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Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

Publisher's Note: There is a Blood Commentary on this article in this issue.

MHC class II deficiency is a rare, but life-threatening, primary combined immunodeficiency. Hematopoietic cell transplantation (HCT) remains the only curative treatment for this condition, but transplant survival in the previously published result was poor. We analyzed the outcome of 25 patients with MHC class II deficiency undergoing first HCT at Great North Children's Hospital between 1995 and 2018. Median age at diagnosis was 6.5 months (birth to 7.5 years). Median age at transplant was 21.4 months (0.1-7.8 years). Donors were matched family donors (MFDs; n = 6), unrelated donors (UDs; n = 12), and haploidentical donors (HIDs; n = 7). Peripheral blood stem cells were the stem cell source in 68% of patients. Conditioning was treosulfanbased in 84% of patients; 84% received alemtuzumab (n = 14) or anti-thymocyte globulin (n = 8) as serotherapy. With a 2.9-year median follow-up, OS improved from 33% (46-68%) for HCT before 2008 (n = 6) to 94% (66-99%) for HCT after 2008 (n = 19; P = .003). For HCT after 2008, OS according to donor was 100% for MFDs and UDs and 85% for HIDs (P = .40). None had grade III-IV acute or chronic graft-versus-host disease. Latest median donor myeloid and lymphocyte chimerism were 100% (range, 0-100) and 100% (range, 64-100), respectively. Latest CD4+ T-lymphocyte number was significantly lower in transplant survivors (n = 14) compared with posttransplant disease controls (P = .01). All survivors were off immunoglobulin replacement and had protective vaccine responses to tetanus and Haemophilus influenzae. None had any significant infection or autoimmunity. Changing transplant strategy in Great North Children's Hospital has significantly improved outcomes for MHC class II deficiency.

Accumulation of CD3(+) T-cell receptor (TCR)αβ(+)CD4(-)CD8(-) double-negative T cells (DNT) is a hallmark of autoimmune lymphoproliferative syndrome (ALPS). DNT origin and differentiation pathways remain controversial. Here we show that human ALPS DNT have features of terminally differentiated effector memory T cells reexpressing CD45RA(+) (TEMRA), but are CD27(+)CD28(+)KLRG1(-) and do not express the transcription factor T-bet. This unique phenotype was also detected among CD4(+) or CD8(+) ALPS TEMRA cells. T-cell receptor β deep sequencing revealed a significant fraction of shared CDR3 sequences between ALPS DNT and both CD4(+) and CD8(+)TEMRA cells. Moreover, in ALPS patients with a germ line FAS mutation and somatic loss of heterozygosity, in whom biallelic mutant cells can be tracked by absent Fas expression, Fas-negative T cells accumulated not only among DNT, but also among CD4(+) and CD8(+)TEMRA cells. These data indicate that in human Fas deficiency DNT cannot only derive from CD8(+), but also from CD4(+) T cells. Furthermore, defective Fas signaling leads to aberrant transcriptional programs and differentiation of subsets of CD4(+) and CD8(+) T cells. Accumulation of these cells before their double-negative state appears to be an important early event in the pathogenesis of lymphoproliferation in ALPS patients.

In this retrospective collaborative study, we have analyzed long-term outcome and donor cell engraftment in 194 patients with Wiskott-Aldrich syndrome (WAS) who have been treated by hematopoietic cell transplantation (HCT) in the period 1980- 2009. Overall survival was 84.0% and was even higher (89.1% 5-year survival) for those who received HCT since the year 2000, reflecting recent improvement of outcome after transplantation from mismatched family donors and for patients who received HCT from an unrelated donor at older than 5 years. Patients who went to transplantation in better clinical conditions had a lower rate of post-HCT complications. Retrospective analysis of lineage-specific donor cell engraftment showed that stable full donor chimerism was attained by 72.3% of the patients who survived for at least 1 year after HCT. Mixed chimerism was associated with an increased risk of incomplete reconstitution of lymphocyte count and post-HCT autoimmunity, and myeloid donor cell chimerism < 50% was associated with persistent thrombocytopenia. These observations indicate continuous improvement of outcome after HCT for WAS and may have important implications for the development of novel protocols aiming to obtain full correction of the disease and reduce post-HCT complications.

Children with primary immunodeficiency diseases, particularly those less than 1 year of age, experience significant toxicity after hematopoietic stem cell transplantation, with busulfan- or melphalan-based conditioning. Treosulfan causes less veno-occlusive disease than busulfan and does not require pharmacokinetic monitoring. We report its use in 70 children. Children received 42 g/m2 or 36 g/m2 with cyclophosphamide 200 mg/kg (n = 30) or fludarabine 150 mg/m2 (n = 40), with alemtuzumab in most. Median age at transplantation was 8.5 months (range, 1.2-175 months); 46 (66%) patients were 12 months of age or younger. Donors were as follows: matched sibling donor, 8; matched family donor, 13; haploidentical, 4; and unrelated, 45. Median follow-up was 19 months (range, 1-47 months). Overall survival was 81%, equivalent in those age less or greater than 1 year. Skin toxicity was common. Veno-occlusive disease occurred twice with cyclophosphamide. Eighteen patients (26%) had graft-versus-host disease, and only 7 (10%) greater than grade 2. Two patients rejected; 24 of 42 more than 1 year after transplantation had 100% donor chimerism. The remainder had stable mixed chimerism. T-cell chimerism was significantly better with fludarabine. Long-term follow-up is required, but in combination with fludarabine, treosulfan is a good choice of conditioning for hematopoietic stem cell transplantation in primary immunodeficiency disease.

Severe combined immunodeficiency (SCID) carries a poor prognosis without definitive treatment by hematopoietic stem cell transplantation. The outcome for transplantation varies and is dependent on donor status and the condition of the child at the time of transplantation. Diagnosis at birth may allow for better protection of SCID babies from infection and improve transplantation outcome. In this comparative study conducted at the 2 designated SCID transplantation centers in the United Kingdom, we show that SCID babies diagnosed at birth because of a positive family history have a significantly improved outcome compared with the first presenting family member. The overall improved survival of more than 90% is related to a reduced rate of infection and significantly improved transplantation outcome irrespective of donor choice, conditioning regimen used, and underlying genetic diagnosis. Neonatal screening for SCID would significantly improve the outcome in this otherwise potentially devastating condition.

CD40 ligand (CD40L) deficiency causes recurrent sinopulmonary infection, Pneumocystis carinii pneumonia, and Cryptosporidium parvum infection. Approximately 40% to 50% of patients survive to the third decade: long-term survival is unclear. Hematopoietic stem cell transplantation (HSCT) is curative. We present a retrospective analysis of 38 European patients undergoing HSCT for CD40L deficiency in 8 European countries between 1993 and 2002. Donor stem cell source included 14 HLA-identical siblings, 22 unrelated donors, and 2 phenotypically matched parental stem cells (12 T-cell depleted). Of the patients, 34 engrafted and 26 (68%) survived; 3 had autologous reconstitution, 22 (58%) were cured, and 1 engrafted but has poor T-cell immune reconstitution. There were 18 evaluated patients who responded to vaccination. Of the patients, 12 (32%) died from infection-related complications, with severe cryptosporidiosis in 6. Grades 2 to 4 graft-versus-host disease (GvHD) associated with infection occurred in 6 of 12 fatal cases. HSCT cured 58% of patients, 72% of those without hepatic disease. Early T-cell function following whole marrow HSCT may limit cryptosporidial disease, but survival was similar after T-cell-depleted HSCT. Preexisting lung damage was the most important adverse risk factor. Further studies will determine optimal timing and type of HSCT.

Human primary immunodeficiency diseases are experiments of nature characterized by an increased susceptibility to infection. In many cases, they are also associated with troublesome and sometimes life-threatening autoimmune complications. In the past few years, great strides have been made in understanding the molecular basis of primary immunodeficiencies, and this had led to more focused and successful treatment. This review has 3 aims: (1) to highlight the variety of autoimmune phenomena associated with human primary immunodeficiency diseases; (2) to explore how primary immunodeficiencies predispose patients to autoimmune phenomena triggered by opportunistic infections; and (3) to consider the rationale for the current treatment strategies for autoimmune phenomena, specifically in relation to primary immunodeficiency diseases. Reviewing recent advances in our understanding of the small subgroup of patients with defined causes for their autoimmunity may lead to the development of more effective treatment strategies for idiopathic human autoimmune diseases.

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 ( STAT3 ) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3 . Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.

The human syndrome of dendritic cell, monocyte, B and natural killer lymphoid deficiency presents as a sporadic or autosomal dominant trait causing susceptibility to mycobacterial and other infections, predisposition to myelodysplasia and leukemia, and, in some cases, pulmonary alveolar proteinosis. Seeking a genetic cause, we sequenced the exomes of 4 unrelated persons, 3 with sporadic disease, looking for novel, heterozygous, and probably deleterious variants. A number of genes harbored novel variants in person, but only one gene, GATA2, was mutated in all 4 persons. Each person harbored a different mutation, but all were predicted to be highly deleterious and to cause loss or mutation of the C-terminal zinc finger domain. Because GATA2 is the only common mutated gene in 4 unrelated persons, it is highly probable to be the cause of dendritic cell, monocyte, B, and natural killer lymphoid deficiency. This disorder therefore constitutes a new genetic form of heritable immunodeficiency and leukemic transformation.

Abstract 2045 The novel syndrome of Dendritic Cell, Monocyte, B and NK lymphocyte (DCML) deficiency has recently been characterised and found to be caused by mutations in the transcription factor GATA-2 (Dickinson et al. Blood 2011, Hsu et al. Blood 2011). In this report we describe a series of 10 patients and show that there is a high risk of death unless curative haematopoietic stem cell transplantation (HSCT) is performed. Our case series includes 10 patients; 4 sporadic cases and 6 cases from 2 pedigrees showing autosomal dominant inheritance. 7 patients died; of the 3 survivors, 2 were transplanted and show marked resolution of their disease with a follow up of 36 and 11 months, respectively. Causes of death in the patients who died include disseminated mycobacterium avium infection (2) H1N1 influenza (1) candidiasis (1) pulmonary alveolar proteinosis (2) and CMV pneumonitis (1). At least 8 patients had chronic HPV infection and 2 patients had autoimmune phenomena including inflammatory arthritis and erythema nodosum. One patient also developed lymphoma. All patients had monocytopenia and decreased B and NK cells with normal T cells at presentation. Haemoglobin, platelets and neutrophil counts were within normal ranges or mildly reduced. In 4 patients, near absolute dendritic cell (DC) deficiency and elevated Flt-3 ligand was also confirmed. BM aspirate showed dysplastic megakaryocytes and increased fibrosis in some instances. Macrophages were present in dermis, bone marrow and lung, epidermal Langerhans cells were largely preserved and plasma cells were found in inflammatory lesions by immunohistochemistry. GATA-2 mutation was confirmed by Sanger sequencing in all cases. Of the 2 patients treated with transplantation, the first patient presented aged 12 with disseminated BCG sepsis 6 months following BCG vaccination. Skin biopsy showed acid fast bacilli but no well-formed granulomata, and ongoing sepsis failed to respond to antitubercular therapy including addition of IFN gamma. The second patient presented to gynaecology services at the age of 20 with vulval intraepithelial neoplasia stage 3 (VIN3), and then to respiratory physicians a year later with breathlessness, productive cough, weight loss and night sweats; pulmonary alveolar proteinosis was diagnosed on biopsy. She continued to deteriorate despite whole lung lavage, and developed respiratory failure requiring 35% oxygen and nocturnal non-invasive ventilation (NIV). The patient's family history included two generations affected by haematological malignancy or respiratory illness in early adulthood, inherited in a fashion suggesting an autosomal dominant trait. Both patients underwent reduced intensity allogeneic haematopoietic stem cell transplantation with PBMC from unrelated adult donors (patient 1 – 10/12 HLA match; patient 2 – 9/12); transplant conditioning was with Fludarabine 150mg/m2, Melphalan 140mg/m2 and Alemtuzumab 60mg (patient 1), and Fludarabine 150mg/m2, Busulphan 6.4mg/kg, Alemtuzumab 60mg (patient 2). GVHD prophylaxis was with Ciclosporin and Mycophenolate Mofetil. Both transplants were uneventful. 32 months post-transplantation, patient 1 is well and off all medication. The monocyte count, lymphocyte subsets and immunoglobulins are normal and there were good responses to tetanus and HIB vaccinations. Chimerism shows 100% donor myeloid and B cells and 85% donor T cells. 9 months post-transplantation patient 2 has improved significantly and no longer requires oxygen or NIV. Lung function tests and radiology have significantly improved. Monocytes are in the normal range and chimerism shows 100% donor CD15 and 85% donor CD3. Follow-up continues with gynaecology for VIN3 associated with HPV16/18 infection. Neither patient has developed GVHD. Here we show that DCML deficiency caused by GATA-2 mutation incurs a high risk of mortality due to infection or respiratory failure unless treated by HSCT. Rapid correction of both immunodeficiency and pulmonary alveolar proteinosis is possible, even in the context of severe infection or respiratory failure, using reduced intensity conditioning. The absence of GVHD in both these cases, despite significant HLA-mismatching, may reflect the absence of recipient DC at transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3460 Autoimmune diseases (AD) have been reported after haematopoetic stem cell transplantation (HSCT) and most commonly autoimmune cytopenias (AIC) are observed. Proposed risk factors are unrelated donor HSCT and presence of chronic graft-versus-host disease (GVHD). Use of CB cells is increasing as an alternative source of adults HSC for allotransplant however incidence and risk factors of AD after CBT have not been described. Since CB lymphocytes are immature and most of the CBT are HLA mismatched, we hypothesize a high incidence and diversity of secondary AD and. Therefore, we conducted a retrospective study in order to describe incidence, nature and risk factors for secondary AD after CBT. We include CBT recipients reported to EUROCORD up to December 2008. All centres were asked to participate to the survey even if they did not observe any case of AD after CBT. For those with AD, detailed information on diagnosis, treatment and outcome were asked. Median follow-up was 44 months (3.2-217). 42 out of 651 patients (pts) from 35 centres had developed at least one secondary AD at a median of 198 days (27-4267) after CBT. The remaining 609 pts without secondary AD served as control group (nonAD). Characteristics of both groups are given in table 1. Cumulative incidence of secondary AD after CBT was 5±1% after 3 years and 6.5±1% after 10 years. Diagnosis of secondary AD were autoimmune haemolytic anemia (AIHA) in 16, autoimmune thrombopenia (ITP) in 9, EVANS syndrome in 6, autoimmune thyroiditis in 3, glomerulonephritis in 2, Graves disease, psoriasis, psoriasis arthritis, immune neutropenia, ulcerative colitis and vasculitis in 1 patient each. Three pts developed ITP followed by AIHA. Median time to develop a AIC was 180 days (27-4267) and non-hematological AD was 322 days (70-1117), respectively (p=0.13). At onset of AD, 3 pts had acute GvHD, 6 chronic GvHD and in 10 pts an infection. In multivariate analysis adjusted for differences between the two groups, the following factors were associated with higher incidence of secondary AD: 1) age Table 1: Characteristics of pts developing AD or not (nonAD) Pts characteristics AD pts nonAD pts P female sex 36% 45% age at HSCT 2.23 (0.2–43.24) years 10.7 (0.11–66.5) years 0.0001 year of HSCT 2006 (95–08) 2006 (92–08) 0.04 Type of donor unrelated 95% 90% 0.26 Indication for HSCT (n) ALL 10 184 AML 2 150 MDS 4 67 AA 5 52 SCID 6 51 CML 3 24 NHL 0 21 Hemoglobinopathy 0 15 Histiocytosis 2 14 Metabolic diseases 9 19 others 1 11 Graft Single cord 85% 77% 0.25 Double cord 14% 23% HLA compatibility 5/6+6/6 68% 48% 0.02 Conditioning RIC 80% 73% 0.34 TBI 20% 43% 0.0004 Serotherapy 73% 74% 0.86 Host CMV status positive 45% 58% 0.11 Disclosures: No relevant conflicts of interest to declare.

Abstract 2301 Poster Board II-278 NK-cell alloreactivity in the setting of allogeneic transplantation is largely determined by the specificity of the killer immunoglobulin receptors (KIR) on donor NK cells for recipient HLA class I. We have recently shown that KIR ligand mismatch in the graft-versus-host (GvH) direction between donor and recipient is associated with improved outcomes after unrelated cord blood transplantation (UCBT) for patients with acute leukemia in complete remission. However, this finding was not confirmed in a series of reduced intensity conditioning and UCBT using 2 unrelated cord blood units. Therefore, those controversial results can raise some questions on the donor choice based on KIR ligand matching status. It is not known whether KIR ligand matching is associated with outcomes after UCBT for non-malignant disorders. To address this question we have analyzed patients with non-malignant disorders who had received a UCBT using a single cord blood unit and were reported to the Eurocord registry with sufficient HLA-A, -B, -C typing to assign KIR ligand groups. In agreement with previous studies in malignant diseases, KIR ligand mismatch (KIR+) in the GvH direction occurred when the donor expressed one or several of the following KIR ligands which were lacking in the recipient: HLA-Bw4, and HLA-C group 1 or 2. In addition patients and cord blood units were grouped according to HLA A-3/A-11 alleles, which are putative KIR ligands. One-hundred-thirty-seven eligible patients were selected, mostly children (n=124). UCBT was performed from January 2000 to December 2008 in 47 EBMT-centers. The frequency of KIR ligand mismatched (KIR+) pairs reported was 23% (n=31). Forty-three percent of patients had bone marrow failure syndromes (BMFS) (n=60), other diagnosis included metabolic disorders (n=41), SCID (n=32), hemoglobinopathies (n=2), autoimmune diseases (n=2). Median follow up was 24 months (4-101). Median recipient age was 3 years. UCB units were HLA matched at 6 of 6 (n=38), 5 of 6 (n=62), 4 of 6 (n=34) and 3 of 6 (n=3). Median cell dose infused was 5.6 ×10E7 TNC/Kg and 2.4 ×10E5 CD34 cells/Kg. Conditioning regimen was myeloablative in 48% of cases and reduced intensity in 52% and included ATG in 90% and fludarabine in 42%. Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporine (CsA) and corticosteroids, CsA alone and CsA and MMF in 47%, 23% and 14% of patients, respectively. Cumulative incidence (CI) of the day-60-neutrophil recovery was 76±3% (median time: 22 days) and it was not associated with KIR mismatch [(74±4% for KIR- and 80±6% for KIR+ group (p=0.9)]. CI of aGvHD was 21±3%, only 3 patients out of 31 with KIR+ presented aGvHD (II-IV). The estimated probability of 2-year overall survival (OS) was 63±4%; it was 64±5% in the group of KIR- and 63±3% in the KIR+ (p=0.5). The main cause of death was graft failure associated with infections (n=16, 32%). There was no statistical difference in frequency of causes of death between the 2 KIR ligand groups. We have also looked the association of KIR ligand mismatch in the host versus graft (HvG) direction. The frequency of KIR mismatched pairs in the HvG direction was 22% and it was not associated with neutrophil recovery (76±4% for KIR ligand matched in HvG direction versus 73±6% for KIR ligand mismatched in HvG direction, p=0.9) or OS (64±5% for KIR ligand matched in HvG direction versus 58±7% for KIR ligand mismatched in HvG direction, p=0.6) In conclusion KIR ligand mismatching in the GvH or HvG direction is not associated with outcomes after UCBT for children with non malignant disease. Therefore it is not useful in the algorithm of donor choice in this setting. Disclosures: No relevant conflicts of interest to declare.

To the Editor: Recent studies by Yamamoto et al[1][1] and Benis and Schneider[2][2] in Blood suggest that Gc-globulin (also known as vitamin D–binding protein) may have an important role in macrophage activation and osteoclast differentiation from monocytes and so may control bone morphogenesis