Jennifer Heimall

A Critical Role for STAT3 Transcription Factor Signaling in the Development and Maintenance of Human T Cell Memory

STAT3 transcription factor signaling in specific T helper cell differentiation has been well described, although the broader roles for STAT3 in lymphocyte memory are less clear. Patients with autosomal-dominant hyper-IgE syndrome (AD-HIES) carry dominant-negative STAT3 mutations and are susceptible to a variety of bacterial and fungal infections. We found that AD-HIES patients have a cell-intrinsic defect in the number of central memory CD4(+) and CD8(+) T cells compared to healthy controls. Naive T cells from AD-HIES patients had lower expression of memory-related transcription factors BCL6 and SOCS3, a primary proliferation defect, and they failed to acquire central memory-like surface phenotypes in vitro. AD-HIES patients showed a decreased ability to control varicella zoster virus (VZV) and Epstein-Barr virus (EBV) latency, and T cell memory to both of these viruses was compromised. These data point to a specific role for STAT3 in human central memory T cell formation and in control of certain chronic viruses.

Pathogenesis of hyper IgE syndrome.

Hyper IgE syndrome (HIES) is a rare primary immunodeficiency characterized by the triad of elevated IgE and eosinophilia, eczema, and recurrent skin and pulmonary infections. The autosomal dominant (AD) form of HIES results from mutations in STAT3 and is characterized by disordered inflammation, connective tissue, and skeletal abnormalities. Tissue-specific STAT3 deficiency in animals, cytokine and transcriptional array data, and careful clinical phenotyping have explained some of the pathophysiology of the immunologic and non-immunologic abnormalities of AD-HIES. In depth study of the role of STAT3 mutations in specific aspects of HIES may lead to better understanding and new approaches to treatment of conditions intrinsic to HIES that are common in the general population, such as staphylococcal infections, scoliosis, osteoporosis, bronchiectasis, and arterial aneurysms. As the genotypes of STAT3 deficiency are further characterized, genotype–phenotype correlations may emerge that will be informative regarding specific molecular interactions. Autosomal recessive forms of hyper IgE (AR-HIES) have also been reported. A single case of homozygous deficiency of the signal protein Tyk2 has been reported as well as a recessive syndrome with some features overlapping AD-HIES, but for which the genetic etiology is unknown. Better understanding of the pathophysiology and mechanisms of dominant and recessive hyper IgE syndromes will shed light on somatic and immune biology and may improve quality of life and survival for HIES patients.

Omalizumab facilitates rapid oral desensitization for peanut allergy

Background: Peanut oral immunotherapy is a promising approach to peanut allergy, but reactions are frequent, and some patients cannot be desensitized. The anti‐IgE medication omalizumab (Xolair; Genentech, South San Francisco, Calif) might allow more rapid peanut updosing and decrease reactions. Objective: We sought to evaluate whether omalizumab facilitated rapid peanut desensitization in highly allergic patients. Methods: Thirty‐seven subjects were randomized to omalizumab (n = 29) or placebo (n = 8). After 12 weeks of treatment, subjects underwent a rapid 1‐day desensitization of up to 250 mg of peanut protein, followed by weekly increases up to 2000 mg. Omalizumab was then discontinued, and subjects continued on 2000 mg of peanut protein. Subjects underwent an open challenge to 4000 mg of peanut protein 12 weeks after stopping study drug. If tolerated, subjects continued on 4000 mg of peanut protein daily. Results: The median peanut dose tolerated on the initial desensitization day was 250 mg for omalizumab‐treated subjects versus 22.5 mg for placebo‐treated subject. Subsequently, 23 (79%) of 29 subjects randomized to omalizumab tolerated 2000 mg of peanut protein 6 weeks after stopping omalizumab versus 1 (12%) of 8 receiving placebo (P < .01). Twenty‐three subjects receiving omalizumab versus 1 subject receiving placebo passed the 4000‐mg food challenge. Overall reaction rates were not significantly lower in omalizumab‐treated versus placebo‐treated subjects (odds ratio, 0.57; P = .15), although omalizumab‐treated subjects were exposed to much higher peanut doses. Conclusion: Omalizumab allows subjects with peanut allergy to be rapidly desensitized over as little as 8 weeks of peanut oral immunotherapy. In the majority of subjects, this desensitization is sustained after omalizumab is discontinued. Additional studies will help clarify which patients would benefit most from this approach.

Filaggrin mutations and atopy: consequences for future therapeutics

Filaggrin is a key component of the epidermal differentiation complex of the stratum corneum in the epidermal layer of human skin. Loss-of-function mutations in filaggrin have been described in patients with atopic eczema and are associated with an increased risk of atopic sensitization in these individuals. Atopic eczema is the first stage of the atopic march that describes the phenomenon of increased rates of allergic rhinitis and allergic asthma observed in individuals with early atopic dermatitis. The skin barrier disruptions of atopic eczema associated with loss-of-function mutations in filaggrin are thought to provide a nidus for allergic sensitization to food and aeroallergens, which can then lead to increased allergic disease. It is on this foundation that therapies aimed at restoration of barrier function are thought to play a role, not only in the effective treatment of atopic eczema, but also in the prevention of further allergic disease development.

Paucity of genotype-phenotype correlations in STAT3 mutation positive Hyper IgE Syndrome (HIES).

Autosomal dominant HIES (AD-HIES) is a primary immunodeficiency caused by dominant negative mutations in STAT3 clustered in the DNA binding and SH2 domains. Although in vitro differences in mutational constructs are observed, clinical phenotypic correlates of these genetic changes have not been described. We reviewed the charts of 65 AD-HIES patients (DNA binding n=35; SH2 n=30), recorded the components of the NIH HIES clinical scoring system as well as brain and coronary artery abnormalities and analyzed data by mutation region in adults and children. Patients with SH2 domain mutations had increased frequency of high palate, broad inter-alar distance, upper respiratory tract infections and, in the pediatric sub-group, significant scoliosis. There was suggestion of increased mortality for patients with DNA binding mutations. Although subtle differences in phenotype were observed to depend on the STAT3 genotype, overall the clinical phenotypes were similar between individuals with DNA binding and SH2 domain mutations.

Efficacy and Safety of AR101 in Oral Immunotherapy for Peanut Allergy: Results of ARC001, a Randomized, Double-Blind, Placebo-Controlled Phase 2 Clinical Trial

BACKGROUND Peanut oral immunotherapy, using a variety of approaches, has been previously shown to induce desensitization in peanut-allergic subjects, but no products have been approved for clinical use by regulatory agencies. OBJECTIVE We performed the first phase 2 multicentered study to assess the safety and efficacy of AR101, a novel oral biologic drug product. METHODS A randomized, double-blind, placebo-controlled trial was conducted at 8 US centers. Eligible subjects were 4 to 26 years old, sensitized to peanut, and had dose-limiting symptoms to ≤143 mg of peanut protein in a screening double-blind, placebo-controlled food challenge (DBPCFC). Subjects were randomized 1:1 to daily AR101 or placebo and gradually up-dosed from 0.5 to 300 mg/day. The primary endpoint was the proportion of subjects in each arm able to tolerate ≥443 mg (cumulative peanut protein) at exit DBPCFC with no or mild symptoms. RESULTS Fifty-five subjects (29 AR101, 26 placebo) were enrolled. In the intention-to-treat analysis, 23 of 29 (79%) and 18 of 29 (62%) AR101 subjects tolerated ≥443 mg and 1043 mg at exit DBPCFC, respectively, versus 5 of 26 (19%) and 0 of 26 (0%) placebo subjects (both P < .0001). Compared with placebo, AR101 significantly reduced symptom severity during exit DBPCFCs and modulated peanut-specific cellular and humoral immune responses. Gastrointestinal (GI) symptoms were the most common treatment-related adverse events (AEs) in both groups, with 6 AR101 subjects (21%) withdrawing, 4 of those due primarily to recurrent GI AEs. CONCLUSIONS In this study, AR101 demonstrated an acceptable safety profile and demonstrated clinical activity as a potential immunomodulatory treatment option in peanut-allergic children over the age of 4, adolescents, and young adults.

Plasma metalloproteinase levels are dysregulated in signal transducer and activator of transcription 3 mutated hyper-IgE syndrome

To the Editor: Hyper-IgE syndrome (HIES) is caused by a heterozygous mutation in signal transducer and activator of transcription 3 (STAT3)1,2 and characterized by increased serum IgE levels along with dermatitis, boils, cyst-forming pneumonias, retained primary dentition, bone abnormalities, and coronary artery aneurysms.3 Because the HIES morphotype suggests abnormal tissue remodeling and STAT3 is known to regulate matrix metal-loproteinases (MMPs) for extracellular matrix turnover and degradation, especially in the lungs,4,5 we investigated MMP levels in plasma from patients with HIES. Patients with heterozygous STAT3 mutations were enrolled in National Institute of Allergy and Infectious Diseases protocols, and they or their parents provided informed consent. The control subjects were healthy blood bank donors on appropriate National Institutes of Health protocols. Plasma samples were analyzed with the Bio-plex (Bio-Rad Laboratories, Hercules, Calif) to determine MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, and MMP-13 concentrations. We tested a single stored plasma sample from each patient with HIES, each of which was collected at a time when the patient did not have a known infection. For statistical analysis, we used the Mann-Whitney U test (GraphPad Prism; GraphPad Software, Inc, La Jolla, Calif). Because of multiple comparisons, we used the Bonferroni correction (dividing the standard significance level of .05 by the 6 comparisons performed), requiring a P value of less than .0083 to achieve statistical significance. Of our 37 patients with HIES, 19 were male subjects; 23 had mutations in the DNA-binding region and 14 had mutations in the SH2-binding region ofSTAT3. Themean agewas 32.6 years (95%CI 27.1-37.69 [SD 6 14.69 years]), and 5 patients were less than 16 years of age. MMP-1 levels (patients, 1,323 pg/mL [SE, 172.3 pg/mL]; control subjects, 803.7 pg/mL [SE, 141.8 pg/mL]) were higher in patients (P 5 .0130, Fig 1), whereas MMP-2 levels (patients, 122,148 pg/mL [SE, 4,044 pg/mL]; control subjects, 139,426 pg/mL [SE, 7,870 pg/mL]; P 5 .0164) were lower, but neither achieved the required level for significance. MMP-3 levels were significantly lower in patients (6,196 pg/mL [SE, 1,240 pg/ mL]) than in control subjects (15,624 pg/mL [SE, 1,596 pg/mL], P < .0001). MMP-7 levels did not differ. MMP-8 levels were significantly higher in patients (6,336 pg/mL [SE, 1,268 pg/mL]) than in control subjects (2,206 pg/mL [SE, 318.4 pg/mL], P 5 .0025). MMP-9 levels were also significantly higher in patients (152,295 pg/mL [SE, 24,717 pg/mL]) than in control subjects (54,975.7 pg/mL [SE, 6,023 pg/mL], P 5 .0004). MMP-12 and MMP-13 were undetectable in all samples. FIG 1 Meanplasma concentrations of MMP-3, MMP-8, and MMP-9 are compared between control subjects and patients with HIES (left panels). MMP-3 levels are significantly lower in patients with HIES, whereas MMP-8 and MMP-9 levels are significantly higher. Changes ... Because there is evidence for age-related changes in plasma MMP levels during childhood,6 we repeated our analyses without the 6 patients younger than 16 years. Even without the youngest patients, the differences remained significant, suggesting that age did not skew the data. Our secondary analyses comparing SH2- and DNA-binding domains showed no differences in MMP levels. We also found no association between patients’ MMP levels and total HIES scores7 using regression analysis for MMP-3, MMP-8, and MMP-9. We found no organ- or system-specific HIES manifestations correlated with MMP levels (eg, bone disease and MMP-3 levels, skin involvement and MMP-8 levels, pneumatoceles and MMP-3 and MMP-9 levels, or aneurysms and tortuosity with MMP-9 levels). We generated a heat map displaying the patient-MMP pattern relative to the mean level for each MMP (Fig 2). Patients with HIES and control subjects were clustered separately. Most patients with HIES with relatively high MMP-8 and MMP-9 levels had low MMP-3 levels. Control subjects had relatively low MMP-8 and MMP-9 levels but higher MMP-3 levels. To investigate the variability or fluctuation of MMP levels over time, we tested multiple samples from 8 patients with HIES collected and stored independent of infection or surgical status over a period of 20 years. Fluctuations were not wide, and there was reasonable stability over time (Fig 1). FIG 2 Heat map analysis of MMP-3, MMP-8, and MMP-9 showing clustering of patients (red) and control subjects (purple). MMP-8 and MMP-9 cluster together, and patients’ levels are significantly higher than those of control subjects. MMP-3 is independent ... Median MMP-8 and MMP-9 levels were approximately 3-fold higher in patients than in control subjects, and both of these MMPs are involved in tissue remodeling after acute lung injury.4 MMP-8 promotes acute inflammation in the lungs, whereas MMP-9 has an important role in vascular smooth muscle cell migration, macrophage infiltration, and elastinolytic activity and has been associated with abdominal aortic aneurysms.8 Therefore increased MMP-8 and MMP-9 levels in patients with HIES might be etiologically linked to the disease-specific predilection to aneurysm development and postinfectious lung injury. In contrast, MMP-3 is an important proteinase for vascular and cardiac matrix remodeling and was detected at only about one third the normal level in patients with HIES. MMP-3 regulation has a wide range of actions influencing fibrinolysis and angio-genesis. In a murine cell line model, mutated STAT3 has been shown to cause a decrease in MMP-3 production.9 Therefore increased MMP-8 and MMP-9 levels and reduced MMP-3 levels in patients with HIES might be implicated in formation of aneurysms or pneumatoceles. Mutations in MMP2 disrupt skeletal and craniofacial development and decrease bone mineralization because of defects in osteoblasts and osteoclasts. The failure to identify significant abnormalities in levels of MMP-2 and other MMPs might reflect that there are no significant differences between patients and control subjects or that resting plasma is not the correct compartment or situation. STAT3 plays a significant role in binding to the MMP2 promoter and can exert transcriptional control over other MMPs as well. Tobetter understand the potential regulatory roles of STAT3 on MMP expression, we used positional weight matrix analysis and identified multiple STAT3-binding sites throughout the MMP promoters.10 However, we only found significant differences in plasma levels of MMP-3, MMP-8, and MMP-9. Although MMP-9 has an experimentally proved STAT3-binding site upstream of exon 1, MMP3 and MMP8 have positional weight matrix–predicted binding sites upstream and downstream of their first exons, respectively. MMP-3, MMP-8, and MMP-9 levels were significantly different between patients with HIES and control subjects and might contribute to some of the characteristic features of HIES. Abnormal STAT3 signaling in HIES and the role for STAT3 in MMP regulation provides an attractive link between the manifestations of HIES and MMP regulation by STAT3. In situ tissue expression and regulation of MMPs in patients with HIES might be more relevant. Understanding MMP regulation in patients with HIES might guide efforts to prevent or mitigate some of the disease’s most threatening end-organ effects.

Immune reconstitution and survival of 100 SCID patients post-hematopoietic cell transplant: A PIDTC natural history study

The Primary Immune Deficiency Treatment Consortium (PIDTC) is enrolling children with severe combined immunodeficiency (SCID) to a prospective natural history study. We analyzed patients treated with allogeneic hematopoietic cell transplantation (HCT) from 2010 to 2014, including 68 patients with typical SCID and 32 with leaky SCID, Omenn syndrome, or reticular dysgenesis. Most (59%) patients were diagnosed by newborn screening or family history. The 2-year overall survival was 90%, but was 95% for those who were infection-free at HCT vs 81% for those with active infection (P = .009). Other factors, including the diagnosis of typical vs leaky SCID/Omenn syndrome, diagnosis via family history or newborn screening, use of preparative chemotherapy, or the type of donor used, did not impact survival. Although 1-year post-HCT median CD4 counts and freedom from IV immunoglobulin were improved after the use of preparative chemotherapy, other immunologic reconstitution parameters were not affected, and the potential for late sequelae in extremely young infants requires additional evaluation. After a T-cell-replete graft, landmark analysis at day +100 post-HCT revealed that CD3 < 300 cells/μL, CD8 < 50 cells/μL, CD45RA < 10%, or a restricted Vβ T-cell receptor repertoire (<13 of 24 families) were associated with the need for a second HCT or death. In the modern era, active infection continues to pose the greatest threat to survival for SCID patients. Although newborn screening has been effective in diagnosing SCID patients early in life, there is an urgent need to identify validated approaches through prospective trials to ensure that patients proceed to HCT infection free. The trial was registered at www.clinicaltrials.gov as #NCT01186913.

Current Knowledge and Priorities for Future Research in Late Effects after Hematopoietic Stem Cell Transplantation (HCT) for Severe Combined Immunodeficiency Patients: A Consensus Statement from the Second Pediatric Blood and Marrow Transplant Consortium International Conference on Late Effects after Pediatric HCT

Severe combined immunodeficiency (SCID) is 1 of the most common indications for pediatric hematopoietic cell transplantation (HCT) in patients with primary immunodeficiency. Historically, SCID was diagnosed in infants who presented with opportunistic infections within the first year of life. With newborn screening (NBS) for SCID in most of the United States, the majority of infants with SCID are now diagnosed and treated in the first 3.5 months of life; however, in the rest of the world, the lack of NBS means that most infants with SCID still present with infections. The average survival for SCID patients who have undergone transplantation currently is >70% at 3 years after transplantation, although this can vary significantly based on multiple factors, including age and infection status at the time of transplantation, type of donor source utilized, manipulation of graft before transplantation, graft-versus-host disease prophylaxis, type of conditioning (if any) utilized, and underlying genotype of SCID. In at least 1 study of SCID patients who received no conditioning, long-term survival was 77% at 8.7 years (range out to 26 years) after transplantation. Although a majority of patients with SCID will engraft T cells without any conditioning therapy, depending on genotype, donor source, HLA match, and presence of circulating maternal cells, a sizable percentage of these will fail to achieve full immune reconstitution. Without conditioning, T cell reconstitution typically occurs, although not always fully, whereas B cell engraftment does not, leaving some molecular types of SCID patients with intrinsically defective B cells, in most cases, dependent on regular infusions of immunoglobulin. Because of this, many centers have used conditioning with alkylating agents including busulfan or melphalan known to open marrow niches in attempts to achieve B cell reconstitution. Thus, it is imperative that we understand the potential late effects of these agents in this patient population. There are also nonimmunologic risks associated with HCT for SCID that appear to be dependent upon the genotype of the patient. In this report, we have evaluated the published data on late effects and attempted to summarize the known risks associated with conditioning and alternative donor sources. These data, while informative, are also a clear demonstration that there is still much to be learned from the SCID population in terms of their post-HCT outcomes. This paper will summarize current findings and recommend further research in areas considered high priority. Specific guidelines regarding a recommended approach to long-term follow-up, including laboratory and clinical monitoring, will be forthcoming in a subsequent paper.

Reduced‐intensity conditioning for hematopoietic cell transplantation of chronic granulomatous disease

Hematopoietic cell transplantation (HCT) is the only available curative therapy for chronic granulomatous disease (CGD), but its use is limited by transplant‐related mortality (TRM) in patients who often come to transplant with existing infections or organ dysfunction. Reduction in the intensity of the preparative regimen mitigates these risks, but increases the potential for mixed donor‐recipient chimerism (MC) that may progress to graft loss. Recently a busulfan‐based reduced‐intensity conditioning (RIC) regimen has been described with excellent survival and little MC. We report our experience with a similar RIC regimen at our institution, demonstrating problems with donor chimerism and graft loss. Pediatr Blood Cancer 2015;62:359–361.