Murine Interleukin Research Articles

Purification of Murine IL-10 + B Cells for Analyses of Biological Functions and Transcriptomics.

B10 cells are the most frequently investigated subset of Breg cells, capable of suppressing immunity through the expression of the immunosuppressive cytokine IL-10. B10 cells are enriched in phenotypically diverse B-cell subsets. Recently, CD9 was identified as a marker of B10 cells in mice (human B10 cells have a separate set of markers that do not overlap with murine B10 cells). Together with a combination of other B10 markers, CD9 can be used to distinguish both mature and immature B10 cells from nonregulatory B cells and support selective purification of B10 cells. Here we provide five methods for the characterization and activity evaluation of CD9+ B cells. The first method is used for the preparation of leukocytes, the second and third are used for the characterization of CD9+ B cells, while the last two methods serve to evaluate CD9+ B-cell activities. Finally, we detail the purification of RNA from B10 cells and the performance of transcriptomic assays.

Inhibition of adjuvant-induced TAM receptors potentiates cancer vaccine immunogenicity and therapeutic efficacy.

Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10+ DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8+ T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.

The role of IL-10 in immune responses against Pseudomonas aeruginosa during acute lung infection.

Pseudomonas aeruginosa is considered an opportunistic pathogen of great clinical importance. The clearance of this bacterium occurs through recognition of the pathogen by innate immune system receptors, leading to a lung inflammatory response. However, this response must be controlled via immunoregulatory pathways. In this study, we evaluate the role of endogenous murine IL-10 after acute infection with the virulent strain P. aeruginosa PA14. To assess the role of IL-10, intratracheal infection with the PA14 strain was performed in C57BL/6 or IL-10 KO mice. The PA14 strain was recovered in both types of animals, although IL-10 KO mice presented a higher number of viable bacteria in the lung when compared to the C57BL/6 group. Histopathological and stereological analyses showed that IL-10 KO mice had higher tissue damage and inflammatory infiltrate when compared to control animals. The activity of MMP-9 but not MMP-2, as well as IL-6 and TNF-α expression, were augmented in the lungs of infected animals and was much more evident in IL-10 KO animals when compared to the other analyzed groups. This work indicates that endogenous IL-10 control P. aeruginosa infection, the expression of pro-inflammatory genes, MMP-9 activity and histopathological processes of the infectious process in question.

Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

The Colony-Stimulating Factor 3 Receptor M696T Mutated in a Patient with Ph+ Acute Lymphoblastic Leukemia

Purpose: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders, such as severe congenital neutropenia (SCN), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and atypical chronic myelogenous leukemia (aCML). Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. We report a case of philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) with the M696T mutation in CSF3R gene and assess the pathogenicity of the CSF3R M696T mutation in Ph+ ALL. Experimental Design: Here we report on a 32-year-old female who presented with asthenia. The initial hematological workup revealed white blood cell (WBC) count of 97 x 109/L (normal range 4-10 x 109/L). There was 84% prolymphocyte in the bone marrow. The immunophenotype of the blasts as judged from flow cytometry was in accordance with a B-ALL. The fusion gene for BCR-ABL P210 was positive. Hot mutation closely related to diseases was: CSF3R (nucleotide change c.2087 T>C, amino acid change p.M696T, mutation frequency 50.4%). Cytogenetic analysis showed 46, XX, t (9;22) (q34;q11). The patient was diagnosed as Ph+ ALL with the CSF3R M696T mutation and achieved Long-term survival after unrelated donor hematopoietic stem cell transplantation. Meanwhile we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. The phosphorylation of STAT3 was analyzed by G-CSF dependence assays and immunoblot analysis to evaluate the CSF3R M696T mutation contribution to the tumor transformation ability of Ba/F3 cells. Results: This patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. We confirmed the presence of a CSF3R M696T germline mutation in this patient, and the mutation was inherited from her mother. The experiments in vitro result showed the CSF3R M696T mutation harbors marginal contribution to the tumor transformation ability of Ba/F3 cells. CSF3R M696T mutation was neutral in tumor transformation ability. Conclusions: We believe that TKI is still effective in patients with the CSF3R M696T mutation in Ph+ ALL. Donor with CSF3R M696T mutation might still be selected. Disclosures No relevant conflicts of interest to declare.

Human Orthogonal IL-2/IL-2Rβ As a Tunable Approach to Enhance CD19-Speific CAR-T Cell Antitumor Activity

Immunotherapy using T cells engineered with a chimeric antigen receptors (CAR) can produce deep and durable control of B-cell malignancies that is associated with the engraftment and persistence of the CAR T cells following adoptive transfer. Interleukin-2 (IL-2) is a central T cell cytokine that promotes T cell proliferation and effector function with a potential to enhance CAR T cell immunotherapy; however, the use of recombinant IL-2 to support T cell immunotherapy is limited by the significant toxicity associated with IL-2 infusion. Previous work has demonstrated the feasibility of engineering murine IL-2 and it's receptor to create an orthogonal (ortho) cytokine-receptor pair capable of delivering IL-2's signal without the associated toxicity (Sockolosky JT et al. Science 2018). In order to translate this novel approach to humans, we engineered a human orthogonal IL-2 (ortho-hIL-2) and human orthogonal IL-2Rβ (ortho-hIL-2Rβ) pair based upon similar mutations to those required in the murine system. We show that hoIL-2 induces JAK-STAT signaling and supports proliferation of the IL-2 dependent cutaneous T-cell lymphoma (CTCL) line, SeAx, and primary human CD8+ primary T cells when hoIL-2Rb is expressed in these cells. HoIL-2 at concentrations 1000-fold higher than wild-type IL-2 failed to induce signaling in the absence of the orthogonal receptor illustrating the high selectivity of ortho-hIL-2 for the ortho-hIL-2Rβ. Using a CD19-specific, 4-1BB-costimulated CAR T cell (CART19), we show that ortho-hIL-2 induced a dose-dependent increase in ortho-hIL-2Rβ CAR-T cell expansion and serum cytokines in vivo by as much as 1000-fold at 2 weeks following adoptive transfer into NSG mice bearing CD19+ Nalm6 leukemia xenografts and daily ortho-hIL-2 treatment. We further demonstrate that hoIL-2 can rescue the anti-leukemic effect of an otherwise suboptimal CAR T cell dose. In addition to augmenting initial CAR T cell engraftment and efficacy, we also show that ortho-hIL-2 administration initiated at the time of leukemic relapse following CAR T cell therapy can rescue an otherwise failed anti-leukemic response. In aggregate, these data demonstrate the ability of the human orthogonal IL-2 cytokine-receptor system to support T cell expansion in vivo and the potential of combining this approach with CAR T cell immunotherapy to augment the anti-tumor efficacy of engineered T cells. Disclosures Sockolosky: Synthekine Therapeutics: Current equity holder in private company. Garcia:Synthekine: Current equity holder in private company, Other: founder. Milone:Novartis: Patents & Royalties.

Retargeted and Multi-cytokine-Armed Herpes Virus Is a Potent Cancer Endovaccine for Local and Systemic Anti-tumor Treatment

Oncolytic viruses (OVs) are novel anti-tumor agents with the ability to selectively infect and kill tumor cells while sparing normal tissue. Beyond tumor cytolysis, OVs are capable of priming an anti-tumor immune response via lysis and cross-presentation of locally expressed endogenous tumor antigens, acting as an “endovaccine.” The effectiveness of OVs, similar to other immunotherapies, can be hampered by an immunosuppressive tumor microenvironment. In this study, we modified a previously generated oncolytic herpes simplex virus (oHSV) retargeted to the human HER2 (hHER2) tumor molecule and encoding murine interleukin-12 (mIL-12), by insertion of a second immunomodulatory molecule, murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), to maximize therapeutic efficacy. We assessed the efficacy of this double-armed virus (R-123) compared to singly expressing GM-CSF and IL-12 oHSVs in tumor-bearing mice. While monotherapies were poorly effective, combination with α-PD1 enhanced the anti-tumor response, with the highest efficacy of 100% response rate achieved by the combination of R-123 and α-PD1. Efficacy was T cell-dependent, and the induced immunity was long lasting and able to reject a second contralateral tumor. Importantly, systemic delivery of R-123 combined with α-PD1 was effective in inhibiting the development of tumor metastasis. As such, this approach could have a significant therapeutic impact paving the way for further development of this platform in cancer immunotherapy.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses

BackgroundThough currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain...

An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice.

Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (Treg) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved Treg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced Treg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective Treg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall Treg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential Treg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of Treg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules.

Abstract LB-082: In vivo efficacy and pharmacodynamic analysis of RTX-321, an engineered allogeneic artificial antigen presenting red cell therapeutic

Abstract Human papilloma virus (HPV)-infection is strongly associated with the risk of cancer, with the high-risk strain HPV16 accounting for ~80% of head and neck cancers. HPV16+ malignancies remain a critical area of high unmet need for new and innovative treatment options. To address this, Rubius Therapeutics has genetically engineered red cells to create an allogeneic artificial antigen-presenting cell (RCT-aAPC) for the potential treatment of HPV16+ cancers. RTX-321 is engineered to express an HPV16 antigen bound to MHC I, 4-1BBL and IL-12 on the cell surface to mimic T cell-APC interactions. RTX-321 is designed to activate and expand tumor-specific T cells present within the patient, bypassing the dependence on host APCs typically required for cancer vaccines and eliminating the need for manufacturing patient-derived T cells as with CAR-T therapies. Previously, a surrogate murine red cell (mRBC-321) with surface conjugated murine MHC I-OVA, 4-1BBL and IL-12 demonstrated in vivo tumor killing in an antigen-specific tumor model (EG7.OVA) with adoptive cell transfer of naïve OT1 cells. We now demonstrate through confocal live cell imaging a direct and tight interaction between mRBC-321 and activated OT1 cells. In addition, mRBC-321 preferentially accumulates in the spleen, directly interacting with OT1 cells, which induces activation and eventual expansion of OT1 cells in the spleen. TCR-sequencing in this tumor model reveals increased TCR clonality (p=0.02) in tumors treated with mRBC-321, accompanied by tumor growth inhibition (TGI) compared to controls, indicating an antigen-specific tumor response. Ongoing analysis will examine TCR diversity in the blood during tumor re-challenge (EG7.OVA or parental EL4) in cured mice to determine the degree of immunological memory and epitope spreading induced by mRBC-321. Follow-up efficacy studies were conducted without OT1 transfer to evaluate endogenous antigen-specific T cells in mediating TGI. Mice treated with mRBC-321 showed significant TGI (p<0.001) and increased overall survival (p<0.001) compared to controls. Immunophenotyping by flow cytometry revealed key mRBC-321 mediated pharmacodynamic changes including increased activation/effector markers PD-1, KLRG-1 and effector memory phenotypes on CD8+ T cells (p<0.05). Additionally, PBMCs from CMV+ donors were used to determine relevant antigen-specific pharmacodynamic readouts. Co-culture of these PBMCs and RCT-aAPC (MHC-I-CMV+4-1BBL+IL12) induced CD137 shedding, IFNγ secretion and expansion of antigen specific T cells with increased CD25, 4-1BB and HLA-DR expression compared to control (p<0.05). Together, these data confirm that RCT-aAPC can induce activation of antigen-specific CD8 T cells which can translate to tumor regression. Rubius plans to file an Investigational New Drug application for RTX-321 in HPV16+ advanced solid tumors in 2020. Citation Format: Mellissa Joy Nixon, Xuqing Zhang, Albert Lee, Mengyao Luo, Annie Khamhoung, Andrea Schmidt, Douglas McLaughlin, Shamael R. Dastagir, Viral Amin, Chris Moore, Jennifer Mellen, Chris Carpenter, Tom Wickham, Tiffany Chen. In vivo efficacy and pharmacodynamic analysis of RTX-321, an engineered allogeneic artificial antigen presenting red cell therapeutic [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-082.

Abstract 4561: Oncolytic vaccinia virus encoding IL-7 and IL-12 systemically sensitizes tumors to anti-PD-1 antibody in a syngeneic mouse model

Abstract Immune checkpoint blockade targeting PD-1, PD-L1 and CTLA-4 have demonstrated remarkable clinical responses in patients with advanced cancers; however, a durable benefit is obtained in only a subset of patients. To overcome this therapeutic limitation, a number of immunotherapies and their combinations have been investigated. Among these, in situ vaccination using tumor-selective oncolytic viruses has proved an attractive approach. Viruses not only directly kill cancer cells through their replication, but also activate antitumor immunity triggered by the release of tumor antigens from killed cells. We have explored this approach using tumor-selective oncolytic vaccinia viruses (VVs) carrying immunomodulatory genes. We previously demonstrated that intratumoral treatment with a VV carrying human IL-7 and murine IL-12 genes (hIL-7/mIL-12-VV) enhanced immune responses in treated tumors and induced complete tumor regression in syngeneic mouse models (AACR-NCI-EORTC 2019). Here, using a bilaterally tumor-bearing mouse model, we examined whether unilateral virus injection affected untreated distant tumors. All tumors that had been directly injected with hIL-7/mIL-12-VV completely regressed. At the same time, untreated tumors in three of six mice also disappeared. We further confirmed significant increases in tumor-infiltrating lymphocytes in both treated and untreated tumors after treatment with hIL-7/mIL-12-VV. Unlike the treated tumors, the untreated distant tumors showed no detectable viral DNA by qPCR. PD-L1 expression was also upregulated in this model following treatment with hIL-7/mIL-12-VV in both treated and untreated tumors. This led us to examine the effects of combining this virotherapy with anti-PD-1 antibody. While monotherapies with anti-PD-1 antibody did not show significant antitumor efficacy, hIL-7/mIL-12-VV prior to this agent elicited improved antitumor activity in the virus-untreated distant tumors, suggesting that hIL-7/mIL-12-VV systemically sensitized tumors to anti-PD-1 antibody. Evidence that intratumoral immune status was changed following hIL-7/mIL-12-VV was found in the upregulation of various immune pathways analyzed using the Nanostring Pancancer Immune Profiling panel, and the increased clonality of T-cell repertoire in tumors. These results support the clinical development of VV carrying IL-7 and IL-12 as both monotherapy and as a combination partner with anti-PD-1 antibody for patients with advanced tumors. Citation Format: Shinsuke Nakao, Midori Yamashita, Mamoru Tasaki, Tatsuya Kawase, Motomu Nakatake, Hajime Kurosaki, Masamichi Mori, Masahiro Takeuchi, Takafumi Nakamura. Oncolytic vaccinia virus encoding IL-7 and IL-12 systemically sensitizes tumors to anti-PD-1 antibody in a syngeneic mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4561.

Oncolytic Measles Virus Encoding Interleukin-12 Mediated Antitumor Activity and Immunologic Control of Colon Cancer In Vivo and Ex Vivo.

Background: An oncolytic measles virus encoding interleukin 12 (IL-12) to treat colon cancer in vivo and ex vivo to investigate its effect on the viability and apoptosis of colon cancer cells in this study. Materials and Methods: A rat model was established to evaluate the immunostimulatory capabilities and therapeutic efficacy of vectors encoding an IL-12 fusion protein (MeVac FmIL-12 vectors). TUNEL staining, Western blot, and enzyme-linked immunosorbent assay were performed to examine the impacts of MeVac FmIL-12 on the expression of inflammatory cytokines. Cell transfection was carried out to validate the anti-tumor role of MeVac FmIL-12 in vitro. Flow cytometry and MTT assay were performed to assess the effects of MeVac FmIL-12 on cell apoptosis and viability. Result: High concentrations (10-1000 ng/mL) of murine IL-12 fusion protein (FmIL-12) decreased the production of interferon γ (IFN-γ) in a concentration-dependent manner and reflected FmIL-12-induced overstimulation. Rats treated with MeVac vectors encoding FmIL-12 showed a significantly increased level of FmIL-12 overtime and a concentration-dependent (0.01-10 ng/mL) increase in IFN-γ production. MeVac FmIL-12 also increased the expression of inflammatory cytokines (IFN-γ, tumor necrosis factor α, and IL-6) both in vivo and in vitro. MeVac FmIL-12 promoted cell apoptosis and reduced cell viability, which helped to trigger a systemic anti-tumor immune response, both in vivo and in vitro. Conclusion: In this study, we suggested that MeVac FmIL-12 enhanced the therapeutic efficacy of tumor treatment by improving anti-tumor immunity.

Targeting interleukin-4 to the arthritic joint

Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.

CORM-3 improves emotional changes induced by hemorrhagic shock via the inhibition of pyroptosis in the amygdala

Hemorrhagic shock and resuscitation (HSR) may lead to long-term neurological dysfunction, such as depression and anxiety. Carbon monoxide (CO) has emerged as an excellent neuroprotective agent against caspase-1-associated pyroptosis, following HSR. We evaluated the effects and determined the mechanism through which CO protects against emotional changes in a model of HSR, in rats. We subjected rats to treatments with an exogenous, CO-releasing compound (CORM-3, 4 mg/kg), in vivo, after HSR. We measured sucrose preference and performed tail suspension and open field tests 7 days after HSR, assessed brain magnetic resonance imaging 12 h after HSR and evaluated pyroptosis, and neuronal and astrocyte death in the amygdala 12 h post-HSR. We also measured changes in behavior and pathology, following an injection of recombinant murine interleukin (IL)-18 into the amygdala. HSR-treated rats displayed increased depression-like and anxiety-like behaviors, increased amygdalar injury, as indicated by T2-weighted magnetic resonance imaging (MRI) and cerebral blood flow with arterial spin labeling (CBFASL), associated with both neuronal and astrocytic death and pyroptosis, and upregulated IL-18 expression was observed in astrocytes. CORM-3 administration after resuscitation, via a femoral vein injection, provided neuroprotection against HSR, and this neuroprotective effect could be partially reversed by the injection of recombinant murine IL-18 into the amygdala. Therefore, CORM-3 alleviated HSR-induced neuronal pyroptosis and emotional changes, through the downregulation of IL-18 in astrocytes.

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

In this study, we present a straightforward approach for functional cell-based screening by co-encapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarose-containing microdroplets. Our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and FACS. This indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins.

Deregulation of miRNAs-cMYC circuits is a key event in refractory celiac disease type-2 lymphomagenesis.

A percentage of celiac disease (CD) patients develop refractory type-2 disease (RCD2), a condition associated with increased risk of enteropathy-associated T-cell-lymphoma (EATL) and without therapeutic option. Therefore, we profiled the miRNome in series of peripheral T-cell lymphomas (PTCLs), CD, RCD1 or 2 and in the murine interleukin-15 (IL15)-transgenic (TG) model of RCD. The transcriptome was analyzed in 18 intestinal T-cell lymphomas (ITLs). Bioinformatics pipelines provided significant microRNA (miRNA) lists and predicted targets that were confirmed in a second set of patients. Our data show that ITLs have a unique miRNA profile with respect to other PTCLs. The c-MYC regulated miR-17/92 cluster distinguishes monomorphic epitheliotropic ITL (MEITL) from EATL and prognosticates EATL outcome. These miRNAs are decreased in IL15-TG mice upon Janus kinase (JAK) inhibition. The random forest algorithm identified a signature of 38 classifier miRNAs, among which, the miR-200 and miR-192/215 families were progressively lost in RCD2 and ITL-CD, whereas miR-17/92 and C19MC miRNAs were up-regulated. Accordingly, SMAD3, MDM2, c-Myc and activated-STAT3 were increased in RCD2 and EATL tissues while JAK inhibition in IL15-TG mice restored their levels to baseline. Our data suggest that miRNAs circuit supports activation of STAT3 and c-Myc oncogenic signaling in RCD2, thus contributing to lymphomagenesis. This novel understanding might pave the way to personalized medicine approaches for RCD and EATL.

Polyinosinic: Polycytidylic Acid and Murine Cytomegalovirus Modulate Expression of Murine IL-10 and IL-21 in White Adipose Tissue.

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli.

Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma

BackgroundTemozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system,...

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.

A Treg-Selective IL-2 Mutein Prevents the Formation of Factor VIII Inhibitors in Hemophilia Mice Treated With Factor VIII Gene Therapy.

Hemophilia A is a genetic disorder that results in the deficiency of functional factor VIII protein, which plays a key role in blood coagulation. Currently, the majority of hemophilia A patients are treated with repeated infusions of factor VIII protein. Approximately 30% of severe hemophilia A patients develop neutralizing antibodies to factor VIII (known as factor VIII inhibitors) due to treatment, rendering factor VIII protein infusions ineffective. Previously, mice receiving murine IL-2 complexed with α-murine IL-2 mAbs (JES6-1A12) showed a lack of factor VIII inhibitor formation after factor VIII treatment, which was associated with the proliferation and the activation of factor VIII-specific regulatory T cells (Tregs). In this paper, we evaluated if an Fc-fused mutated protein analog of mouse IL-2, named Fc.Mut24, engineered to selectively promote the expansion of Tregs in vivo can modulate factor VIII-specific immune responses. The mice received one intraperitoneal injection of Fc.Mut24. When the regulatory T cell population reached its highest frequency and peak activation, the mice received a hydrodynamic injection of factor VIII plasmid (day 4) followed by a second Fc.Mut24 dose (day 7). Peripheral blood was collected weekly. Flow cytometry was used to characterize the peripheral blood cell populations, while ELISA and Bethesda assays were used to assess the inhibitor concentrations and the functional titers in plasma. The activated partial thromboplastin time assay was used to assess the functional activities of factor VIII in blood. The mice receiving Fc.Mut24 showed a dramatic and transient increase in the population of activated Tregs after Fc.Mut24 injection. Factor VIII gene therapy via hydrodynamic injection resulted in high anti-factor VIII inhibitor concentrations in control PBS-injected mice, whereas the mice treated with Fc.Mut24 produced no inhibitors. Most significantly, there were no inhibitors generated after a second hydrodynamic injection of factor VIII plasmid administered at 19 weeks after the first injection in Fc.Mut24-treated mice. The mice receiving Fc.Mut24 maintained high levels of factor VIII activity throughout the experiment, while the control mice had the factor VIII activity dropped to undetectable levels a few weeks after the first factor VIII plasmid injection. Our data show that human therapies analogous to Fc.Mut24 could potentially provide a method to prevent inhibitor formation and induce long-term immune tolerance to factor VIII in hemophilia patients.