ALBERT

Description: IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4+ T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4+ T cells. RNA-Seq analysis of CD4+ T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21–mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4+ T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21–mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4+ T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.

Description: Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital- and community-acquired pathogen, but the mechanisms underlying host-defense to MRSA remain poorly understood. Here, we investigated the role of IL-21 in this process. When administered intra-tracheally into wild-type mice, IL-21 induced granzymes and augmented clearance of pulmonary MRSA but not when neutrophils were depleted or a granzyme B inhibitor was added. Correspondingly, IL-21 induced MRSA killing by human peripheral blood neutrophils. Unexpectedly, however, basal MRSA clearance was also enhanced when IL-21 signaling was blocked, both in Il21r KO mice and in wild-type mice injected with IL-21R-Fc fusion-protein. This correlated with increased type I interferon and an IFN-related gene signature, and indeed anti-IFNAR1 treatment diminished MRSA clearance in these animals. Moreover, we found that IFNβ induced granzyme B and promoted MRSA clearance in a granzyme B-dependent fashion. These results reveal an interplay between IL-21 and type I IFN in the innate immune response to MRSA.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.