Katelyn McCann

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.

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

Spontaneous food allergy in Was−/− mice occurs independent of FcεRI‐mediated mast cell activation

Food allergies are a growing health problem, and the development of therapies that prevent disease onset is limited by the lack of adjuvant‐free experimental animal models. We compared allergic sensitization in patients with food allergy or Wiskott‐Aldrich syndrome (WAS) and defined whether spontaneous disease in Was−/− mice recapitulates the pathology of a conventional disease model and/or human food allergy.

363 Human Hematopoietic Stem Cells With a Defined Immunodeficiency and Enteropathy Transfer Clinical Phenotype to a Novel Humanized Mouse Strain

Investigation into the pathogenesis of IBD has been limited by the absence of humanized murine IBD models. The development of immunodeficient mouse strains has facilitated the study of human immune reconstitution in murine hosts. However, adaptive immune responses and antibody class switching are generally weak in models that lack autologous fetal thymic grafts, possibly due to human CD4+ T cell selection in the mouse thymus occurring on murine major histocompatibility complex II (MHCII). To address this, we modified the widely utilized NOD.Prkdc.Il2rγ (NSG) immunocompromised strain to be deficient for murine MHCII (NSGAbo) and instead expressed human leukocyte antigen-DR1 under the control of the murine MHCII promoter (NSGAboDR1). We tested whether CD4+ T cell development and adaptive immune responses are improved following reconstitution using human HLA-matched CD34+ hematopoietic stem cells (HSCs) and whether immune reconstitution using HSCs from a patient with IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked), a well-defined immunodeficiency, that is caused by a mutation in the transcription factor FOXP3, would transfer the clinical phenotype to NSGAboDR1 mice. Results: At 20 weeks following HSC injection into radiation-conditioned neonates, we found that the development of human T cells and dendritic cells were increased in NSGAboDR1 mice and that these mice displayed delayed-type hypersensitivity response to a secondary recall antigen challenge. There was an increase in T cell clonotype diversity as determined by CDR3 sequencing of the TCRβ gene in CD4+ T cells in NSGAboDR1 mice compared to NSG mice. In addition, NSGAboDR1 mice had an increase in mature B cells and antibody class switching compared to NSG and NSGAbo mice. We then reconstituted NSGAboDR1 mice using HSCs from a patient with IPEX that is associated with functional defects in Treg and assessed if the clinical phenotype would be transferred to NSGAboDR1 mice. All mice reconstituted with IPEX CD34+ HSCs were deceased by 18 weeks while all control mice receiving healthy bone marrow CD34+ cells remained viable. IPEX mice developed lymphocytic infiltrate in lung, portal tract and bile ducts of the liver, and slight accumulation of lymphocytes in the small intestine similar to scurfy mice, which also harbor a mutation in Foxp3. CD4+ T cell in IPEX mice had an expanded effector memory T cell population and concomitant decrease in Naive CD4+ T cells. Conclusions: We demonstrate that human HSCs from a patient with a defined genetic immunodeficiency and enteropathy can transfer the disease phenoytpe in a novel humanized mouse strain. This strain does not require grafts of human fetal tissue nor is it restricted to autologous fetal HSCs allowing for reconstitution using human HSCs with defined genetics to study human immune cells and IBD pathogenesis in vivo.

O-01: IL-10 receptor dependent signals regulate the generation and function of anti-inflammatory macrophages in mice and humans

O-01 IL-10 receptor dependent signals regulate the generation and function of anti-inflammatory macrophages in mice and humans S.B. Snapper1 *, D. Shouval1, A. Biswas1, J.A. Goettel1, K. McCann1, J.C. Escher2, J.N. Samsom2, R.S. Somech3, B. Weiss3, R. Beier4, L. Conklin5, C.L. Ebens6, F.G.M. Santos7, M. Sherlock8, C. Klein9, A.M. Muise10, B.H. Horwitz11. 1Boston Children’s Hospital, Boston, United States of America, 2Erasmus Medical Center Sophia Children’s Hospital, Rotterdam, The Netherlands, 3Edmond and Lily Safra Children’s Hospital, Tel Hashomer, Israel, 4Hannover Medical School, Hannover, Germany, 5Children’s National Medical Center, Washington DC, United States of America, 6University of Michigan, Ann Arbor, United States of America, 7Hospital das Clinicas, Federal University of Minas Gerais, Minas Gerais, Brazil, 8McMaster Children’s Hospital, West Hamilton, Canada, 9Dr von Hauner Children’s Hospital, Munich, Germany, 10Hospital for Sick Children, Toronto, Canada, 11Harvard Medical School, Boston, United States of America

P-200 Human Hematopoietic Stem Cells with a Defined Immunodeficiency Transfer Clinical Phenotype to Novel Humanized Mouse Strain

BACKGROUND: Immunopathologies resulting from immunodeficiencies can be caused by a single mutation in a critical regulatory gene as seen in immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) which harbors a mutation in the forkhead transcription factor, foxP3. One remaining hurdle for advances in personalized medicine is the lack of a robust model system to study patient cells in vivo. The development of immunodeficient mouse strains have allowed for human immune reconstitution within murine hosts. Unfortunately adaptive immune responses and antibody class switching are generally weak, possibly due to T cell education in the thymus occurring on murine MHC molecules, thus hindering the ability to assess patient genetics and immune homeostasis in this setting. We generated NOD/SCID.Il2r&ggr;-/- (NSG) mice that lack murine major histocompatibility complex II (MHCII) (NSGAbo mice) and instead express a human MHCII (HLADR1) transgene under the control of the murine MHCII promoter (NSGAboDR1 mice) to see if CD4+ T cell development and adaptive immune responses are improved and if immune reconstitution using stem cells from a patient with IPEX, a well-defined genetic disease, would transfer the clinical phenotype to a mouse. METHODS: CD34+ HSCs were isolated from cord blood and screened for the HLADR1*01:01 allele. 5 × 104 allelically matched HSCs were injected intrahepatically into 1-day old NSG, NSGAbo, and NSGAboDR1 neonates pre-conditioned with 100 rads137Cs. For delayed-type hypersensitivity, 16-week old reconstituted mice were immunized with OVA/CFA emulsion at the base of the tail and 14 days later were challenged with OVA or PBS in the footpad. Footpad swelling was measured with digital calipers 24 hours post footpad injection. Immunophenotyping and serum antibody quantification were performed between 18 and 24 weeks of age. RESULTS: The percent of human T cells and human dendritic cells were elevated in the MLN of NSGAboDR1 mice. Compared to NSG mice, HLADR1 promoted 4-fold greater TCR&bgr; sequence diversity and NSGAboDR1 mice developed DTH compared to little or no footpad swelling in NSG or NSGAbo mice. We quantified antibody class switching and found increased levels of IgM, IgG, and IgE in NSGAboDR1 mice. We then tested whether reconstituting NSGAboDR1 mice using HSCs from an IPEX patient would transfer disease to mice. All mice reconstituted with IPEX CD34+ HSCs were deceased by 18 weeks of age while all control mice remained healthy. Histological examination showed that IPEX mice developed lymphocytic infiltrate in lung, portal tract and bile ducts of the liver, and slight accumulation of lymphocytes in the small intestine similar to scurfy mice which also harbor a mutation in foxP3. CD4+ T cell in IPEX mice had an expanded antigen experienced effector memory T cell population and concomitant decrease in naïve CD4+ T cells. CONCLUSIONS: We demonstrate that human HSCs with a defined genetic mutation can transfer clinical phenotype in a novel humanized mouse strain that displays better T cell responses and antibody class switching. This strain does not require human fetal tissue grafts nor restricted to autologous fetal HSCs allowing reconstitution using human HSCs with defined genetics to study human immune cells in vivo. Figure. No caption available.