Christen L. Ebens

Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function

Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

Blockade of individual Notch ligands and receptors controls graft-versus-host disease

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell-mediated human disorders.

Functional magnetic resonance imaging studies of eye movements in first episode schizophrenia: Smooth pursuit, visually guided saccades and the oculomotor delayed response task

Schizophrenia patients show eye movement abnormalities that suggest dysfunction in neocortical control of the oculomotor system. Fifteen never-medicated, first episode schizophrenia patients and 24 matched healthy individuals performed eye movement tasks during functional magnetic resonance imaging studies. For both visually guided saccade and smooth pursuit paradigms, schizophrenia patients demonstrated reduced activation in sensorimotor areas supporting eye movement control, including the frontal eye fields, supplementary eye fields, and parietal and cingulate cortex. The same findings were observed for an oculomotor delayed response paradigm used to assess spatial working memory, during which schizophrenia patients also had reduced activity in dorsolateral prefrontal cortex. In contrast, only minimal group differences in activation were found during a manual motor task. These results suggest a system-level dysfunction of cortical sensorimotor regions supporting oculomotor function, as well as in areas of dorsolateral prefrontal cortex that support spatial working memory. These findings indicate that a generalized rather than localized pattern of neocortical dysfunction is present early in the course of schizophrenia and is related to deficits in the sensorimotor and cognitive control of eye movement activity.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Cytogenetic Abnormalities in Attention-Deficit/Hyperactivity Disorder

OBJECTIVE To systematically assess the prevalence of fragile X syndrome, velocardiofacial syndrome, and other cytogenetic abnormalities in a group of children with attention-defict/hyperactivity disorder (ADHD). METHOD Blood samples were obtained from 100 children (64 boys) with combined type ADHD and normal intelligence and analyzed for the presence of fragile X mutation expansions, the 22q11.2 microdeletion associated with velocardiofacial syndrome, and cytogenetic abnormalities that would be detected with high resolution chromosomal banding. RESULTS One girl with ADHD had a sex chromosome aneuploidy (47,XXX). One boy had a premutation-sized allele for fragile X; no subjects showed the full mutation. Testing for 22q11.2 microdeletion was negative for all subjects with ADHD screened. None of these differences exceeded those expected by chance. CONCLUSIONS In the absence of clinical signs or positive family history, these relatively expensive laboratory assessments are not clinically indicated for children with ADHD and normal intelligence, and are not recommended as a component of other genetic investigations of this disorder.

Hematopoietic cell transplantation in Fanconi anemia: current evidence, challenges and recommendations

ABSTRACT Introduction: Hematopoietic cell transplantation for Fanconi Anemia (FA) has improved dramatically over the past 40 years. With an enhanced understanding of the intrinsic DNA-repair defect and pathophysiology of hematopoietic failure and leukemogenesis, sequential changes to conditioning and graft engineering have significantly improved the expectation of survival after allogeneic hematopoietic cell transplantation (alloHCT) with incidence of graft failure decreased from 35% to <10% and acute graft-versus-host disease (GVHD) from >40% to <10%. Today, five-year overall survival exceeds 90% in younger FA patients with bone marrow failure but remains about 50% in those with hematologic malignancy. Areas covered: We review the evolution of alloHCT contributing to decreased rates of transplant related complications; highlight current challenges including poorer outcomes in cases of clonal hematologic disorders, alloHCT impact on endocrine function and intrinsic FA risk of epithelial malignancies; and describe investigational therapies for prevention and treatment of the hematologic manifestations of FA. Expert commentary: Current methods allow for excellent survival following alloHCT for FA associated BMF irrespective of donor hematopoietic cell source. Alternative curative approaches, such as gene therapy, are being explored to eliminate the risks of GVHD and minimize therapy-related adverse effects.

Large B-Cell Lymphoma in an Adolescent Patient With Interleukin-10 Receptor Deficiency and History of Infantile Inflammatory Bowel Disease.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine1, 2 and loss of function mutations in IL-10 or the IL-10 receptor (IL-10R) have been implicated as a common cause of infantile IBD3, 4, 5, 6. These patients typically present in the first months of life with severe colitis, perianal disease, folliculitis, and, on occasion, arthritis, and are classically refractory to various immunosuppressive agents. Hematopoietic stem cell transplantation (HSCT) has been shown to be curative, but is not available for all patients4, 5, 6. The long-term risks of IL-10R-deficiency are unclear; however B-cell lymphomas have recently been reported in untransplanted young children with a known diagnosis of IL-10R deficiency7. Here we report the discovery of IL-10R deficiency in a patient who presented with severe IBD as an infant and developed a mature B cell lymphoma in adolescence. Through our interNational Early Onset Pediatric IBD Cohort Study (NEOPICS)/Care-for-Rare IBD Alliance we were referred a 15 year-old female patient with history of infantile IBD. She developed bloody diarrhea and anal fissures in the first weeks of life, failure to thrive and anemia requiring several blood transfusions by 6 months, and a rectovaginal fistula at 8 months of age. Endoscopic evaluation revealed severe pan-colitis, a distal colonic stricture, and pseudopolyps; biopsies demonstrated patchy areas of cryptitis, ulcerations, and lymphocytic infiltration. Her disease was resistant to various medications including steroids and aminosalicylates for the first 5 years, Imuran for the next 3 years, and combination of anti-TNF antibodies and methotrexate for 3 more years. Persistent symptoms led to a partial colectomy and colostomy at 8 months of age and at 5 years of age a subtotal colectomy, Hartmanns pouch construction, and permanent ileostomy. Despite these interventions, the patient continued to suffer from severe perianal fistulizing disease. At 12 years of age the patient presented with two months history of right-sided abdominal pain and hepatosplenomegaly. Blood tests demonstrated mild thrombocytopenia, hyperuricemia and abnormal liver tests (Table 1). An abdominal CT showed hepatosplenomegaly with multiple focal liver lesions accompanied by enlarged mesenteric lymph nodes, all confirmed to be hypermetabolic on PET scan (Figure 1). Liver biopsy revealed CD20 positive, EBV-encoded RNA (EBER)-negative small round blue cells leading to a diagnosis of mature large B cell lymphoma. Despite successful initial treatment with cyclophosphamide, vincristine, prednisone, ritixumab, cytarabine, doxorubicin as well as intrathecal methotrexate, cytarabine, and hydrocortisone, remission was maintained for only two years. At time of relapse at age 15, salvage chemotherapy of rituximab, ifosfamide, carboplatin, and etoposide was initiated and consolidation with autologous HSCT was being considered at the time of referral. Figure 1 Imaging and histology results at time of diagnosis with diffuse large B cell lymphoma Table 1 Laboratory results at presentation with diffuse large B cell lymphoma We performed IL-10R functional testing on freshly isolated peripheral blood mononuclear cells obtained from the patient and her father who served as a healthy control. Our flow cytometry-based assay measures IL-10-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is a key transcription factor down-stream of the IL-10 receptor; IL-6-induced STAT3 phosphorylation serves as an internal positive control. While the patients IL-6-dependent STAT3 phosphorylation was intact, IL-10-dependent phosphorylation of STAT3 was completely abrogated (Figure 2A), suggesting abnormalities in the IL-10 receptor or downstream signaling components. Targeted sequencing of IL10R genes revealed novel compound heterozygous mutations in IL10RB (NM000628.4) comprised of a variant on exon 2 (c. 172 A>G; p. S58R) leading to a serine to arginine substitution, predicted by PolyPhen analysis as highly deleterious, and a variant on exon 5 (c. 611 G>A; p. W204X) leading to a stop codon (Figure 2B). Additional functional assays with monocytes isolated from this patient, as well as other IL-10R deficient patients, demonstrating an increase in proinflammatory macrophage function and a defect in anti-inflammatory macrophage generation and function have been recently published 2. Since autologous HSCT would be predicted to be ineffective in patients with IL-10R deficiency given the broad requirement of IL-10-dependent signalling in the hematopoietic compartment, the patient was referred for allogeneic HSCT. Four months after matched unrelated allogeneic transplantation, the patient is fully engrafted without any signs of active colitis or lymphoma recurrence, and the rectovaginal fistula is resolving. Figure 2 Identification of loss of function mutations in IL10RB

Notch signaling in hematopoietic cell transplantation and T cell alloimmunity

Notch signaling can regulate both hematopoietic progenitors and alloimmune T cells in the setting of allogeneic bone marrow or hematopoietic cell transplantation (allo-HCT). Ex vivo culture of multipotent blood progenitors with immobilized Delta-like ligands induces supraphysiological Notch signals and can markedly enhance progenitor expansion. Infusion of Notch-expanded progenitors shortened myelosuppression in preclinical and early clinical studies, while accelerating T cell reconstitution in preclinical models. Notch also plays an essential role in vivo to regulate pathogenic alloimmune T cells that mediate graft-versus-host disease (GVHD), the most severe complication of allo-HCT. In mouse allo-HCT models, Notch inhibition in donor-derived T cells or transient blockade of Delta-like ligands after transplantation profoundly decreased GVHD incidence and severity, without causing global immunosuppression. These findings identify Notch in T cells as an attractive therapeutic target to control GVHD. In this review, we discuss these contrasting functions of Notch signaling with high translational significance in allo-HCT patients.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD

Background: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+ T-cell function. Methods: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4+ T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Results: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4+ T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1&bgr; leads to enhanced production of IL17A. Conclusions: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Comparable Outcomes after HLA-Matched Sibling and Alternative Donor Hematopoietic Cell Transplantation for Children with Fanconi Anemia and Severe Aplastic Anemia

Fanconi anemia (FA)-associated severe aplastic anemia (SAA) requires allogeneic hematopoietic cell transplantation (HCT) for cure. With the evolution of conditioning regimens over time, outcomes of alternative donor HCT (AD-HCT) have improved dramatically. We compared outcomes of HLA-matched sibling donor HCT (MSD-HCT; n = 17) and AD-HCT (n = 57) performed for FA-associated SAA at a single institution between 2001 and 2016. Overall survival at 5 years was 94% for MSD-HCT versus 86% for AD-HCT, neutrophil engraftment was 100% versus 95%, platelet recovery was 100% versus 89%, grade II-IV acute graft-versus-host disease (GVHD) was 6% versus 12%, grade III-IV acute GVHD was 6% versus 4%, and chronic GVHD was 0 versus 7%, with no statistically significant differences by type of transplant. The use of UCB was associated with decreased rates of neutrophil recovery in AD-HCT and platelet recovery in both MSD-HCT and AD-HCT. A trend toward a higher serious infection density before day +100 post-HCT was observed in AD-HCT compared with MSD-HCT (P = .02). These data demonstrate that AD-HCT should be considered at the same time as MSD-HCT for patients with FA-associated SAA.