Interleukin-2 Receptor Gamma Research Articles

Genetic relationship between Hashimoto`s thyroiditis and papillary thyroid carcinoma with coexisting Hashimoto`s thyroiditis.

Hashimoto's thyroiditis (HT) is present in the background of around 30% of papillary thyroid carcinomas (PTCs). The genetic predisposition effect of this autoimmune condition is not thoroughly understood. We analyzed the microarray expression profiles of 13 HT, eight PTCs with (w/) coexisting HT, six PTCs without (w/o) coexisting HT, six micro PTCs (mPTCs), and three normal thyroid (TN) samples. Based on a false discovery rate (FDR)-adjusted p-value ≤ 0.05 and a fold change (FC) > 2, four comparison groups were defined, which were HT vs. TN; PTC w/ HT vs. TN; PTC w/o HT vs. TN; and mPTC vs. TN. A Venn diagram displayed 15 different intersecting and non-intersecting differentially expressed gene (DEG) sets, of which a set of 71 DEGs, shared between the two comparison groups HT vs. TN ∩ PTC w/ HT vs. TN, harbored the relatively largest number of genes related to immune and inflammatory functions; oxidative stress and reactive oxygen species (ROS); DNA damage and DNA repair; cell cycle; and apoptosis. The majority of the 71 DEGs were upregulated and the most upregulated DEGs included a number of immunoglobulin kappa variable genes, and other immune-related genes, e.g., CD86 molecule (CD86), interleukin 2 receptor gamma (IL2RG), and interferon, alpha-inducible protein 6 (IFI6). Upregulated genes preferentially associated with other gene ontologies (GO) were, e.g., STAT1, MMP9, TOP2A, and BRCA2. Biofunctional analysis revealed pathways related to immunogenic functions. Further data analysis focused on the set of non-intersecting 358 DEGs derived from the comparison group of HT vs. TN, and on the set of 950 DEGs from the intersection of all four comparison groups. In conclusion, this study indicates that, besides immune/inflammation-related genes, also genes associated with oxidative stress, ROS, DNA damage, DNA repair, cell cycle, and apoptosis are comparably more deregulated in a data set shared between HT and PTC w/ HT. These findings are compatible with the conception of a genetic sequence where chronic inflammatory response is accompanied by deregulation of genes and biofunctions associated with oncogenic transformation. The generated data set may serve as a source for identifying candidate genes and biomarkers that are practical for clinical application.

Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy.

Immune checkpoint blockade (ICB) monotherapy shows early promise for the treatment of nasopharyngeal carcinoma (NPC) in patients. Nevertheless, limited representative NPC models hamper preclinical studies to evaluate the efficacy of novel ICB and combination regimens. In the present study, we engrafted NPC biopsies in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice and established humanized mouse NPC-patient-derived xenograft (NPC-PDX) model successfully. Epstein–Barr virus was detected in the NPC in both NSG and humanized mice as revealed by Epstein–Barr virus-encoded small RNA (EBER) in situ hybridization (ISH) and immunohistochemical (IHC) staining. In the NPC-bearing humanized mice, the percentage of tumor-infiltrating CD8+ cytotoxic T cells was lowered, and the T cells expressed higher levels of various inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) than those in blood. The mice were then treated with nivolumab and ipilimumab, and the anti-tumor efficacy of combination immunotherapy was examined. In line with paired clinical data, the NPC-PDX did not respond to the treatment in terms of tumor burden, whilst an immunomodulatory response was elicited in the humanized mice. From our results, human proinflammatory cytokines, such as interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were significantly upregulated in plasma. After treatment, there was a decrease in CD4/CD8 ratio in the NPC-PDX, which also simulated the modulation of intratumoral CD4/CD8 profile from the corresponding donor. In addition, tumor-infiltrating T cells were re-activated and secreted more IFN-γ towards ex vivo stimulation, suggesting that other factors, including soluble mediators and metabolic milieu in tumor microenvironment may counteract the effect of ICB treatment and contribute to the tumor progression in the mice. Taken together, we have established and characterized a novel humanized mouse NPC-PDX model, which plausibly serves as a robust platform to test for the efficacy of immunotherapy and may predict clinical outcomes in NPC patients.

Acquired Immunity Is Not Essential for Radiation-Induced Heart Dysfunction but Exerts a Complex Impact on Injury.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG−/−)) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p < 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

Effects of yolkin on the immune response of mice and its plausible mechanism of action

Yolkin is a product of proteolytic degradation of vitellogenin, a protein contained in eggs’ yolk, with already described procognitive properties. Here, we investigated effects of yolkin on the humoral and cellular immune response in mice, phenotype of cells from lymphoid organs and function of innate immunity cells. In vitro studies included effects of yolkin on mitogen-induced thymocyte proliferation, percentage of CD19 cells in bone marrow cells culture, expression of signaling molecules in Jurkat cells, interleukin 2 receptor (IL-2R) subunits in WEHI 231 cells and susceptibility of these cells to anti-Ig-induced cell death. The results showed that repeatable i.p. injections of yolkin stimulated the humoral immune response to sheep red blood cells (SRBC) irrespective of the time of the treatment. On the other hand, yolkin inhibited contact sensitivity to oxazolone. Treatment of mice with yolkin diminished the percentage of double positive cells and increasing the content of single positive CD4+ and CD8+ cells in the thymus. At the same time an increase of percentage of CD19 + B cells in the spleen and mesenteric lymph nodes was observed. In addition, the protein, given i.p., diminished ex vivo ability to synthesize nitric oxide by resident, peritoneal macrophages, stimulated with lipopolisaccharide (LPS). In vitro studies showed that yolkin increased CD19+ cell content in bone marrow cell population. The protein also enhanced proliferation of thymocytes to concanavalin A and stimulated expression of MAP kinases in Jurkat cells. In WEHI 231 B cell line yolkin caused a loss of IL-2R gamma chain expression, correlated with an increased resistance of these cells to proapoptotic action of anti-Ig antibodies. In conclusion, this is a first demonstration of immunotropic properties of yolkin in in vitro and in vivo tests. The results provide evidence for induction of maturation and stimulatory signals in immature T and B cells by the protein, suggesting its potential role in the development of an embryo’s immune system.

Generation of novel Il2rg-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9.

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Low-Dose Busulfan Reduces Human CD34+ Cell Doses Required for Engraftment in c-kit Mutant Immunodeficient Mice

Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2 × 105 to 2 × 106 cells/mouse) with or without busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (non-obese diabetic [NOD]/B6/severe combined immunodeficiency [SCID]/ interleukin-2 receptor gamma chain null (IL-2rγ−/−) KitW41/W41 [NBSGW]). We then tested a range of BU (5–37.5 mg/kg) using 2 × 105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using 10-fold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10 mg/kg) was sufficient to allow human engraftment using 2 × 105 CD34+ cells, whereas higher doses (≥37.5 mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome-editing studies, especially for modeling in disorders, such as sickle cell disease, where access to HSCs is limited.

Abstract 3185: CAR-T cells can eradicate human uveal melanoma and immune-therapy resistant malignant melanoma in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout mice

Abstract Immune therapies including checkpoint blockade and adoptive T cell transfer show great promise for the treatment of melanoma, with long-term effects in some patients. However, not all patients respond to current immune therapies and are in need of other treatment strategies. For example, around half of the patients with metastatic malignant melanoma will not be cured with available therapies today. For metastatic uveal melanoma (a rare melanoma of the eye) available immune therapies have less effect, and there is currently no approved therapy for these patients. Our objective is to find novel immune therapies with the potential to treat immune-therapy resistant melanoma, with the use of chimeric antigen receptor T cells (CAR-T). So far, no CAR-T therapy is approved for use in solid tumors. We have previously developed a humanized mouse model, where tumor cells and T cells from the same patient are grafted in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout (NOG) mice, and found that responses in the mouse model correlated to responses in the corresponding patients in a clinical trial of adoptive T cell transfer (Jespersen et al, Nature Communications, 2017). In order to test the potential for CAR-T therapy in immune-therapy resistant melanoma in this mouse model, we used TCGA to determine the expression in melanoma biopsies of targets for commercially available CAR-T cells. Interestingly, we were able to use this model to treat immune-therapy resistant malignant melanoma and uveal melanoma patient-derived xenografts with human CAR-T cells and obtain curative responses, but only in IL-2 transgenic mice and not in regular NOG mice. These findings prove potential for the use of CAR-T cell therapy in immune-therapy resistant melanoma, and indicate the utility of this mouse model in the translation of CAR-T therapies. We are currently working on the development of CAR-T cells derived from patient-specific T cells. Citation Format: Elin Forsberg, Mattias Lindberg, Henrik Jespersen, Samuel Alsén, Roger Olofsson Bagge, Marco Donia, Inge Marie Svane, Ola Nilsson, Lars Ny, Lisa Nilsson, Jonas Nilsson. CAR-T cells can eradicate human uveal melanoma and immune-therapy resistant malignant melanoma in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3185.

T Lymphocyte Development and Activation in Humanized Mouse Model.

Humanized mice, containing engrafted human cells and tissues, are emerging as an important in vivo platform for studying human diseases. Since the development of Nod scid gamma (NSG) mice bearing mutations in the IL-2 receptor gamma chain, many investigators have used NSG mice engrafted with human hematopoietic stem cells (HSCs) to generate functional human immune systems in vivo, results in high efficacy of human cell engraftment. The development of NSG mice has allowed significant advances to be made in studies on several human diseases, including cancer and graft-versus-host-disease (GVHD), and in regenerative medicine. Based on the human HSC transplantation, organ transplantation including thymus and liver in the renal capsule has been performed. Also, immune reconstruction of cells, of the lymphoid as well as myeloid lineages, has been partly accomplished. However, crosstalk between pluripotent stem cell derived therapeutic cells with human leukocyte antigen (HLA) mis/matched types and immune CD3 T cells have not been fully addressed. To overcome this hurdle, human major histocompatibility complex (MHC) molecules, not mouse MHC molecules, are required to generate functional T cells in a humanized mouse model. Here, we briefly summarize characteristics of the humanized mouse model, focusing on development of CD3 T cells with MHC molecules. We also highlight the necessity of the humanized mouse model for the treatment of various human diseases.

Biological Characteristics of Severe Combined Immunodeficient Mice Produced by CRISPR/Cas9-Mediated Rag2 and IL2rg Mutation.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 is a novel and convenient gene editing system that can be used to construct genetically modified animals. Recombination activating gene 2 (Rag2) is a core component that is involved in the initiation of V(D)J recombination during T- and B-cells maturation. Separately, the interleukin-2 receptor gamma chain gene (IL2rg) encoded the protein-regulated activity of natural killer (NK) cells and shared common receptors of some cytokines. Rag2 and IL2rg mutations cause immune system disorders associated with T-, B-, and NK cell function and some cytokine activities. In the present study, 2 single-guide RNAs (sgRNAs) targeted on Rag2 and IL2rg genes were microinjected into the zygotes of BALB/c mice with Cas9 messenger RNA (mRNA) to create Rag2/IL2rg-/- double knockout mice, and the biological characteristics of the mutated mice were subsequently analyzed. The results showed that CRISPR/Cas9-induced indel mutation displaced the frameshift of Rag2 and IL2rg genes, resulting in a decrease in the number of T-, B-, and NK cells and the destruction of immune-related tissues like the thymus and spleen. Mycobacterium tuberculosis 85B antigen could not induce cellular and humoral immune response in mice. However, this aberrant immune activity compromised the growth of several tumor heterogenous grafts in the mutated mice, including orthotopic and subcutaneous transplantation tumors. Thus, Rag2/IL2rg-/- knockout mice possessed features of severe combined immunodeficiency (SCID), which is an ideal model for human xenograft.

HER2 CAR-T Cells Eradicate Uveal Melanoma and T-cell Therapy-Resistant Human Melanoma in IL2 Transgenic NOD/SCID IL2 Receptor Knockout Mice.

Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR-T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner. SIGNIFICANCE: These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.

IL2RG hypomorphic mutation: identification of a novel pathogenic mutation in exon 8 and a review of the literature

BackgroundAtypical X-linked severe combined immunodeficiency (X-SCID) is a variant of cellular immunodeficiency due to hypomorphic mutations in the interleukin 2 receptor gamma (IL2RG) gene. Due to a leaky clinical phenotype, diagnosis and appropriate treatment are challenging in these patients.Case presentationWe report a 16-year-old patient with a Tlow B+ NK+ cellular immunodeficiency due to a novel nonsense mutation in exon 8 (p.R328X) of the IL2RG gene. Functional impairment of the IL2RG was confirmed by IL2-Janus kinase 3-signal transducer and activator of transcription signaling pathway investigation. In addition, the characteristics of the mutations previously described in 39 patients with an atypical phenotype were reviewed and analyzed from the literature.ConclusionThis is the first report of an atypical X-SCID phenotype due to an exon 8 mutation in the IL2RG gene. The variability in the phenotypic spectrum of classic X-SCID associated gene highlights the necessity of multi-disciplinary cooperation vigilance for a more accurate diagnostic workup.

Interleukin 4 Induces Macrophage-Mediated Phagocytosis of Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive disease associated with poor prognosis, hence, new therapeutic strategies are needed. The AML cells reside in the bone marrow niche and depend on external signals such as cytokines for their growth and survival. In contrast to providing a supporting role, cytokines can act as negative regulators of AML cells. We previously identified that interleukin 4 (IL-4) induces apoptosis of AML cells in a Stat6-dependent manner and demonstrated strong anti-leukemic activity of IL-4 in a murine AML model driven by MLL-AF9 (Peña-Martínez et al., Leukemia 2017). Ectopic expression of IL-4 in AML cells resulted in prolonged survival in C57BL/6 mice, with mice almost devoid of leukemia cells in bone marrow and spleen upon the time of sacrifice. However, the IL-4-induced apoptosis of AML cells did not fully explain the strong antileukemic activity of IL-4 in vivo.To investigate whether the anti-leukemic effect of ectopic IL-4 expression in vivo was microenvironment dependent, we used IL-2 receptor gamma(-/-) immunodeficient mice, which lack a functional IL-4 receptor type I complex, as recipients for transplantation of murine MLL-AF9 AML cells. Notably, there was no antileukemic activity of IL-4 in these mice, suggesting that the in vivo anti-leukemic effect of IL-4 was predominantly microenvironment-dependent. To identify the cell population that was responsible for the antileukemic effect of IL-4 stimulation, we performed flow cytometry analysis of bone marrow and spleen cells in mice exposed to high IL-4 levels. We found that the anti-leukemic activity of IL-4 was associated with a significant expansion of F4/80+ macrophages in both the bone marrow and spleen, which was confirmed by immunohistochemistry.To determine whether macrophages were responsible for the anti-leukemic effect of IL-4, we depleted macrophages with clodronate liposomes in mice receiving IL-4-secreting AML cells. Depletion of macrophages partially rescued the anti-leukemic effect of IL-4, indicating that macrophages stimulated with IL-4 were responsible for the antileukemic activity. Consistent with these findings, in macrophage differentiation assays with either murine or human monocytes, IL-4 enhanced the phagocytosis of leukemia cells.To characterize the IL-4-induced macrophages, we performed RNA sequencing of F4/80+ macrophages from animals exposed to high IL-4 levels. Gene expression analysis revealed IL-4-induced M2 polarization of the macrophages, as key markers of M2 activation such as Arg1, Retnla and Chil3 were upregulated. Moreover, consistent with the in vitro findings, gene set enrichment analysis revealed an enrichment of phagocytosis signatures in the macrophages stimulated with IL-4.Strikingly, we found that exposure of AML cells to IL-4 resulted in strong upregulation of CD47, a ‘don't eat me signal’, which protects cells from macrophage-mediated phagocytosis. By CRISPR/Cas9-mediated disruption of Stat6 in IL-4 treated AML cells, we found that the CD47 upregulation was Stat6 dependent, recognizing a previously unknown link between IL-4 signalling and CD47 upregulation. Consistent with this finding, combined antibody blockade of CD47 on leukemia cells and IL-4 stimulation of macrophages resulted in enhanced phagocytosis of AML cells.Taken together, this study shows that IL-4 has anti-leukemic activity by activating macrophages, findings that might translate into new immunotherapeutic opportunities in AML and possibly other malignancies. DisclosuresNo relevant conflicts of interest to declare.

The Vascular Bone Marrow Niche Influences Outcome in Chronic Myeloid Leukemia

The endosteal bone marrow niche is known to protect leukemic stem cells (LSC) from chemotherapy, but the role of the vascular niche in the bone marrow microenvironment in leukemia is largely unknown. E-selectin, which is expressed on bone marrow endothelial cells, has been described to regulate the dormancy of normal hematopoietic stem cells (HSC) by increasing HSC proliferation (Winkler IG, 2012). In chronic myeloid leukemia (CML), one of the myeloproliferative neoplasias caused by the oncogene BCR-ABL1, E-selectin (Krause DS, 2014) and the adhesion molecule CD44, which is overexpressed on CML-initiating cells (Krause DS, 2006) and to which E-selectin binds (Videira PA, 2018), was shown to be essential for homing and engraftment of LSC. However, exact mechanisms of the interactions between BCR-ABL1, CD44 expression and interactions with the vascular niche are unclear.Hypothesizing that E-selectin influences the cell cycle of leukemic stem cells (LSC) in CML leading to improved eradication of LSC in case of treatment with the tyrosine kinase inhibitor imatinib, we treated murine recipients of CML-initiating cells in the retroviral transduction/transplantation model with the E -selectin inhibitor GMI-1271 (uproleselan, GlycoMimetics, Inc.) and imatinib, considered standard of care in CML. This increased the survival of mice compared to animals treated with imatinib alone and decreased the engraftment of CML-initiating cells in this model, as well as in the majority of mice in a xenotransplantation model of NOD SCID IL2 receptor gamma knockout (NSG) mice injected with human CML cells and treated with uproleselan. Treatment of NSG mice with uproleselan also decreased the contact time of injected human CML cells with the bone marrow endothelium. As shown in in vitro and in vivo experiments treatment with GMI-1271 and non-adhesion to E-selectin increased the cell cycle of BCR-ABL1+ LSC with a concomitant increase in expression of the transcriptional regulator and protooncogene Scl/Tal1. SCL/TAL1 was shown to negatively regulate CD44 expression by binding to the CD44 regulatory element, which was increased in presence of imatinib. In addition, we demonstrated SCL/TAL1 to be an indirect phosphorylation target of BCR-ABL1 and a negative transcriptional regulator of CD44 expression.In confirmation of our findings in mice, we also showed a negative correlation between SCL/TAL1 and CD44 expression in leukocytes of human patients with CML and that increased SCL/TAL1 expression is associated with decreased probability of relapse and increased relapse-free survival in human CML patients after allogeneic hematopoietic stem cell transplantation.In summary, inhibition of E-selectin in murine CML may lead to ‘non-adherence’ of LSC to the vascular niche and improved eradication by imatinib. This is likely due to an increase in cell cycle and an increase of Scl/Tal1 expression, which is shown to be regulated by BCR-ABL1. These data connect the adhesion of LSC in CML to the vascular niche via CD44 with the regulation of CD44 expression by SCL/TAL1 and BCR-ABL1, offering a potential new avenue for treatment. DisclosuresKrause:Glycotope: Consultancy; Glycomimetics Inc. until 06/2016: Consultancy, Research Funding; Merck KgGA in future: Research Funding; European Patent: Patents & Royalties: European Patent No. 16187926.7-1401 “FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA” .

Abstract A19: Establishment of in vitro and in vivo models of inflammatory myofibroblastic tumor

Abstract Inflammatory myofibroblastic tumors (IMTs) are sarcomas of soft tissue that most commonly arise in children, adolescents, and young adults. If not cured by surgery, IMT has a high propensity for local recurrence as multifocal nodules, and may rapidly progress to fatal disease. Anaplastic Lymphoma Kinase (ALK) driven tumorigenesis is implicated in approximately 50% of IMT cases. IMT patients with ALK rearrangement have been reported to respond to the ALK inhibitor, crizotinib, but with the possibility of relapse with resistant disease. The second-generation ALK inhibitor ceritinib showed improved specificity and potency toward crizotinib-resistant tumors; however, resistant disease may still emerge. Thus, there is an urgent need for identifying alternative treatment options for ALK-driven IMT. In this study, we utilized fresh malignant ascites collected from a 9-year-old boy (IMT-1) with epithelioid inflammatory myofibroblastic sarcoma (eIMS), at diagnosis (IMT-1A) and third relapse (IMT-1D) to establish the in vitro cell culture and in vivo orthotopic patient-derived xenograft (PDX) model. Primary cell cultures were successfully established from both samples with confirmed ALK rearrangement. The PDX was established by intraperitoneal injection of cultured IMT-1D cells into nonobese diabetic severe combined immune-deficient mice with IL-2 receptor gamma chain deficiency (NSG) mice. Within 6-8 weeks of inoculation, 100% of the mice became moribund due to robust multifocal intraperitoneal tumors that grew in close association with abdominal organs, including the pancreas and small bowel. The xenograft recapitulated both morphologic and immunohistochemical features of the tumor within the patient. Positive immunohistochemical staining for ALK in a characteristic perinuclear membrane pattern and ALK gene rearrangement were also demonstrated in tumor cells cultured from peritoneal fluid of the xenografted mice. Our in vitro cultures and in vivo PDX models provide a valuable opportunity to undertake a detailed characterization of ALK-driven IMT at diagnosis and following ceritinib failure. They are ideal models for preclinical evaluation of new therapeutic approaches to the treatment of ALK-driven IMT. Citation Format: Jinhan (Angela) Xie, Emily V. A. Mould, Ashleigh Fordham, Andrew J. Gifford, Toby Trahair, Karen L. Mackenzie. Establishment of in vitro and in vivo models of inflammatory myofibroblastic tumor [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A19.

Production and rearing of germ-free X-SCID pigs.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20–100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=−0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/− pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

Transgenic expression of human cytokines in immunodeficient mice does not facilitate myeloid expansion of BCR-ABL1 transduced human cord blood cells.

Several attempts have been made to model chronic myeloid leukemia (CML) in a xenograft setting but expansion of human myeloid cells in immunodeficient mice has proven difficult to achieve. Lack of cross-reacting cytokines in the microenvironment of the mice has been proposed as a potential reason. In this study we have used NOD/SCID IL2–receptor gamma deficient mice expressing human SCF, IL-3 and GM-CSF (NSGS mice), that should be superior in supporting human, and particularly, myeloid cell engraftment, to expand BCR-ABL1 expressing human cells in order to model CML. NSGS mice transplanted with BCR-ABL1 expressing cells became anemic and had to be sacrificed due to illness, however, this was not accompanied by an expansion of human myeloid cells but rather we observed a massive expansion of human T-cells and macrophages/histiocytes. Importantly, control human cells without BCR-ABL1 expression elicited a similar reaction, although with a slight delay of disease induction, suggesting that while BCR-ABL1 contributes to the inflammatory reaction, the presence of normal human hematopoietic cells is detrimental for NSGS mice.

Murine Th17 cells utilize IL-2 receptor gamma chain cytokines but are resistant to cytokine withdrawal-induced apoptosis.

Adoptive cellular therapy (ACT) with the Th17 subset of CD4+ T cells can cure established melanoma in preclinical models and holds promise for treating human cancer. However, little is known about the growth factors necessary for optimal engraftment and anti-tumor activity of Th17 cells. Due to the central role of IL-2 receptor gamma chain (IL2Rγ-chain) cytokines (IL-2, IL-7, and IL-15) in the activity and persistence of many T cell subsets after adoptive transfer, we hypothesized that these cytokines are important for Th17 cells. We found that Th17 cells proliferated in response to IL-2, IL-7, and IL-15 in vitro. However, in contrast to many other T cell subsets, including conventionally activated CD8+ T cells, we found that Th17 cells were resistant to apoptosis in the absence of IL2Rγ-chain cytokines. To determine whether Th17 cells utilize IL2Rγ-chain cytokines in vivo, we tracked Th17 cell engraftment after adoptive transfer with or without cytokine depletion. Depletion of IL-7 and/or IL-2 decreased initial engraftment, while depletion of IL-15 did not. Supplementation of IL-2 increased initial Th17 engraftment. To assess the clinical relevance of these findings, we treated melanoma-bearing mice with Th17 cell adoptive transfer and concurrent cytokine depletion or supplementation. We found that simultaneous depletion of IL-2 and IL-7 decreased therapeutic efficacy, depletion of IL-15 had no effect, and IL-2 supplementation increased therapeutic efficacy. Our results show that Th17 cells are responsive to IL2Rγ-chain cytokines, and provide insight into the application of these cytokines for Th17-based therapeutic strategies.

C75 Fatty Acid Synthesis (FAS) Inhibitor Has Potent Immunosuppressive Activity

Fatty acid synthesis (FAS) or oxidation (FAO) are important regulatory pathways in immune response. In fact, FAS plays a pivotal role in antigen presentation and T cells activation and FAO leads to fatty acid degradation which has been previously shown to regulate hematopoietic stem cell maintenance. Here we hypothesized that FAS can be a new target to suppress T cell alloimmune responses in solid organ or stem cell transplantations. Therefore, we tested if the FAS inhibitor C75 could suppress T cell alloreactivity without impairing normal hematopoiesis.The immuno-suppressive (IS) effect of moderate FAS inhibition was demonstrated in mixed leukocyte cultures (MLC) where C75 at 10 mkg/ml significantly reduced T cell proliferation and prevented the expansion of CD3+CD25+ and CD3+CD69+ T cells. In T cells stimulated by alloantigen, C75 also induced the downregulation of NF-kB gene expression and the upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene involved in ubiquitination and degradation of NF-kB protein.When compared to other standard IS agents, such as anti-thymocyte globulin (ATG), Cyclosporine A, Rapamycin or inhibitor of FAO Etomoxir, C75 showed similar anti-T cell activity.The same dose of C75 (10 mkg/ml) did not cause apoptotic death of human CD34+ cells in vitro, nor affected CD34+ cell clonogenicity in vitro. In fact, C75 increased the number of BFU-E and CFU-GM colonies (P < 0.05). We observed that the expression of de novo DNA methyltrasferases DNMT3A and DNMT3B, which are important regulators of stem cell renewal, was strongly reduced in CD34+ cells co-cultured for 3 days with allogeneic T cells. On the contrary, in the presence of C75 the expression of DNMT3A and DNMT3B was not different from baseline control. To test the in-vivo effect of C75 we utilized a xenograft model of stem cell rejection where 2 x 105 human CD34+ cells and HLA incompatible T lymphocytes were injected in immunodeficient nonobese diabetic/ltsz-scid/scid - IL2 receptor gamma chain knockout (NSG) mice at 1:1 ratio. Four weeks after transplantation, control NSG mice showed complete rejection of huCD45+CD34+ cells and the expansion of T cells in the marrow and spleen. NSG mice treated with intra-peritoneum injections of C75 every 3 days for 2 weeks, instead, showed 10-15% human CD45+ myeloid cells in the marrow and spleen at week 4 after transplant, suggesting at least a partial effect on preventing rejection of incompatible stem cells.We showed here that moderate FAS inhibition may represent a novel immunosuppressive strategy and our findings will prompt preclinical investigations exploiting the effect of FAS inhibitors alone or in combination with standard IS agents in models of allogeneic transplantation or bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Rapid Expansion of Gene-Marked Lymphocytes in X-SCID Dogs after AMD3100+G-CSF-Based Hematopoietic Stem/Progenitor Cell Mobilization and Intravenous Injection of a Common γ-Chain Expressing Foamy Viral Vector

In both humans and canines, X-linked severe combined immunodeficiency disease (XSCID) is caused by mutations in the interleukin-2 receptor gamma chain gene (IL2RG) which results in a lack of response to common gamma-chain (gammaC) dependent cytokines and abnormal development of T and B lymphocytes, and natural killer (NK) cells. Death from infections usually occurs before 1 year of age unless allogeneic hematopoietic cell transplantation (HCT) is performed. While HCT is successful if an HLA-matched sibling donor is available, transplants from mismatched and unrelated donors are associated with greater morbidity and overall survival can be as low as 50%. To circumvent these complications, several clinical trials are testing the possibility of utilizing blood and marrow stem cells from the patient for ex vivo gene therapy to treat X-SCID. Although these trials show promising results, they require expensive GMP cell manufacturing that are not accessible to many patients, and may also necessitate low level of conditioning to improve engraftment of gene-corrected cells. With these limitations in mind, we have explored in vivo gene therapy as a treatment for X-SCID. We previously showed that foamy virus vectors (FVs), exhibit a potentially more favorable integration profile compared to lenti- and gamma-retroviral vectors. In vivo delivery of a gammaC-FV in dogs resulted in immune reconstitution with gene-corrected T cells in dogs but the treated animals still developed infections and had low levels of immunoglobulin levels. We hypothesized that an increased transduction of hematopoietic stem/progenitor cells in vivo might result in more rapid and sustained immune reconstitution. Thus, in the current study, we used cG-CSF and AMD3100 to mobilize hematopoietic stem/progenitor cells into the peripheral blood prior to in vivo injection with a FV expressing the gammaC gene driven by a PGK promoter (PGK-gammaC-FV). We mobilized two X-SCID dogs at ~3 weeks of age with 5ug/kg of cG-CSF bi-daily from day -4 to -1 prior to FV injection, and with 4mg/kg of AMD3100 on the morning of the injection with 4x10e8 IU of PGK-gammaC-FV. Our mobilization protocol resulted in a 10-fold increase in CD34+ cells in the peripheral blood of mobilized X-SCID dogs as compared to a unmobilized normal littermate control (Figure 1 A). Lymphocyte recovery and gene marking in the mobilized animals was significantly improved as compared to animals that were previously injected with similar doses of either PGK-gammaC-FV or EF1a-gammaC-FV but without mobilization. As illustrated in Figure 1B-C, lymphocyte counts expanded to ~3000 cells/uL with ~75% gene marking in the mobilized animals treated with PGK-gammC-FV within 30 days, as compared to <1500 cells/uL with <5% gene marking in unmobilized dogs treated with EF1a-gammaC-FV and to <1000 cells/uL with <50% gene marking in unmobilized dogs treated with PGK-gammaC-FV at all time points post-therapy. The expansion of CD3+ T-cells at 6 weeks post injection for the mobilized dogs was about 2700 cells/uL, as compared to <380 cells/uL in the PGK-gammaC-FV and <210 cells/uL in the EF1a-gammaC-FV unmobilized dogs. Notably, in human clinical trials, CD3 T cell counts were <250 cells/uL following transplantation with autologous CD34+ cells modified with EF1a-gammaC-SIN gamma-retrovirus (Hacein-Bey-Abina, NEJM, 2014). In conclusion, mobilization with cG-CSF and AMD3100 prior to in vivo injection of PGK-gammaC-FV substantially improved the lymphocyte expansion and immune reconstitution in X-SCID dogs and resulted in a higher level of gene marking in myeloid cells (about 1%) at one-month post injection than seen in our previous studies in unmobilized dogs. These results suggest remarkable potential for an accessible and portable approach for treatment of human X-SCID clinical trials using combination of hematopoietic stem/progenitor cells mobilization and in vivo foamy viral vector delivery. [Display omitted] [Display omitted] [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership. Scharenberg:bluebird bio: Consultancy, Equity Ownership, Research Funding; Alpine Immune Sciences: Consultancy. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

MiRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity

Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.