develop MDS/AML spontaneously. We hypothesized that crossing these mice to other MDS/AML prone strains would accelerate the progression of MDS/AML. Specific HOX genes are implicated in this disease process. GATA2-deficient patients often develop monosomy 7 and ASXL1 mutations, both of which lead to the overexpression of HOX genes including HOXA9. The known AML translocation t(2;11)(q31;p15) results in the fusion of NUP98 and HOXD13 (NHD13) and the upregulation of HOX gene expression. Gata2 was identified in a mouse retroviral insertion screen as a potential collaborator in NHD13-mediated leukemogenesis. We hypothesized that hematopoietic specific Hox gene overexpression in a mouse with germline Gata2 haploinsufficiency would lead to the development of MDS/AML. This was done using the established NHD13 transgenic strain crossed to the Gata2+9.5+/- strain. The NHD13 mice express the fusion protein under the control of the hematopoietic specific vav promoter, and have up-regulation of Hoxa5, Hoxa7, Hoxa9, and Hoxa10. Secondly, elevated Flt3 ligand levels have been detected in GATA2-deficient patients as they progress from normal bone marrow morphology to MDS/AML. To model the hyper-activation of the receptor Flt3 by Flt3 ligand, we used a model of constitutively active Flt3, the Flt3-ITD mouse. Gata2 +9.5+/- mice were bred to the NHD13 and the Flt3-ITD transgenic strains and compound heterozygotes were analyzed. Gata2+9.5+/-;NHD13 compound heterozygous mice were born normally and at Mendelian ratios. No significant differences in blood cell counts were noted in Gata2+9.5+/-;NHD13 compound heterozygotes until disease progression. These mice developed aggressive AML at 9-14 months of age, similar ages and rates as the NHD13 only mice. Whereas compound Gata2+9.5+/-;Flt3-ITD mice are also born in Mendelian ratios, starting at 10 weeks of age they developed B-cell lymphopenia, similar to that which is seen in human GATA2 deficiency patients displaying B-cell loss early in disease. The Gata 2+9.5+/-; Flt3-ITD mice exhibited increased circulating c-Kit-positive cells in the peripheral blood. Studies to further characterize hematopoiesis in the Gata2+9.5+/-; Flt3-ITD mice, and analyze for the development of MDS/AML are in progress. The Gata 2+9.5+/-; Flt3-ITD mouse model holds promise for the further study of MDS/AML in the context of GATA2 haploinsufficiency. Disclosures Aplan: NIH Office of Technology Transfer: Patents & Royalties.

Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency. Methods: Eleven patients with GATA2 deficiency received a myeloablative-conditioning regimen (2 matched related donors or MRD, 4 matched unrelated donors or URD, and 5 haploidentical related donors. MRD and URD received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on days -6, -5, -4, and -3. Haploidentical related donors received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. MRD and URD recipients received tacrolimus and short course methotrexate post-transplant, while haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for graft-versus-host disease. Results: Ten of the 11 (91%) of patients are alive and disease-free at a mean follow-up of 12 months (range 1 mo to 24 mo). One URD recipient died from persistent acute myelogenous leukemia. Four patients developed graft-versus-host disease, one case Grade 4. All 10 patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant. All 10 patients had reversal of the infection susceptibility phenotype. In particular, there were no recurrences of NTM infections. Importantly, all 10 patients had correction of the cytogenetic abnormalities present pre-transplant (5 patients with trisomy 8 and 1 patient with monosomy 7). Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of GATA2 deficiency. There was a low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular myelodysplastic syndrome. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood , Drewniak et al[1][1] demonstrate that neutrophil killing of Candida depends on caspase recruitment domain–containing protein 9 (CARD9) function but not on the production of reactive oxidants. Furthermore, although CARD9 is required for Candida killing, it is dispensable

Interferon-γ receptor-1 (IFNγR1) deficiency is a rare inherited immunodeficiency. We performed a nonmyeloablative allogeneic stem cell transplantation on a boy with complete IFNγR1 deficiency and refractory disseminated Myco-bacterium avium infection. Despite the patient's profound immune defect, early donor stem cell engraftment was low. Full donor engraftment was accomplished only following multiple donor lymphocyte infusions. Detection of IFNγR1 expression on peripheral blood monocytes and neutrophils corresponded with establishment of stable, complete donor hematopoietic chimerism. However, expression of, and signaling through IFNγR1 disappeared shortly thereafter. Disseminated Mycobacterium avium infection persisted and the patient died. Coculture of Myco-bacterium avium with normal myeloid cells resulted in an IFNγ signaling defect similar to that observed in vivo. Active disseminated Mycobacterium avium infection may significantly compromise normal immune reconstitution following allogeneic stem cell transplantation. Patients with IFNγR1 deficiency should receive transplants before developing refractory mycobacterial infections. (Blood. 2003;102:2692-2694)

Culture-based methods have been the primary techniques used to study microbes inhabiting humans; however, many species are not successfully grown in culture. We performed high throughput genomic sequencing surveys to investigate the topographical and temporal complexity of skin microbial communities from 20 skin sites in healthy adults. Significant differences were observed in the bacterial species predominating in particular microenvironments: sebaceous, moist, and dry. Surveying fungal diversity with genomic sequencing, we determined that core body and arm sites were dominated by Malassezia fungi, with species-level classifications revealing greater topographical resolution between sites. Three foot sites, plantar heel, toenail, and toeweb, exhibited tremendous fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that skin physiological attributes and topography differentially shape these two microbial communities. While activated and shaped by microbiota, little is known of how the human immune system regulates the human microbiome, and in turn, how this can result in a disease phenotype. We describe the microbial characteristics of the skin of primary immunodeficiency (PID) patients who share a common phenotype of skin eczema yet have different syndromes arising from monogenic mutations leading to loss of distinct lymphocytic populations. We surveyed the skin microbiomes of 41 individuals with Hyper IgE, Wiskott-Aldrich, and Dedicator of Cytokinesis 8 syndromes and compared them against classical atopic dermatitis (AD) patients and healthy controls at skin sites characteristically affected by eczema, a control site, and a site of pathogen carriage (nares). We found that primary immunodeficiency increases the permissiveness of skin microbial colonization not observed in healthy controls or AD patients. We observed decreased site specificity and longitudinal stability in the PID patients as well as unique colonization by environmental microbiota very rare in healthy or AD controls. We identified taxa correlated and anti-correlated with clinical metamarkers in the PID patients; while Staphylococcus aureus, a known pathogen, was most strongly correlated with disease severity, other staphylococci such as S. haemolyticus and S. epidermidis were significantly overrepresented in the PID individuals. These data provide the first illustration of how PID affects microbial prevalence, diversity, and dynamics in relation to skin disease, gaining insight into host-microbiome interactions and how environmental microbes can uniquely colonize PID patients. This comprehensive survey of the skin microbiome also provided the foundation for analyzing changes in the microbial community associated with common forms of AD, which affect ~15% of U.S. children and ~2% of adults and is associated with Staphylococcus aureus colonization and infection. We studied 10 children with moderate to severe AD at baseline, flare, and post-flare, and healthy controls. Severity was quantified using scoring atopic dermatitis (SCORAD). Samples were obtained from characteristically affected areas, a control site, and nares. Bacterial diversity was dramatically reduced during flare as compared to post-flare and controls. Our studies provide comprehensive characterization of skin microbes in AD and controls confirm the frequent culture-based isolation of S. aureus in AD flares and represent one of the earliest longitudinal investigations of the skin microbiome in a dermatologic disorder. Disclosures No relevant conflicts of interest to declare.

Background Autosomal dominant germline mutations in the phosphatidylinositol-3-OH kinase (PIK3CD) encoding for the PI(3)K catalytic subunit p110δ, lead to combined immunodeficiency with increased incidence of B-cell lymphomas. (Lucas CL et.al. Nature Immunology 2014). While p110δ is selectively expressed in leukocytes, it is critical for TCR and BCR signaling and lymphocyte homeostasis. Clinically, these patients may present with sinopulmonary infections, bronchiectasis, cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) viremia, lymphoproliferation and autoimmune cytopenias. Immune phenotype includes naïve CD4+ T cell lymphopenia, expanded terminally differentiated or exhausted T cells, increased circulating transitional B cells and reduced class-switched memory B cells. Herein we report immunophenotypic abnormalities in B-lymphoid maturation in the bone marrow (BM) of 5 patients with PIK3CD mutations. Methods BM from 5 patients with PIK3CD mutations (2 males, 3 females, age range: 4–15 years, median 11.5 years) were studied by flow cytometry (FC), morphology and immunohistochemistry (IHC). BM aspirate from 5 healthy age matched pediatric patients were used as controls for flow cytometric analysis of B-cell subsets and maturation. Antibodies against CD45, CD3, CD4, CD8, CD19, CD10, CD34, CD20, and surface kappa and lambda light chains were used for FC. B-lymphocyte subsets were defined as: early stage precursor B-cells (CD34+/CD19+/CD10+bright/CD20-); intermediate precursor B-cells (CD45+moderate/ CD19+/CD10+moderate/CD34-); and late stage and mature B cells (CD34-/CD10-/CD19+/CD45+bright/CD20+). The intermediate subset corresponds to transitional B cells (developmentally intermediate between immature and mature naive B cells). IHC and in situ hybridization staining were applied to biopsy sections using standard methods. Prism software was used for statistical analyses (Mann-Whitney test). Results There was no significant difference in the median percentage of early B-cell precursors (among all B-lymphocytes) between the PIK3CD patients and the age-matched controls (3.6% vs. 3.7%; p=0.8). However, all PIK3CD marrows showed expanded CD10+ intermediate precursor B-cells which were overall 2.5 times more abundant in PIK3CD marrows than in controls (94.6% vs. 37.4% of all B-cells; p BM core biopsies showed overall normal cellularity with increased lymphocytes (20-30% of the cellular marrow). IHC revealed increased CD20+ lymphocytes (15-20% of all nucleated cells) and CD10+ lymphocytes showed similar distribution suggesting coexpression with CD20. TdT and CD34 highlighted approximately 5% of all nucleated cells. CD138, and kappa and lambda light chains showed unremarkable scattered polytypic plasma cells. CD3+ and CD8+ T-cells accounted for 5-10% of BM cells and CD4+ lymphocytes were reduced. EBV was positive in one case. CMV was negative in all cases. Conclusions For the first time, we report B-cell maturation abnormalities in the bone marrow of patients with germline mutations in PIK3CD. All marrows showed an abnormal pattern of B cell maturation characterized by an absolute increase in CD10+ intermediate precursor B-cells and a marked decrease in mature B-cells. The findings suggest either a partial block in B-cell late stage maturation or other mechanism leading to increased CD10+ B-cell precursors and markedly reduced mature B-cells. Lymphoid hyperplasia and lymphoma have been described in PIK3CD patients. The increased CD10+ B cell precursors and the abnormal maturation pattern noted by flow cytometry may mimic CD10+ B-cell neoplasia (e.g. acute lymphoblastic leukemia or Burkitt lymphoma) but detailed analysis showed no morphologic or immunophenotypic evidence of B-cell neoplastic involvement in any of the five patients studied. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Patients with GATA2 deficiency specifically lack CD56bright/CD16- natural killer (NK) cells, while the CD56dim/CD16+ (DP) subset is preserved. CD56bright/CD16- cells have been postulated to be the precursor population for more mature and cytotoxic CD16+NK cells. The mechanism by which an immature population disappears while a putatively more mature population is preserved is not understood. Our group has recently shown that CD16+/CD56dim/- NK cells have a distinct ontogeny, showing little overlap with CD56+/CD16- NK cells, which share clonal derivation with myeloid, T and B lineages (Wu et al, Cell Stem Cell 2014). We hypothesize that the missing CD56bright NK population in GATA2 deficiency is due to a maturation defect in a specific hematopoietic stem/progenitor population, separate from those relatively preserved progenitors producing CD16+ NK cells. We performed ex vivo expansion of NK cells from 4 healthy volunteers and 5 patients with GATA2 mutations and compared phenotypes at baseline and upon expansion. Sorted peripheral blood total NK (TNK) cells (CD3, CD20, CD14 depleted and expressing CD16 and/or CD56) and double negative (DN) cells (CD3-/CD20-/CD14- and CD16-/CD56-) were cultured in the presence of IL2 and irradiated Epstein-Barr virus-transformed lymphoblastoid cells. After 14 days of culture, each cell culture was characterized and re-sorted into TNK cells and DN cells, and cultured for an additional 14 days with restimulation. Subject 1 had GATA2 c.229+1 G>A mutation and presented with pancytopenia, recurrent warts, classic B/NK lymphopenia/monocytopenia and MDS with trisomy 8. Subjects 2 and 5, and subjects 3 and 4 were from the same families, respectively, and all shared the same intron 5 GATA2 c.1017+572C>T mutation. The proband, subject 2 had low grade MDS, recurrent warts and classic B/NK lymphopenia and monocytopenia. However, subjects 3, 4 and 5 had normal peripheral blood counts and bone marrow morphology, with only mild B or NK lymphopenia. TNK cells from subject 1 had profound lack of CD56bright cells at baseline, and showed decreased ex vivo expansion efficiency; 18.5-fold increase at day 14 compared to a 39.4-fold increase for control TNK cells. Even after expansion, the CD56bright population was still absent. When DN cells from subject 1 were placed into culture, they did not give rise to a CD56brightpopulation after 14 or 28 days, while control DN cells did give rise to a clear CD56bright population, also expressing additional NK-defining markers such as NKG2D and NKp46. TNK cells from patients with GATA2 intron 5 mutations had a less striking phenotype and were more variable. Subject 2 had greatly decreased CD56bright NK cells at baseline, in contrast to her asymptomatic father, subject 5, and to subjects 3 and 4 from another family. Ex vivo expansion kinetics for the TNK cells from all 4 subjects were similar to controls. By day 14 in culture, their TNK cells showed preserved generation of CD56bright/+ cells and DP cells. DN cells from subject 2 showed no CD56 expression upon expansion at day 14 and only a minor CD56dim population at day 28. Subjects 3, 4 and 5 showed comparable phenotypes at baseline and upon expansion until day 14 (Figure 1). GATA2 DN cells sorted and expanded twice from DN cells at days 1 and 14, at day 28 had much lower CD56bright NK cells (1.1±0.6% vs. 10.1±3.9%; p CD107a functional analysis showed impaired NK degranulation capacity upon K562 co-incubation in expanded GATA2 TNK cells and DN cells compared to their controls at day 14 (12.4±0.6 vs. 3.5±0.1 vs. 6.7±0.1 (p Our findings suggest that NK differentiation defects in GATA2 deficiency may be due to a differentiation block from a precursor present in the DN subset which is impaired in giving rise to CD56bright/CD16- cells upon prolonged culture, and also potentially in vivo. The differences between the GATA2c.229+1 G>A and GATA2 c.1017+572C>T (intron 5) samples in both phenotype and expansion characteristics correlate with the clinical severity of patients, and suggest a mutation-specific, dose-dependent role of GATA2 in NK development, as well as other modifying factors. Disclosures Townsley: GSK: Research Funding.

Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) is the only definitive therapy for GATA2 deficiency. Methods: We used matched related donors (MRD), matched unrelated donors (URD), umbilical cord blood (UCB), and haploidentical related donors in allogeneic HSCT for GATA2 deficiency. Fourteen patients received a nonmyeloablative conditioning regimen (4 MRD, 4 URD, 4 UCB, and 2 haplo donors). Five patients received a myeloablative conditioning regimen (1 MRD, 2 URD, and 2 haplo donors). In the nonmyeloablative group, MRD and MUD recipients received fludarabine and 200cGy of total body irradiation (TBI), UCB recipients received cyclophosphamide 50mg/kg, fludarabine 150 mg/m2, and 200cGy of TBI, and haploidentical related donor recipients received cyclophosphamide 29 mg/kg, fludarabine 150 mg/m2, and 200 cGy TBI. In the myeloablative group, MRD and URD received busulfan 12.8 mg/kg and fludarabine 160 mg/m2, and haploidentical related donors received the same regimen as in the nonmyeloablative regimen except for the addition of two days of busulfan 6.4 mg/kg total dose. Nonmyeloablative MRD and URD recipients received tacrolimus and sirolimus post-transplant, and myeloablative MRD and URD recipients received tacrolimus and short course methotrexate post-transplant. All haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil. Three patients in the nonmyeloablative cohort required one or more rounds of pre-transplant chemotherapy because of an increased number of blasts, whereas none of the 5 patients in the myeloablative arm required pre-transplant chemotherapy. Results: In the nonmyeloablative cohort, 8 of 14 (57%) of patients are alive at a median follow-up of 3.7 years (range 12 months to 5 years). One MRD recipient died of GVHD and one relapsed, one URD recipient rejected the donor stem cells and died, three UCB recipient died (one rejection, one early death, and one donor cell leukemia), and one haploidentical recipient died from regimen-related toxicity. All 5 patients (100%) in the myeloablative group, including two recipients of haploidentical related donors, are alive at a median follow-up of 9.2 months (range 6 to 12 months). All patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant, and all had reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of non-tuberculous mycobacterial (NTM) infections. Conclusions: Nonmyeloablative HSCT results in reversal of the hematologic and clinical manifestations of GATA2 deficiency. However, a more intensive conditioning regimen with busulfan resulted in more uniform engraftment, a reduced risk of relapse, avoidance of pre-transplant chemotherapy, and a low regimen-related toxicity. We anticipate that with the use of a high-dose regimen with busulfan, the replacement of UCB with haploidentical related donors, and HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Disclosures No relevant conflicts of interest to declare.

Background Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of excessive immune activation. Herpesvirus infection is a common trigger of HLH, and in some patients may indicate an underlying immunodeficiency. Primary immunodeficiencies associated with HLH are often characterized by impaired cytotoxic function of NK cells and/or cytotoxic T lymphocytes and include Chediak-Higashi, Hermansky-Pudlak, and Griscelli syndromes and the X-linked lymphoproliferative disorders, SAP and XIAP deficiency. GATA2 deficiency, also known as MonoMAC syndrome, is a primary immunodeficiency resulting from haploinsufficiency of the GATA2 transcription factor, a master regulator of hematopoiesis. Clinically, GATA2 deficiency is characterized by progressive cytopenias of monocytes, dendritic cells, and B and NK lymphocytes; susceptibility to infection with human papillomavirus, herpesviruses, nontuberculous mycobacteria, and invasive fungi; and bone marrow failure with risk for clonal evolution to myelodysplastic syndrome and acute myelogenous leukemia. Additional manifestations may include pulmonary alveolar proteinosis, congenital lymphedema, and sensorineural hearing loss. We report two patients with GATA2 deficiency/MonoMAC syndrome who developed aggressive HLH in the setting of marked NK cell dysfunction and disseminated herpesvirus infection. Case 1 The patient was an African-American female with severe bilateral lymphedema since age 9 requiring multiple hospitalizations for lymphedema cellulitis. At age 12, she was hospitalized for severe pulmonary blastomycosis with necrotizing pneumonia. An immunologic workup revealed profound monocytopenia and B and NK lymphocytopenia, and NK functional testing revealed markedly reduced cytotoxicity and near absence of the CD56(bright) subset. Genetic testing for mutations in PRF1, UNC13D, and STX11 was negative. At age 18, she presented with septic shock and persistent fevers despite empiric antibiotics, and HSV-1 DNA was detected in the blood. The diagnosis of MonoMAC syndrome was strongly suspected in light of her congenital lymphedema and highly characteristic cytopenias and infections. Unfortunately, she rapidly decompensated and developed massive hyperferritinemia, hypofibrinogenemia, pancytopenia, and fulminant hepatitis concerning for HSV-1-driven HLH. An autopsy following her death revealed disseminated HSV-1 infection in the lungs, liver, and genitalia, and a hypocellular bone marrow with hemophagocytosis. The cause of death was HLH in the setting of NK cell deficiency and widespread HSV-1 infection. Genetic testing for GATA2 mutation was performed posthumously and is currently in progress. Case 2 A Chinese female presented at age 20 with persistent fevers, cervical lymphadenopathy, and cutaneous ulcers. Mycobacterium avium complex was identified in the sputum and urine, and she was treated with clarithromycin, rifampin, and ethambutol. HIV testing was negative. Biopsy of her skin lesions revealed an EBV+ hydroa vaccineforme-like cutaneous T-cell lymphoma, and she was found to have significant EBV viremia with over 1 million copies/mL detected by quantitative PCR. An immunologic workup revealed profound B and NK lymphocytopenia. Monoallelic GATA2 mRNA expression was demonstrated leading to haploinsufficiency. She developed persistent fevers, worsening hyperferritinemia, hypofibrinogenemia, pancytopenia, and transaminitis. Bone marrow biopsy revealed a hypocellular marrow with EBV+ T cells and abundant hemophagocytosis. She was diagnosed with EBV-driven HLH and was treated with etoposide and dexamethasone followed by nonmyeloablative, haploidentical related donor stem cell transplant. She is currently 12-months post-transplant with an undetectable EBV viral load and complete resolution of her T cell lymphoma and HLH. Conclusion GATA2 deficiency is associated with NK cell dysfunction and disseminated herpesvirus infection, which can lead to aggressive HLH. Genetic testing for GATA2 mutation should be considered in patients with HLH and underlying NK cell deficiency and/or herpesvirus infection. NK functional testing typically shows markedly reduced cytotoxicity and near absence of the CD56(bright) subset. Allogeneic transplant represents a potentially curative approach in this setting. Disclosures No relevant conflicts of interest to declare.

Background: GATA2 deficiency results in a clinical syndrome known variably as: MonoMAC for the lack of monocytes and atypical mycobacterial infections (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Reconstitution of the deficient cell compartments in GATA2 deficiency with allogeneic hematopoietic stem cell transplant (HSCT) results in reversal of the infection susceptibility and represents the only curative therapy for both the myeloid disease and the virally driven malignancies seen in this disorder. However, only about one-half of patients that require HSCT will have an HLA-matched related or unrelated donor. Moreover, the use of umbilical cord blood has been reported to be suboptimal in this cohort of patients in whom infection susceptibility underlies much of the need for a transplant. Methods: We performed haploidentical, related donor HSCT with high dose post-transplant cyclophosphamide (PT/CY) in 3 patients with GATA2 deficiency in whom a suitable HLA matched donor was not available. All three patients had a first degree relative (sibling) who shared at least 1 HLA-haplotype. The first patient, a 21 year-old Chinese woman presented with Hydroa Vacciniforme like T-cell lymphoma involving her lung, bowel (resulting in multiple small bowel resections and ileostomy), and skin (with large ulcerative lesions); multiple thrombotic cerebral events; and macrophage activation syndrome requiring etoposide and high dose prednisone in the intensive care unit (ICU). The second patient, a 20 year-old Hispanic female, presented with myelodysplasia (MDS), profound neutropenia, an invasive fungal sinusits due to Fusarium sp., and recurrent bacteremias. The third patient, a 45 year-old woman, presented with Emberger’s Syndrome, multiple episodes of bacterial and fungal sepsis and hypocellular MDS. All three patients received cyclophosphamide 14.5mg/kg on day’s -6 and-5; fludarabine 30mg/m2 on day’s -6 through -2; 200cGy of total body irradiation (TBI) on day -1. Patients 2 and 3 also received busulfan 3.2 mg/kg on day’s -4 and -3 because of MDS. All 3 patients received high dose PT/CY 50mg/kg on day’s +3 and +4, and mycophenolate mofetil and tacrolimus starting on day +5. Results: All 3 patients engrafted at a mean of 18 days and all are alive at a median follow-up of 9 months (range, 5 to 13 months). All three patients achieved 100% donor myeloid and lymphoid chimerim by day +100 post-transplant. The first patient had complete resolution of her T-cell lymphoma and macrophage activation syndrome, the second patient has had complete reversal of her hematopoietic and infectious phenotype, and the third patient has had resolution of her infectious complications that required continuous IV Daptomycin for 5 years prior to transplant. In addition, all three patients had complete reconstitution of the monocyte, NK cell, and B-lymphocyte compartments that were severely deficient prior to transplant. The main toxicity was mucositis, which required intravenous narcotics in all three patients. Lastly, the first patient developed late grade I liver graft-versus-host disease (GVHD) that was responsive to cyclosporine, the second patient developed grade I acute GVHD of skin that was responsive to topical steroids, and the third patient had no evidence of acute or chronic GVHD. Conclusions: Haploidentical, related donor HSCT represents a safe and effective viable alternative for patients with GATA2 deficiency who lack an HLA-matched donor, including patients such as our first patient who was in the ICU at the time of transplant. Disclosures No relevant conflicts of interest to declare.

Background DOCK8 deficiency— a combined immunodeficiency characterized by recurrent sinopulmonary infections and severe cutaneous DNA viral infections, eczema, food and drug allergies, and increased risk of viral driven malignancies— results from homozygous or compound heterozygous mutations in the dedicator of cytokinesis 8 protein. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a potential curative therapy for DOCK8 deficiency. Methods We treated 4 patients with DOCK8 deficiency with allogeneic HSCT using a reduced toxicity, myeloablative-conditioning regimen consisting of busulfan 3.2 mg/kg/day IV for 4 days and fludarabine 40 mg/m2/day for 4 days. Two patients received matched related donor allogeneic HSCT, and 2 received 10/10 HLA matched unrelated donor allogeneic HSCT. All patients received tacrolimus, and short methotrexate on days 1,3, 6, and 11, for graft-versus-host-disease (GVHD) prophylaxis. Indication for transplant was severe recurrent infections with end organ damage in three patients, and a refractory EBV-virus driven lymphoma at the time of unrelated donor PBSC transplant in the fourth patient. Results The ages of the patients at the time of transplant were 27, 25, 18, and 16. The median follow-up for patients was 7 months (range 5-18 months). All 4 patients engrafted at a median of 12 days, and all 4 had complete reconstitution of the CD3/CD4 and CD3/CD8 compartments with donor cells. Clinical correction of the DOCK8 clinical phenotype occurred within 3-6 months of engraftment and correlated with lymphocyte reconstitution. Mucositis was the major side effect of the transplant-conditioning regimen. All had transient worsening of their pre-transplant infections 1-3 months post-transplant. In the patient with an EBV-virus driven lymphoma that had been refractory to chemotherapy, there was complete resolution of the disease on PET scanning at 100 days post-transplant. One patient, who received unrelated donor bone marrow cells, had skin GVHD that responded to a short course of steroids. There was no chronic GVHD. Conclusions Allogeneic HSCT in DOCK8 deficiency results in reconstitution of the deficient lymphocyte compartments that are present pre-transplant and complete reversal of the infection susceptibility phenotype. There was minimal regimen-related toxicity, and the incidence of GVHD was low despite complete donor chimerism. With genetic testing for DOCK8 deficiency now more widely available, we anticipate that earlier diagnosis will enable patients to be transplanted earlier in their clinical course, before significant organ damage or the development of viral-driven malignancies Disclosures: No relevant conflicts of interest to declare.

Introduction Signal transducer and activator of transcription 3 (STAT3) is an important regulator of cellular immunity, and in some animal models inhibition of STAT3 has been effective in improving natural killer (NK) cell antitumor efficacy. However, there has been little direct assessment of the STAT3 signaling role in human NK cells. In our previous work we found that (i) human NK cells stimulated with K562-based artificial antigen presenting cells (aAPC) genetically modified to express membrane bound IL-21 (mbIL21), which predominantly activates STAT3, resulted in greater proliferation, longer telomere length, and less senescence than NK cells expanded with mbIL15, which predominantly activates STAT5 (ii) NK cell proliferation, cytotoxicity and expression of the activating receptor NKG2D were enhanced by STAT3 activating cytokines and diminished by STAT3 inhibitors. Based on these results we hypothesized that activation of STAT3 plays a critical role in NK cell expansion and anti-tumor activity and tested our hypothesis by studying NK cells from Job syndrome patients. Hyper-immunoglobulin E syndrome (HIES / Job syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses primarily caused by staphylococcal infection. Both, sporadic and autosomal dominant Job syndromes are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development, proliferation and function in Job syndrome patients has not been studied yet. Methods We obtained peripheral blood from healthy donors and from HIES patients with documented STAT3 mutations. NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by Student’s t test using GraphPad Prism. Results Compared to normal donors (1) Flow-cytometric analysis of PBMCs showed a significantly lower percentage of NK cells in Job syndrome patients (2) We observed impaired proliferation of Job syndrome patients’ NK cells upon stimulation of PBMCs not only with mbIL21 but also with mbIL15 in complex with its receptor α (mbIL15Rα), a physiologically relevant presentation of a physiologically relevant cytokine involved in NK cell survival and proliferation (3) Lower percentage of mature, CD56+CD16+ NK cells in Expanded NK cells from Job syndrome patients and (4) Reduced basal expression of NKG2D receptor as well as lower induction of NKG2D receptor expression upon stimulation with STAT3 activating cytokines IL10 and IL21, on Job syndrome patients’ NK cells. Conclusions Our finding of a deficient NK cell number in Job syndrome patients carrying dominant negative STAT3 mutations is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job syndrome patient’s NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job syndrome patients are also more prone to viral infections and lymphoma. As NK cells perform surveillance of virally infected and tumorigenic cells through NKG2D receptor, low NK cell number with reduced NKG2D expression may explain the proclivity of Job syndrome patients to viral infections and lymphoma. Work is in progress to assess cytokine generation and anti-tumor activity of Job syndrome patients’ NK cells. Disclosures: No relevant conflicts of interest to declare.

Myelodysplastic syndrome (MDS) is rare in children. Certain inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia, severe congenital neutropenia and Shwachman Diamond syndrome, markedly increase the risk of MDS during childhood. However, the genetic factor(s) underlying sporadic pediatric MDS are unknown. Germline mutations in GATA2, a hematopoietic transcription factor, explain four MDS-predisposing conditions: monocytopenia and mycobacterial infection (MonoMAC); dendritic cell, monocyte, B and NK cell lymphoid deficiency (DCML); primary lymphedema with myelodysplasia progressing to acute myeloid leukemia (Emberger syndrome); and a subset of familial MDS. Cases of pediatric MDS have been observed in some of the reported pedigrees. In addition, three individuals have been reported with large, de novo deletions encompassing GATA2 and surrounding genes and manifesting developmental delay, intellectual disability and dysmorphic features alongside their hematologic abnormalities. We identified a novel GATA2 splice site variant (c.1018-2A>C) in a teenager with MDS, WHO classification refractory cytopenia of childhood (RCC). Although he was found to have monocytopenia and B and NK cell deficiencies, he had no history of infections associated with MonoMAC or pertinent family history. We, therefore, hypothesized that mutations in GATA2 might be present in additional cases of pediatric MDS that were neither associated with an IBMFS nor relevant personal or family history. Two Baylor College of Medicine biology studies open to children with hematologic disease were queried for patients with the diagnosis of MDS. Exclusion criteria included treatment-related MDS, diagnosis of an IBMFS, prior diagnosis of severe aplastic anemia or infections suspicious for MonoMAC or DCML, and known or suspected family history of a GATA2-associated disorder. Cases lacking a pre-hematopoietic stem cell transplantation (HSCT) tissue sample available for study were also excluded. In addition to the patient described above, six children were identified who met eligibility criteria. DNA was isolated from banked peripheral blood or bone marrow cells and GATA2 sequencing performed, including upstream and intronic regulatory regions. Array comparative genomic hybridization was also performed on one sample that lacked GATA2 sequence variants, but was notable for complete absence of heterozygosity (AOH), including 6 polymorphic sites with minor allele frequencies of 0.20 or greater. Pertinent clinical and laboratory features were extracted by medical record review blinded to GATA2 status. We found heterozygous GATA2 mutations in three of the six additional patient samples. Thus, four of this seven patient, pediatric MDS cohort had mutated GATA2. Two of the newly identified mutations were splice site variants: a previously described c.1018-1G>A and a novel variant altering the exon 7 splice site acceptor (c.1114-1G>C). The third mutation was a de novo 3.1-3.3 Mb deletion encompassing the entire GATA2 locus and contiguous genes, and was established to be germline by analysis of skin fibroblasts. Notably, the patient had normal neurocognitive development and was without dysmorphic features. Their ages of presentation were 5, 9, 12 and 15 years. With the exception of the initial case, peripheral blood T and B cell phenotyping was not obtained prior to HSCT. Monocytopenia of less than 200/µL was present in five of seven patients, three of whom had a GATA2 mutation. All four GATA2 mutation cases had RCC and three of the four had monosomy 7 at diagnosis. In contrast, the three cases lacking GATA2 mutation presented with the MDS classification refractory anemia with excess blasts (RAEB-2), with either a normal karyotype, complex karyotypic changes or chromosome 13.q12q14 deletion. GATA2 mutation may explain a significant portion of sporadic, seemingly nonsyndromic pediatric MDS, particularly cases with monosomy 7. Evaluation of larger cohorts is warranted to ascertain the true prevalence. Although this cohort is small, we recommend GATA2 sequencing be performed as part of the initial evaluation of pediatric MDS as the identification of a germline mutation has critical implications for related donor selection and genetic counseling. AOH in GATA2 sequencing should prompt deletion analysis, even in cases without infections, dysmorphic features or neurocognitive impairment. Disclosures: No relevant conflicts of interest to declare.

Background Heterozygous sporadic or inherited mutations in the GATA2 gene result in a syndrome known variably as: “MonoMAC” for monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome, lymphedema and MDS with monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Life-threatening opportunistic infections and myeloid transformation constitute the rationale for allogeneic hematopoietic stem cell transplant (HSCT) for GATA2 deficiency. Methods We treated 14 patients with GATA2 deficiency using a nonmyeloablative-conditioning regimen and matched related donors (MRD) (n=4), matched unrelated donors (URD) (n=4), umbilical cord blood (UCB) (n=4), and haploidentical related donor (HD) (n=2) sources. There was considerable pre-transplant morbidity in this cohort of patients. MRD and URD recipients received 200 cGy of total body irradiation (TBI) and 3 days of fludarabine; UCB recipients received 200cGy TBI, cyclophosphamide 50 mg/kg on day -6, and 5 days of fludarabine; HD recipients received 200cGy TBI, cyclophosphamide 14.5 mg/kg on days -6 and -5, and fludarabine for 5 days. MRD, URD, and UCB recipients received tacrolimus and sirolimus for GVHD prophylaxis. HD recipients received cyclophosphamide 50 mg/kg on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil. MRD and URD donor recipients received peripheral blood stem cells (PBSC) and HD donor recipients received bone marrow stem cells. Results (table 1): The median follow-up in the MRD cohort was 32 months, excluding one early death in a patient on a ventilator at the time of transplant. One patient relapsed one year post-transplant and required re-transplant using a myeloablative regimen. All 4 patients in this cohort developed acute GVHD. In the URD cohort, all patients are alive, however one patient rejected the PBSC graft and required a second URD transplant from a different donor. Two patients developed acute GVHD. In the UCB cohort, there was one early death from sepsis, one graft rejection, and one donor cell leukemia that occurred 2.5 years post-transplant. The remaining patient had a complicated course, but at 3 years post-transplant he is fully engrafted, on no medications, and has no GVHD. In the two patients who received HD transplants, one had progressed to proliferative CMML prior to transplant and died in the immediate post-transplant period. The second patient engrafted and is doing well two months post-transplant with resolution of an EBV-driven T cell lymphoma. All patients who engrafted had complete reconstitution of the monocyte, NK, and B lymphocyte compartments; all had correction of the underlying myeloid malignancy, and reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of NTM. Conclusions Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B and NK cell populations and correction of the infection susceptibility phenotype. However, 1 of 4 MRD recipients developed a relapse of the original clone, and 1 of 4 URD recipients rejected the donor PBSC. The incidence of relapse and rejection, as well as the unfavorable cytogenetics in many patients, suggests that a more intense conditioning regimen is required to treat these patients. In this regard, we are now using a myeloablative regimen with busulfan and fludarabine. The poor outcome with UCB in this cohort of immunocompromised patients supports the use of HD transplants when a MRD or URD is not available. We anticipate that with increasing use of genetic testing for GATA2 mutations, patients will be transplanted earlier in the course of disease, before significant organ damage or clonal evolution of MDS to AML and CMML occurs, and that the outcome of allogeneic HSCT in these patients will continue to improve with these modifications in the transplant approach. Disclosures: No relevant conflicts of interest to declare.

WHIM, a dominant immunodeficiency caused by mutations in the chemokine receptor CXCR4, leads to myeloid and lymphoid retention in tissues producing its cognate ligand, CXCL12 (SDF-1), by virtue of an exaggerated chemotactic and adhesion response to ligand. Gulino and colleagues (page [444][1]) have

Abstract 1226 How transcriptional and post-transcriptional mechanisms control the levels/activities of master developmental regulators has fundamental importance for understanding complex developmental processes such as hematopoiesis and associated pathological disorders. GATA-2 is an essential regulator of hematopoiesis, and GATA-2 mutations characterize heritable disease associated with myelodysplastic syndrome and acute myeloid leukemia, including MonoMAC (syndrome of monocytopendia, B and NK cell lymphopenia, and mycobacterial, fungal and viral infection). However, many questions remain unanswered regarding mechanisms underlying GATA-2 regulation and function. We demonstrated that a MonoMAC patient harbors a 28 bp deletion within GATA2 intron 5 that eliminates a conserved E-box and 5 base pairs of an 8 base pair spacer between the E-box and a conserved GATA motif, which constitutes an E-box-GATA composite element. This composite element resides within the +9.5 kb “GATA switch site” that binds GATA-2 and GATA-1 in the transcriptionally active and repressed states, respectively, and confers hematopoietic and vascular endothelial enhancer activities in transgenic mouse embryos. Importantly, this patient lacked mutations in the GATA2 coding sequence characteristic of other MonoMAC patients, but exhibited prototypical MonoMAC. To elucidate the mechanism underlying the function of the +9.5 composite element, we generated a targeted deletion of the murine element, which yielded embryonic lethality at E13 to E14. Prior to death, +9.5−/− mice exhibit reduced liver size, hemorrhaging, and edema. Nucleated primitive red cells are abundant in the +9.5−/− embryos, in contrast to Gata2 knockout mice, which die at approximately E10.5 from anemia due to failure of primitive and definitive hematopoiesis. Furthermore, primitive erythroid (EryP) colony assays conducted with yolk sacs revealed that the mutation does not affect primitive erythroid precursor functionality. However, the +9.5 deletion strongly reduced Gata2 expression at sites of definitive hematopoiesis, including the fetal liver (8.1 fold, P < 0.004) and cultured explants of the hematopoietic stem cell-generating Aortic Gonadal Mesonephric (AGM) region (4.0 fold, P < 0.001). The homozygous mutant animals exhibited a nearly complete loss of hematopoietic stem cells as determined by flow cytometry (20-fold reduction of Lin-Mac1+CD41-CD48-CD150+Sca+Kit+ cells, P < 0.005) and competitive repopulation (complete loss, P < 0.02) assays, as well as progenitors as determined by colony assays (BFU-E, 60-fold reduction, P < 0.002; CFU-GM, 8.8-fold reduction, P < 0.0001; CFU-GEMM, 19-fold reduction, P < 0.001). To investigate the underlying mechanisms, we developed an allele-specific Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay with heterozygous fetal liver cells to test whether the deletion influences Gata2 chromatin accessibility at the +9.5 region. The deletion significantly reduced (8.4 fold reduction, P < 0.001) chromatin accessibility at this region within the mutant allele, while the wild type allele was unaffected. Thus, any potential remaining cis-elements are insufficient to confer chromatin accessibility, supporting a model in which the transcription factors that normally occupy this GATA switch site lose the capacity to access their respective cis-elements in the context of the mutant allele. Our human and murine studies have therefore revealed a cis-element indispensable for the regulation of Gata2 expression in multiple developmental contexts and necessary for the generation of the definitive hematopoietic stem/progenitor cell compartment. As additional elements are likely to confer Gata2 expression in distinct contexts, including primitive erythropoiesis, we have implemented a multi-faceted effort to identify such elements and to compare their mechanisms with that of the +9.5 site, which will provide fundamental insights into genetic mechanisms controlling normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 3488 Germline heterozygous mutations in GATA2 have been reported to cause familial myelodysplasia–acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infection (monoMAC syndrome), dendritic cell, myeloid and NK cell lymphopenia (DCML), and Emberger syndrome (lymphedema and MDS). GATA2 is a zinc finger transcription factor that plays a crucial role in regulating growth of hematopoietic progenitors. In some pedigrees, patients or family members have manifestations of bone marrow failure. We hypothesized that patients with aplastic anemia (AA) may harbor mutations in GATA2. The coding regions and regulatory regions of GATA2 were sequenced in 99 patients with confirmed AA. Sequences from 100 normal individuals as well as published human genomes from unaffected individuals (dbSNP build 135 and 1000 Genomes Project) were used as controls. Genetic variants were confirmed in hematopoietic and somatic tissues. We identified 4 heterozygous mutations in regulatory regions of GATA2 in 5 patients. In two patients, a mutation at nucleotide 59T>G in exon 1 of isoform 2 was identified; both had severe AA in early adulthood refractory to immunosuppressive therapy. We noted this 59T>G mutation in two unrelated individuals with severe disseminated mycobacterial disease. We identified a mutation at nucleotide 20G>A in exon 2 of isoform 1, in a 3 year-old male with hepatitis-associated severe AA whose disease was refractory to multiple rounds of immunosuppressive therapy. Another mutation was present in 38G>A in exon 2 of isoform 1 in a 32 year old male with moderate AA and paroxysmal nocturnal hemoglobinuria (PNH). We also identified the exon 2 38G>A mutation in a patient with disseminated mycobacterial disease where reduced transcription of the mutant 38G>A allele was noted on RT-PCR. Finally, an intron 5, c.512+573 G>A variant was identified in an 18 year old male with severe AA who progressed after immunosuppressive therapy to MDS/AML. This variant, which causes a disruption of the FLI1 binding site, has also been found to be pathogenic in monoMAC syndrome. In summary, a subset of patients with AA were found to have mutations in GATA2 suggesting a role for the gene in the pathogenesis of bone marrow failure. It also may identify patients at higher risk of infectious complications, those who may have less advantageous responses to immune suppression, and command earlier bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3807 Background: Somatic and germline mutations in GATA2 were recently identified in patients diagnosed with MonoMAC, the hallmarks of which include mono cytopenia, B-cell and NK-cell lymphopenia, susceptibility to opportunistic infections (e.g. MAC), and a strong propensity to develop hypocellular MDS/AML or CMML. GATA2 mutations were also recently identified in other related disorders: Emberger syndrome (primary lymphedema with myelodysplasia), Familial MDS/AML, and DCML (Dendritic Cell, Monocytes, Lymphoid Deficiency). Family members with GATA2 mutations show variable penetrance and expressivity indicating that other factors may be required for development of disease and phenotype. GATA2 mutations are thought to result in loss of function or haploinsufficiency, but the precise mechanism for the development of cytopenias, immunodeficiency, and susceptibility to MDS remains to be elucidated. MicroRNA (miR) represents a unique mechanism of post-transcriptional gene regulation. In this study we generated microRNA profiles of patient derived MonoMAC cell lines followed by functional studies to identify aberrant miRs and their targets, which could potentially cooperate with GATA2 deficiency in generating hematologic disease. Inducible deletion of Myeloid Leukemia Cell 1 (Mcl1), a member of the Bcl2 family, in mice results in the loss of hematopoietic stem cells (HSCs) and progenitors, and in development of cytopenias. Design: RNA was isolated from EBV-immortalized B cells of 10 healthy controls and 13 MonoMAC patients with MDS and defined mutations in GATA2. microRNA expression profiles were generated using the Agilent high density human microRNA array. Array data were normalized to the data point of 75th percentile signal strength and to a set of spike-in and control probes. The differences between the means of experimental groups were analyzed by Mann-Whitney rank sum test. The miRs with significant p values (p≤ 0.05) and fold change (≥ 2-fold) in both normalization methods were selected for further analysis. TargetScan was utilized to predict the mRNA targets of aberrantly expressed miRs. miR targets were validated by functional studies in the Ly8 cell line. Results: Eight miRs were significantly differentially expressed (≥ 2-fold; p ≤ 0.05) as determined by microRNA microarray profiles. Six miRs showed increased expression in monoMAC cell lines compared to controls (miR-9, −181a-2–3p, −181c, −181c-3p, −486–3p, −582–5p) while two miRs showed significantly decreased expression (miR-223, −424–3p). Among the differentially expressed miRs that were validated by quantitative RT-PCR was miR-181c, which demonstrated a 2.2 fold increase in expression in MonoMAC cell lines (p = 0.013). Among the target transcripts potentially regulated by miR-181c, MCL1 expression was significantly decreased (2 fold; p = 0.018) in monoMAC cell lines in comparison to control cell lines. Transient transfection of miR-181c in Ly8 cells resulted in 40% decrease of MCL1 mRNA level, suggesting that miR-181c negatively regulates MCL1 in MonoMAC. Conclusions: These findings indicate that MonoMAC/GATA2 deficiency is associated with significantly decreased expression of MCL1 possibly through negative regulation involving miR-181c. Deletion of Mcl1 is known to cause apoptosis, loss of HSCs, and cytopenias in murine studies. Thus, down-regulation of MCL1 seen in MonoMAC/GATA2 deficiency may similarly favor unregulated apoptosis and the depletion of hematopoietic progenitors resulting in cytopenias, immunodeficiency, and risk of MDS/AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1264 Patients with familial myelodysplastic syndrome (MDS) associated with mutations in GATA2 are at increased risk of MDS and acute myeloid leukemia (AML). Specific clinical syndromes recently found to be due to mutations in GATA2 include MonoMAC (monocytopenia and mycobacterial infection), Emberger (MDS with severe lymphedema), and DCML (defects in dendritic cells, monocytes, and B and NK lymphoid cells). Features shared by many patients with these GATA2-associated syndromes include monocytopenia, markedly decreased B and NK cells, and clinical immunodeficiency manifested as warts and mycobacteria and fungal infections. MDS and/or AML occur with multilineage dyspoieses, particularly prominent in the megakaryocyte lineage (micromegakaryocytes, small mononuclear megakaryocytes, and large megakaryocytes with multiple separated nuclei). Several reports mention family members who are “asymptomatic,” without further details. We identified mutations in GATA2 in two of three families with familial MDS. In both families, one apparently healthy parent was found to have a GATA2 mutation; only in-depth laboratory examinations uncovered subtle findings consistent with familial GATA2 mutation in these clinically silent carriers. Family 1: The proband presented at age 15 with pancytopenia, and was found to have MDS and monosomy 7; he died from post-BMT complications including aspergillosis. His brother was found to have leukopenia, neutropenia and macrocytosis at age 13 during an infection with H1N1 influenza; the leukopenia and macrocytosis persisted. Six months later, repeat bone marrow showed early refractory anemia; the next year his marrow had myeloid dyspoiesis and dysplastic megakaryocytes; FISH showed −7 in 2.3% of cells, leading to classification as MDS-RCC. In retrospect, both boys had absolute monocytopenia ( Family 2: Three children in this family were diagnosed with MDS. The oldest had a history of warts and pancytopenia at age 18; his marrow showed MDS with trisomy 8. His brother was a compatible transplant donor, but he had mild pancytopenia and monocytopenia; his marrow had MDS and trisomy 8. Their sister was diagnosed at age 14 with MDS and trisomy 8; she, too, had monocytopenia. All 3 were transplanted. Subsequently, a mutation - c.1187G>A, p.R396Q - was found in GATA2, in all 3 brothers and their healthy father. He had normal blood counts (monocytes 500/uL) and immunoglobulins, but low B-cells in peripheral blood (CD20 23/uL) and bone marrow. His normocellular marrow had occasional atypical megakaryocytes with separated lobes, hypolobulation, and mononuclear and micromegakaryocytes. He, too, would not have been suspected to have GATA2-related MDS, and is also a clinically silent carrier. These two families indicate that familial GATA2-related MDS is a dominantly-inherited syndrome. In our two families, dominant inheritance was not initially considered, in part because the genetically affected parent was clinically asymptomatic. It is unclear whether GATA2 MDS shows “anticipation,” in which the younger generation is more severely affected than the parental generation. It is important that GATA2 be evaluated in families with apparently inherited childhood MDS, since the variable expression might lead inadvertently to selecting an asymptomatic GATA2 mutation carrier as a stem cell transplant donor. Genetic counseling needs to be provided with regard to risk to other family members. In addition, only long-term follow-up and surveillance of the clinically silent carriers will determine whether they remain unaffected. Disclosures: No relevant conflicts of interest to declare.

Abstract 405 Background: Recently, monoallelic mutations in the zinc-finger transcription factor GATA2 have been shown to be responsible for GATA2-deficiency, a syndrome characterized by opportunistic infections, frequently atypical mycobacterial infections or MAC, and a hypocellular myelodysplastic syndrome (MDS) that transforms into acute myelogenous leukemia (AML). GATA2-deficiency was previously known by several other names: MonoMAC (Monocytopenia and Atypical Mycobacterial Infection), DCML (Dendritic Cell Monocyte, Lymphoid Deficiency), Familial MDS/AML, or Emberger syndrome (lymphedema with monosomy 7). Heterogeneous genetic defects in GATA2 result in haploinsufficiency in both spontaneous and familial forms of the disease. Predicting the transformation from MDS to AML in GATA2-deficiency has clinical implications for both prognosis and the timing of hematopoietic stem cell transplantation. ASXL1, a gene related to the Drosophilia additional sex combs gene, encodes a chromatin binding/transcription repressor protein that is frequently mutated in MDS/AML. Mutations in ASXL1 are associated with reduced time to progression to AML and poor overall survival, independent of IPSS score. Methods: We sequenced the critical region of the ASXL1 gene in 20 patients with GATA2- deficiency to determine the frequency of ASXL1 mutations, and to correlate the presence of ASXL1 mutations with hematopoietic transformation. Since the ASXL1 mutations described in hematopoietic malignancies are located within the coding sequence of the two 3Õ-terminal exons (COSMIC: Catalogue of Somatic Mutations in Cancer), this ∼4.3kb region of ASLX1 was amplified by PCR and sequenced using five overlapping primer sets with substrate DNA isolated from mononuclear cell and granulocyte cell preparations from peripheral blood or bone marrow aspirates, or from extracts prepared from unfixed, unstained bone marrow aspirates. Mutations were confirmed with at least two independent PCR reactions with two unique primer sets. Results: Somatic ASXL1 mutations were detected in 8 of 20 patients with GATA2 mutations, 19/20 of whom had MDS. Five of these ASXL1 mutations have been previously associated with MDS/AML, including four independent cases of the most frequently described ASXL1 mutation (G646fs*12insG). The other four mutations were found once each; two of these were previously unreported (G652S(G>A) and L817fs*1delT). The patient cohort included two sisters with the same germline GATA2R398W mutation, but different somatic ASXL1 mutations (G464fs*12insG and R693X(C>T)). ASXL1 mutations were found in 4/5 GATA2- deficiency patients whose MDS had transformed into chronic myelomonocytic leukemia (CMML). Overall survival was lower for GATA2-deficiency patients with ASXL1 mutation (50% survival) compared to patients without ASXL1 mutation (83% survival), and was independent of IPSS score. Conclusions: ASXL1 is frequently mutated in patients with GATA2-deficiency with at least 40% of patients having a mutation in ASXL1 compared to a 10–15% mutation rate reported for all MDS/AML patients. ASXL1 mutation correlates with the development of CMML and with poor overall survival, as reported previously for MDS/AML patients. There was no correlation between the presence and type of ASXL1 mutation and the specific GATA2 mutation: the eight different ASXL1 mutation events were found in six different GATA2 mutant backgrounds. These results are directly relevant to the prognosis and the timing of hematopoietic stem cell transplantation for GATA2-deficiency. Disclosures: No relevant conflicts of interest to declare.

Abstract 3091 Background: Recently, sporadic and heritable mutations in the zinc finger transcription factor GATA2 were shown to be responsible for four different syndromes in young adults coupling opportunistic infection with a predilection to develop myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). These four syndromes are: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome, lymphedema and MDS with monosomy 7; and familial MDS/AML. Life-threatening infections, and the transformation to AML, either alone or together, constitute a rationale for allogeneic hematopoietic stem cell transplant (HSCT) for GATA2 deficiency. Methods: We evaluated matched related, unrelated, and umbilical cord blood as donor sources for nonmyeloablative conditioning for HSCT in GATA2 deficiency. Twelve patients with GATA2 deficiency underwent allogeneic transplant: 4 received peripheral blood stem cells (PBSCs) from matched related-donors (MRD), 4 received PBSC from matched unrelated-donors (MUD), and 4 received umbilical cord blood (UCB) units. Recipients of MRD and MUD transplant received fludarabine and 200cGy of total body irradiation (TBI), while UCB recipients received cyclophosphamide 50 mg/kg, fludarabine, and 200cGy of TBI. All patients received tacrolimus and sirolimus for GVHD prophylaxis. This cohort of patients had considerable pre-transplant morbidity: two patients required baseline oxygen for pulmonary alveolar proteinosis, one of whom was on a ventilator at the time of transplant; one patient had active hepatitis C that was not responding to therapy; one patient had RAEB-2; two patients were platelet transfusion-dependent; one patient had recurrent strokes and culture negative endocarditis two months before transplant. Results: Median follow-up for patients was 14.4 months (range 0.2–38.2 months). Ten of 11 patients engrafted at a median of 10 days (range 0–76); engraftment was not evaluable in a recipient of an UCB transplant due to death early in the post-transplant period. One rejection occurred in a recipient of a double UCB transplant who had been heavily transfused pre-transplant. All patients who engrafted had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant, and all had reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of NTM infection and extensive human papilloma virus infections regressed starting around 6 months post transplant. Two patients required a single cycle of pre-transplant chemotherapy for RAEB-1 and RAEB-2, respectively. In both patients the monosomy 6 and monosomy 7 clones have not recurred, now 2.5 years and 9 months following single UCB and MUD transplant, respectively. Three patients died, two early after transplant. One recipient of UCB, who had pre-transplant hepatitis C, died on day+7 of fulminant liver failure and sepsis. A second patient, who was intubated at the time of transplant because of severe pulmonary alveolar proteinosis and pulmonary hypertension, died on day + 88 of grade IV GVHD after MRD transplant. The third patient died 9 months after transplant of sepsis. One recipient of a MRD relapsed from her MDS at 1 year following transplant and was retransplanted using a myeloablative conditioning regimen. She is alive with no evidence of MDS at 3 months. Acute GVHD developed in 6 patients, five of which were steroid-responsive. One patient developed chronic GVHD. Other complications included immune mediated cytopenias that responded to rituximab and eltrombopag. Conclusions: Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B and NK cell populations and reversal of the infection susceptibility phenotype. The TRM in this cohort of patients with severe comorbidities was 25%. Now that genetic testing for GATA2 mutations is available, we anticipate that earlier diagnosis will enable patients to be transplanted earlier in the course of disease, before significant organ damage or clonal evolution of MDS to AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3293 Background: Hyper-immunoglobulin E syndrome (Job's syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses from inappropriately mild immune responses to staphylococcal infection. Both sporadic and autosomal dominant Job's syndrome patients are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job's syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development and function in Job's syndrome patients has not been studied yet. Previous work performed in our lab employing STAT3 activating cytokines and STAT3 inhibitors showed that STAT3 activation plays a role in NK cell proliferation, survival, cytolytic activity, and regulation of expression of the activating receptor NKG2D. This finding prompted us to test whether aberrant STAT3 activation affects NK cell proliferation, survival and cytolytic activity, in NK cells of Job's syndrome patients. Objective: To assess proliferation, cytolytic activity and receptor expression on NK cells from Job's syndrome patients with dominant negative STAT3 mutations. Methods: NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cytolytic activity was evaluated by Calcein release assay. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by paired Student's t test using GraphPad Prism. Results: Flow-cytometric analysis of PBMCs showed significantly lower percentage of NK cells, identified as CD3− CD56+, in Job's syndrome patients compared to normal donors. The expansion in response to membrane bound IL21 stimulation was found to be impaired in NK cells obtained from Job's syndrome patients compared to those from normal donors. Expanded NK cells from Job's syndrome patients had lower percentage of mature, CD56+CD16+NK cells compared to those from normal donors. We also found significantly reduced cytolytic activity in NK cells from Job's syndrome patients. STAT3 activating cytokines IL10 and IL21 were also found to be impaired in their ability to induce NKG2D expression on NK cells obtained from Job's syndrome patients. Conclusions: Our finding of deficient NK cell number and cytolytic activity in Job's syndrome patients is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job's syndrome patient's NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job's syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job's syndrome patients are also more prone to viral infections and lymphoma. Given the role of NK cells in the immune surveillance of virally infected and tumorigenic cells, low NK cell function may explain the proclivity of Job's syndrome patients to viral infections and lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract SCI-20 Nontuberculous mycobacteria are relatively ubiquitous organisms of low virulence, disseminated infection with which suggests an underlying defect in host defense. Dimorphic molds (e.g., histoplasmosis, coccidioidomycosis, penicilliosis) are regionally limited and cause severe infections only in the immune compromised. We have focused on severe cases of these infections to identify genetic and acquired syndromes of immune deficiency that have not been previously recognized. 1. The syndrome of monocytopenia, NK, and B cell lymphopenia and lack of circulating dendritic cells is associated with severe infections with mycobacteria, dimorphic molds, human papilloma virus, and molluscum cantagiosum. These patients also develop myelodysplasia and pulmonary alveolar proteinosis and may progress to frank leukemia. Mutations in GATA2 are responsible for the syndrome (monoMAC for monocytopenia and M. avium complex infection). Hematopathologically, patients have micromegakaryocytes and cytogenetic abnormalities, most commonly monosomy 7 and trisomy 8. Both protein-positive and protein-negative heterozygous mutations have been identified, suggesting that haploinsufficiency is a unifying cause of disease. The spectrum of pathologic mutations is broad and likely extends into phenotypes that are distinct from those recognized initially. 2. Mutations in STAT1 have been associated with mycobacterial disease (dominant negative inhibitory), fatal viral and bacterial disease (recessive), and mucocutaneous candidiasis (dominant gain of function). We have identified distinct and independent STAT1 mutations in three patients that have led to severe, refractory disseminated coccidioidomycosis or histoplasmosis. These mutations lead to hypomethylation of STAT1, which leads to its persistent hyperphosphorylation, and impairment of restimulation activity. These mutations are important because they lie in the same plane of the molecule and cause severe invasive dimorphic yeast infections. 3. Autoantibodies are common, especially in adult women. We have previously recognized East Asia-born adult women living in the United States who have high titer autoantibodies to interferon gamma (IFNγ). We have recently recognized large cohorts of similar patients in Thailand and Taiwan and conducted a prospective trial of their phenotype and the presence and type of anti-IFNγ autoantibodies. We found a significant population of patients with disseminated nontuberculous mycobacterial infections (but not tuberculosis), salmonella, cryptococcosis, histoplasmosis, or varicella zoster in Asia whose sole identifiable risk factor was extremely elevated anti-IFNγ autoantibodies. We have characterized the severity of the blockade and the subclass of the antibodies and explored the rate in several control populations as well. These apparently adult-onset acquired autoantibodies profoundly inhibit IFNγ signaling, leading to infections with IFNγ-responsive organisms. These emerging syndromes cross the lines of infectious disease, cancer, and rheumatology. Disclosures: No relevant conflicts of interest to declare.