Murine Interleukin Research Articles

An IL-2 mutein promotes Foxp3+ Treg-mediated suppression of dendritic cell activation in response to inflammatory stimuli

Abstract Foxp3+ regulatory T (Treg) cells are essential for maintaining immune tolerance and the development of strategies that boost Tregs is a promising approach for the treatment of autoimmune disorders. We previously developed a murine Fc-fused IL-2 mutein (Fc.Mut24) with decreased affinity for IL-2Rβ resulting in increased CD25 dependency and potent Treg selectivity. High-dose Fc.Mut24 administration prevents autoimmunity in the non-obese diabetic (NOD) mouse; however, the mechanisms by which Fc.Mut24 affects Treg suppressive function remain unclear. In this study, we have performed a phenotypic analysis of Treg and classical dendritic cells (DC) from IL-2 mutein-treated mice. We have found that Fc.Mut24 treatment increases CTLA-4 recycling/expression on Treg with a concurrent decrease in PD-1 expression specifically on effector regulatory T (eTreg) cells. Both CD8+ cDC1 and DCIR2+ cDC2 subsets from Fc.Mut24-treated mice have reduced expression of the co-stimulatory molecules CD80/CD86 following lipopolysaccharide (LPS) injection. Furthermore, RNA-sequencing reveals that Tregs regulate the transcriptional response of DCs to LPS and may preferentially suppress cDC1 activation in vivo. These results suggest that a major mechanism of action by IL-2 mutein may be promoting CTLA-4-mediated transendocytosis of CD80/CD86 while simultaneously reducing PD-1: PD-L1 co-inhibitory signaling between eTreg and DCs. Our findings address unanswered questions about the role of IL-2 in modulating Treg suppressive function and have important implications for the use of Treg-selective IL-2 therapeutics in the clinic. B.L.J. is supported by a postdoctoral fellowship from the Washington Research Foundation.

Oncolytic Viral Therapy Mediates its Effectiveness by Inducing and Enhancing CD4 cytotoxic T-cells

Abstract Malignant glioma remains a fatal disease with a dismal survival rate. To address this urgent medical need, we pioneered the concept of engineered oncolytic viruses for cancer. Using the herpes simplex virus (HSV)-1 as an experimental virus, our published Phase I trials have demonstrated the safety and efficacy of such oHSV-based therapy for treating brain tumor patients. In searching for biomarkers for better responses to this therapy, our preclinical studies using intracranial glioma models and an oHSV secreting murine IL-12 (M002) revealed that a subset of CD4+ T-cells expressing the cytolytic signatures, including granzyme B, were increased in M002-treated mice compared to vehicle-treated controls. Correspondingly, CD4+ T-cells isolated from brain tumors of M002-treated mice killed tumor cells better than cells isolated from control tumors. However, the factors and temporal requirements that direct the development of this CD4 cytotoxic T-cell (CTL) subset are not fully understood. To address this question, we performed single-cell RNA sequencing analysis of CD4+ T-cells isolated from brain tumors of M002-treated and control mice. The results revealed a complex change of the CD4+ T-cell compartment with CD4 CTLs being induced from two different precursors. The first CTL subset was derived from T helper-1 (Th1) cells and retained Th1 gene signatures while expressing high levels of CTL associated molecules. The second CTL subset was a reprogrammed regulatory T-cell population capable of producing high levels of cytokines and granzymes. Both CD4 CTL subsets were more prominent in M002-treated mice, suggesting that M002 therapy could induce CD4 CTLs with specialized functionality, which may account for its improved outcome. Supported by grants from DoD W81XH-18-1-0315 and UAB Start up Funds

Evaluation of the long-term anti-Spike immune response following a novel coronavirus vaccine (CORVax): electroporation of SARS-CoV-2 Spike plasmid DNA plus IL12 plasmid DNA

Abstract The global SARS-CoV-2 (CoV2) pandemic has focused efforts to generate effective vaccines that induce potent and persistent immunity. Recently, we described a novel vaccine approach using electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax) plus plasmid Interleukin-12 (IL-12). IL-12 is an effective vaccine adjuvant that increases Th1 and Th2 antibodies in the serum. Here we examine the persistence of anti-Spike antibodies present in the serum of mice one year following CORVax vaccination. C57BL/6 and BALB/c were vaccinated intramuscular (IM) and/or intradermal (ID) with a plasmid encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12. Mice received plasmid EP immediately following injection. Splenocytes and serum were harvested at various time points to interrogate virus-specific cellular responses as well anti-spike antibody titers. Anti-Spike IgG antibodies were elicited by EP of CORVax (IC50 = 1/2112), as well as EP of CORVax combined with IL-12 (IC50 = 1/4214) approximately 40 days after the booster vaccination. These anti-Spike IgG titers decayed over time but were still present 1 year after vaccination: CORVax (IC50 = 1/351 day 146, IC50 = 1/208 day 383); CORVax + IL12 (IC50 = 1/590 day 146, IC50 = 1/266 day 383). Our data shows that EP of CORVax induces IgG responses to CoV2 Spike and the CoV2 Spike receptor binding domain. At one year following vaccination the anti-Spike IgG titers were higher in mice that received CORVax plus IL-12, however the rate at which the titers waned from their initial peak was comparable whether the mice received IL-12 or not. Additional studies are ongoing to determine whether the addition of IL-12 will enhance an anti-Spike memory response. Generous support from The Chiles Foundation, Nancy Lematta, and the Providence Medical Foundation.

Synthesis, Characterization, and In Vivo Cytokinome Profile of IL-12-Loaded PLGA Nanospheres.

We report the successful encapsulation and elution of recombinant murine IL-12 (rmIL-12) from poly(lactide-co-glycolic) acid (PLGA) nanospheres (IL-12-NS) synthesized using the double emulsion/solvent evaporation (DESE) technique with microsphere depletion through ultracentrifugation. Images obtained with scanning electron microscopy (SEM) showcased a characteristic spherical shape with a mean particle diameter of 138.1 ± 10.8 nm and zeta potential of −15.1 ± 1.249 mV. These values suggest minimal flocculation when in solution, which was reflected in an in vivo biodistribution study that reported no observed morbidity/mortality. Encapsulation efficiency (EE) was determined to be 0.101 ± 0.009% with average particle concentration obtained per batch of 1.66 × 109 ± 4.45 × 108 particles/mL. Disparate zeta (ζ) potentials obtained from both protein-loaded and protein-unloaded batches suggested surface adsorption of protein, and confocal microscopy of BSA-FITC-loaded nanospheres confirmed the presence of protein within the polymeric shell. Furthermore, elution of rmIL-12 from IL-12-NS at a concentration of 500 million particles/mL was characterized using enzyme-linked immunosorbent assay (ELISA). When IL-12-NS was administered in vivo to female BALB/c mice through retroorbital injection, IL-12-NS produced a favorable systemic cytokine profile for tumoricidal activity within the peripheral blood. Whereas IFN-γ nadir occurred at 72 hours, levels recovered quickly and displayed positive correlations postburst out to 25 days postinjection. IL-12-NS administration induced proinflammatory changes while prompting minimal counterregulatory increases in anti-inflammatory IL-10 and IL-4 cytokine levels. Further, while IL-6 levels increased to 30 folds of the baseline during the burst phase, they normalized by 72 hours and trended negatively throughout the sill phase. Similar trends were observed with IL-1β and CXCL-1, suggesting a decreased likelihood of progression to a systemic inflammatory response syndrome-like state. As IL-12-NS delivers logarithmically lower amounts of IL-12 than previously administered during human clinical trials, our data reflect the importance of IL-12-NS which safely create a systemic immunostimulatory environment.

Interleukin-11 drives human and mouse alcohol-related liver disease

ObjectiveAlcoholic hepatitis (AH) reflects acute exacerbation of alcoholic liver disease (ALD) and is a growing healthcare burden worldwide. Interleukin-11 (IL-11) is a profibrotic, proinflammatory cytokine with increasingly recognised toxicities in...

IFIT2-depleted metastatic oral squamous cell carcinoma cells induce muscle atrophy and cancer cachexia in mice.

BackgroundInterferon‐induced protein with tetratricopeptide repeat 2 (IFIT2) is a reported metastasis suppressor in oral squamous cell carcinoma (OSCC). Metastases and cachexia may coexist. The effect of cancer metastasis on cancer cachexia is largely unknown. We aimed to address this gap in knowledge by characterizing the cachectic phenotype of an IFIT2‐depleted metastatic OSCC mouse model.MethodsGenetically engineered and xenograft tumour models were used to explore the effect of IFIT2‐depleted metastatic OSCC on cancer cachexia. Muscle and organ weight changes, tumour burden, inflammatory cytokine profiles, body composition, food intake, serum albumin and C‐reactive protein (CRP) levels, and survival were assessed. The activation of the IL6/p38 pathway in atrophied muscle was measured.ResultsIFIT2‐depleted metastatic tumours caused marked body weight loss (−18.2% vs. initial body weight, P < 0.001) and a poor survival rate (P < 0.01). Skeletal muscles were markedly smaller in IFIT2‐depleted metastatic tumour‐bearing mice (quadriceps: −28.7%, gastrocnemius: −29.4%, and tibialis: −24.3%, all P < 0.001). Tumour‐derived circulating granulocyte‐macrophage colony‐stimulating factor (+772.2‐fold, P < 0.05), GROα (+1283.7‐fold, P < 0.05), IL6 (+245.8‐fold, P < 0.001), IL8 (+616.9‐fold, P < 0.001), IL18 (+24‐fold, P < 0.05), IP10 (+18.8‐fold, P < 0.001), CCL2 (+439.2‐fold, P < 0.001), CCL22 (+9.1‐fold, P < 0.01) and tumour necrosis factor α (+196.8‐fold, P < 0.05) were elevated in IFIT2‐depleted metastatic tumour‐bearing mice. Murine granulocyte colony‐stimulating factor (+61.4‐fold, P < 0.001) and IL6 (+110.9‐fold, P < 0.01) levels were significantly increased in IFIT2‐depleted metastatic tumour‐bearing mice. Serum CRP level (+82.1%, P < 0.05) was significantly increased in cachectic shIFIT2 mice. Serum albumin level (−26.7%, P < 0.01) was significantly decreased in cachectic shIFIT2 mice. An assessment of body composition revealed decreased fat (−81%, P < 0.001) and lean tissue (−21.7%, P < 0.01), which was consistent with the reduced food intake (−19.3%, P < 0.05). Muscle loss was accompanied by a smaller muscle cross‐sectional area (−23.3%, P < 0.05). Muscle atrophy of cachectic IFIT2‐depleted metastatic tumour‐bearing mice (i.v.‐shIFIT2 group) was associated with elevated IL6 (+2.7‐fold, P < 0.05), phospho‐p38 (+2.8‐fold, P < 0.05), and atrogin‐1 levels (+2.3‐fold, P < 0.05) in the skeletal muscle. Neutralization of IL6 rescued shIFIT2 conditioned medium‐induced myotube atrophy (+24.6%, P < 0.01).ConclusionsOur results suggest that the development of shIFIT2 metastatic OSCC lesions promotes IL6 production and is accompanied by the loss of fat and lean tissue, anorexia, and muscle atrophy. This model is appropriate for the study of OSCC cachexia, especially in linking metastasis with cachexia.

Targeting High-Risk Neuroblastoma Patient-Derived Xenografts with Oncolytic Virotherapy.

Simple SummaryMore than half of children with high-risk neuroblastoma will not survive despite aggressive treatment regimens. The discovery of suitable novel therapies that target high-risk disease remain limited. Oncolytic virotherapy employs common viruses, such as herpes simplex virus. These viruses are modified to solely target cancer cells and avoid damage to healthy cells. The previous work of others and ourselves has shown oncolytic herpes simplex viruses (oHSVs) are effective in targeting cultured neuroblastoma cancer cell lines, but to our knowledge, no one has demonstrated the effects of oHSV in neuroblastoma patient-derived xenograft cells. Herein, we show that oHSV works well in high-risk neuroblastoma patient-derived xenografts and provide critical preclinical evidence to translate this therapy to a clinical setting.Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

Resveratrol Treatment Prevents Increase of Mast Cells in Both Murine OVA Enteritis and IL-10-/- Colitis.

Mast cells are involved in allergic and other inflammatory diseases. The polyphenol resveratrol is known for its anti-inflammatory properties and may be used as nutraceutical in mast cell associated diseases. We analyzed the effect of resveratrol on mast cells in vivo in ovalbumin-induced allergic enteritis as well as experimental colitis in IL-10−/− mice which received resveratrol via drinking water. Treatment with resveratrol prevented the increase in mast cells in both allergic enteritis and chronic colitis in duodenum as well as in colon. Further, it delayed the onset of diseases symptoms and ameliorated diseases associated parameters such as tissue damage as well as inflammatory cell infiltration in affected colon sections. In addition to the findings in vivo, resveratrol inhibited IgE-dependent degranulation and expression of pro-inflammatory cytokines such as TNF-α in IgE/DNP-activated as well as in LPS-activated bone marrow-derived mast cells. These results indicate that resveratrol may be considered as an anti-allergic and anti-inflammatory plant-derived component for the prevention or treatment of mast cell-associated disorders of the gastrointestinal tract.

Chemo-Enzymatic PEGylation/POxylation of Murine Interleukin-4.

Interleukin-4 (IL-4) is a potentially interesting anti-inflammatory therapeutic, which is rapidly excreted. Therefore, serum half-life extension by polymer conjugation is desirable, which may be done by PEGylation. Here, we use PEtOx as an alternative to PEG for bioconjugate engineering. We genetically extended murine IL-4 (mIL-4) with the d-domain of insulin-like growth factor I (IGF-I), a previously identified substrate of transglutaminase (TG) Factor XIIIa (FXIIIa). Thereby, engineered mIL-4 (mIL-4-TG) became an educt for TG catalyzed C-terminal, site-directed conjugation. This was deployed to enzymatically couple an azide group containing peptide sequence to mIL-4, allowing C-terminal bioconjugation of polyethylene glycol or poly(2-ethyl-2-oxazoline). Both bioconjugates had wild-type potency and alternatively polarized macrophages.

Abstract P064: Novel engineering of therapeutic Fusobacterium nucleatum phage for colorectal cancer treatment

Abstract Fusobacterium nucleatum (FN) is enriched in human colorectal tumors [1] and its presence is correlated with poor prognosis [2]. Bacteriophages ("phages") are naturally occurring, self-amplifying viruses that are highly specific for particular bacterial species and strains and are recognized to have a high intrinsic safety. They offer a promising treatment strategy to both target FN associated with colorectal cancer (CRC) and, by phage engineering, to deliver an anti-tumoral immune-stimulating payload that may enhance the effectivity of other anti-cancer therapies such as checkpoint inhibitors in unresponsive colorectal cancer patients. The major challenge in engineering phages that target FN is the optimization of the eukaryotic payloads for high expression in this bacterial host. Since no data is available about codon usage and non-coding genetic sequences in the genome of FN bacteria, two computational approaches were used to optimize the codon usage for elevated payload expression of the first selected payload, murine IL-15. These approaches involved identifying ribosome binding sites and elongation speeds to optimize codon usage. Following design of an IL-15 expression vector, expression of this eukaryotic payload in FN bacteria was tested in vitro. For phage engineering, a plasmid containing the designed payload sequence was used to introduce the selected IL-15 encoding sequences into the phage genome. FN phage was successfully engineered to deliver a payload encoding the sequence of murine cytokine IL-15. Targeting of FN with engineered phage resulted in IL-15 protein expression in phage-infected bacteria in vitro. Given the specificity of FN for CRC tumors and the ability to locally express a eukaryotic protein by using FN targeting phage to deliver a payload, phage therapy may offer novel treatment approaches for patients with CRC. Citation Format: Lihi Ninio-Many, Yael Zigelman, Nufar Buchshtab, Yifat Elharar, Gal Eylon, Eliya Gidron, Dikla Berko-Ashur, Julian Nicenboim, Lior Zelcbuch, Einav Safyon Gartman, Sharon Kredo-Russo, Noga Kowalsman, Ilya Vainberg Slutskin, Iddo Weiner, Inbar Gahali-Sass, Naomi Zak, Sailaja Puttagunta, Merav Bassan. Novel engineering of therapeutic Fusobacterium nucleatum phage for colorectal cancer treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P064.

Helminth-induced CD9+ B-cell subset alleviates obesity-associated inflammation via IL-10 production

It has been shown that helminth infection can protect against obesity and improve insulin sensitivity to a certain extent, based on epidemiological investigations and animal experiments. Meanwhile, helminths induce a network of regulatory immune cells, including regulatory B cells (Bregs). However, the molecule characteristics and function of these Bregs in improving whole-body metabolic homeostasis remains largely unclear. We established a mouse model with chronic Schistosoma japonicum infection, and compared the differences in B10 cells (CD19+CD5+CD1dhi) and B10− cells (CD19+CD5−CD1d−) from splenic B cells of infected mice using RNA-seq. A unique Breg population was identified. Furthermore, these Bregs were evaluated for their ability to produce inhibitory cytokines in vitro and suppress obesity when adoptively transferred into mice on a high-fat diet. We found that schistosome infection could expand Breg cell populations in mice. CD9 was demonstrated to be a key surface marker for most murine IL-10+ B cells in spleen. CD19+CD9+ B cells produced more IL-10 than conventional B10 cells. Adoptive transfer of CD9+ B cells had the capacity to alleviate obesity-associated inflammation via promoting Tregs, Th2 cells and decreasing Th1, Th17 cells in high-fat diet mice. In conclusion, schistosome infection can induce regulatory CD9+ B cell production, which plays a critical role in the regulation of metabolic disorders through IL-10 production.

DAPK1 may be a potential biomarker for arterial aneurysm in clinical treatment and activated inflammation levels in arterial aneurysm through NLRP3 inflammasome by Beclin1.

Death-associated protein kinase (DAPK1) is one of the positive regulators of apoptosis, and it is widely involved in apoptosis induced by multiple pathways. We examined that the function of DAPK1 in Clinical treatment of arterial aneurysm and its underlying mechanisms. Arterial aneurysm is a common cerebrovascular disease with high disability and fatality rate. Male C57BL/6 mice or DAPK1-/- mice were injected with 50mg/kg pentobarbital sodium and then were injected with angiotensin II (AngII) infusion for vivo model. hASMCs (Human artery smooth muscle cell) were treated with murine recombinant IL-6 (20 ng ml-1; Cell Signaling) for vitro model. DAPK1 gene, mRNA expression, and protein expression were induced in mice of arterial aneurysm. DAPK1 mRNA expression was increased and Area Under Curve was 0.9075 in patients with arterial aneurysm. Knockout of DAPK1 decreased inflammation and vascular injury in mice model of arterial aneurysm. Beclin1/NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) signal pathway is a critical downstream effector of DAPK1 by TAP production. The regulation of Beclin1 participated in the effects of DAPK1 on inflammation of arterial aneurysm by ATP-dependent NLRP3 inflammasome. The regulation of NLRP3 participated in the effects of DAPK1 on inflammation of arterial aneurysm. Collectively, our data indicated that DAPK1 may be a potential biomarker for arterial aneurysm in clinical treatment and activated inflammation levels in arterial aneurysm through NLRP3 inflammasome by Beclin1. DAPK1 might be a key pathogenic event underlying excess inflammation of arterial aneurysm.

DDRE-48. COMPARTMENT LOCKED IL-12 - INCREASED TISSUE RETENTION AND MINIMAL PERIPHERAL EXPOSURE ALLOW HIGHER TREATMENT EFFICACY AND TOLERABILITY IN LOCAL GLIOBLASTOMA THERAPY

Abstract Recent clinical studies in glioblastoma (GBM) highlight the potential of local IL-12 therapy, but they also bring back tolerability concerns due to leakage into the periphery. This leakage might thus hamper exploiting the full potential of local IL-12 therapy. Fusion with an IgG4 Fc portion increases the tissue retention of IL-12; but could also confer export into the blood and subsequent systemic recycling through the neonatal Fc receptor (FcRn), ultimately leading to potentially toxic IL-12 serum levels. We assessed the expression of FcRn in human and murine GBM and its role in IL-12Fc tissue retention and systemic exposure upon local delivery. Human or murine IL-12Fc was injected in GBM-bearing or naïve wt or FcRn-humanized mice continuously or as bolus via convection-enhanced delivery (CED). We screened combinations of amino-acid substitutions at the (IL-12)Fc:FcRn binding interface to abolish this interaction. Brain and blood concentrations were assessed via ELISA or cytokine bead arrays. FcRn affinity was measured by SPR/ELISA and bioactivity tested on PBMCs and human GBM explant cultures. Treatment efficacy and immunological correlates were assessed in GBM bearing mice. FcRn is upregulated in human and mouse GBM and contributes to brain export and subsequent peripheral recycling of IL-12Fc in the blood. IL-12Fc with abrogated FcRn binding due to a unique set of substitutions is fully functional and appears brain compartment locked (CL IL-12) as it exhibits enhanced tissue retention and reduced serum levels upon local injection, reaching up 100x higher brain to serum concentration ratios than regular IL-12. Compared to its non-modified counterpart, murine CL IL-12 shows significantly higher treatment efficacy at negligible systemic footprint in late stage murine GBM. In patient explant cultures, human CL IL-12 leads to successful inflammatory conditioning. Compartment locked IL-12 should thus allow a wide dosing window to fully harness its therapeutic potential for local GBM therapy.

Rescue of Murine IL-7 Receptor Deficiency with Human IL-7 Receptor Gene Therapy

▪In humans, interleukin 7 (IL-7) receptor (IL-7R) deficiency causes approximately 10% of cases of severe combined immunodeficiency (SCID). IL-7R deficient SCID is a T-B+NK+ SCID and is caused by autosomal recessive deficiency of the IL-7R alpha chain gene (IL7R). IL-7R is a heterodimeric receptor comprised of the alpha chain and the IL-2 receptor common gamma chain (IL2RG). In both mouse and human, IL-7R is a marker of the common lymphoid progenitor cell, and IL-7 signaling leads to STAT5 phosphorylation and proliferation of developing T and B cells. Mice lacking IL7R, Il7r -/-, lack both T and B cells (Peschon, JJ, et al. J Exp Med. 1994). T cells do not progress to TCR beta chain rearrangement and B cell development is halted at the pre-pro-B cell stage. Similar to the mouse, IL-7 signaling in humans is required for T cell receptor beta gene rearrangement and T cell maintenance, however humans lacking IL-7R can develop B cells.A prior attempt to rescue murine IL-7R deficiency utilized a retroviral vector (mouse stem cell virus, MSCV), the MSCV retroviral promoter, and the murine Il7r gene (Jiang, Q, et al. Gene Therapy. 2005). This strategy did restore T cells and had variable restoration of B cells. However, retroviral-based gene addition of Il7r led to a myeloproliferative condition with significant neutrophilia and splenomegaly. Transduced bone marrow cells formed myeloid progenitors in response to IL-7 in vitro. We evaluated a novel gene therapy for IL-7R deficient SCID that utilizes the human IL7R gene. To prevent lineage skewing, we sought to limit ectopic expression of IL7R in non-lymphoid cells by utilizing the endogenous enhancers and promoters of IL7R. These sequences were identified as sites of high sequence conservation across species and DNA accessibility/hypersensitivity (DHS) in human lymphocytes. We are testing these sequences alone or in combination with the constitutive phosphoglycerate kinase promoter (PGK) in VSV-G pseudotyped lentiviral vectors (LV): vPGK_DHS_hIL7R and vDHS_hIL7R. Here we present the first data evaluating the ability of the human IL-7R protein to functionally replace the murine IL-7R protein and the ability of IL7R gene addition to rescue the murine Il7r -/- immunodeficient phenotype in vivo.Transduction of Il7r -/- bone marrow cells with IL7R encoding LV rescued the formation of lymphocyte precursors from murine bone marrow cells in colony forming unit (CFU) assays (pre-B CFU with human IL-7), with the most robust response seen with vPGK_DHS_hIL7R. Mouse bone marrow from Il7r -/- animals transduced ex vivo engrafted in lethally irradiated (8 Gy) Il7r -/-oppositegender recipients and there were no significant aberrations in absolute neutrophil count, hemoglobin or platelet count. Absolute lymphocyte counts in mice receiving transduced Il7r -/-bone marrow cells was higher (mean 2555/μL) than in mice receiving untransduced bone marrow (mean 1410/μL). The proportion of leukocytes that were T cells was 4.2-fold and 9.8-fold higher at 1 and 2 months post-transplant, respectively. B cells were only seen in mice receiving vPGK_DHS_hIL7R: 7.4% of leukocytes versus 1.5% in controls. A reciprocal decrease in the fraction of Gr1+ cells (neutrophils and monocytes) was seen at two months post-transplant in transduced marrow recipients compared to untransduced controls: 36.5% versus 63% Gr1+, respectively. Lymphocyte subsets are being further analyzed, bone marrow and thymic lymphoid precursors assessed, and T and B cell function in response to immunizations are in progress. Further evaluation in human derived IL7R deficient human cells is warranted.For individuals with IL-7R deficient SCID, but no HLA matched hematopoietic stem cell (HSC) donor, there is a difficult choice between the risks of GVHD with a mismatched HSC donor and supportive care with the hope of identifying a matched HSC donor in the future. In immunodeficiencies however age and serious infection are both associated with increased mortality (Pai, SY, et al. NJEM. 2014). This novel approach to IL7R gene replacement has the potential to be a therapeutic and expedient option for those without a matched donor. Additionally, this would be an ideal disorder for HSC conditioning with less toxic, HSC-targeted strategies given gene corrected lymphocytes and progenitors will preferentially expand post-transplant. DisclosuresRivella: Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy.

Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study.

Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.

Interactions of thymic stromal lymphopoietin with interleukin-4 in adaptive immunity during Aspergillus fumigatus keratitis.

To investigate the potential interactions of thymic stromal lymphopoietin (TSLP) with interleukin-4 (IL-4) in adaptive immunity during fungal keratitis (FK). An FK mouse model was induced with Aspergillus fumigatus (AF) hyphal infection. Mice were divided into several groups: untreated, phosphate buffer saline (PBS), infected with AF, and pretreated with a scrambled siRNA, a TSLP-specific siRNA (TSLP siRNA), murine recombinant TSLP (rTSLP), immunoglobulin G (IgG), murine recombinant IFN (rIFN-γ), murine recombinant IL-4 (rIL-4), rIL-13, murine recombinant IL-17A (rIL-17A), and murine recombinant IL-17F (rIL-17F) groups. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) or Western blot were performed to determine mRNA and protein levels in the inflamed cornea. Cytokine locations were observed by immunofluoresence staining after AF hyphal infection. Compared to those in the untreated group, TSLP and T helper type 1 (Th1) cytokine levels in the AF group were upregulated at 24h post infection (hpi), and those of T helper type 2 (Th2) and T helper type 17 (Th17) cytokines were increased at 5d post infection (dpi). Th2 cytokine levels were decreased in the TSLP siRNA-pretreated group and increased in the rTSLP-pretreated group compared with the AF group. The TSLP level was increased in the rIL-4-pretreated group, but there were no significant changes among the other groups. Immunofluorescence staining showed cytokine locations after AF hyphal infection. TSLP induces a Th2 immune response and promots Th2 T cell differentiation in vivo. IL-4 promotes TSLP secretion. Therefore, TSLP with IL-4 regulates adaptive immunity in FK.

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine–cytokine receptor binding are important to develop novel therapeutic strategies.

Interleukin-4 Programmed Macrophages Suppress Colitis and Do Not Enhance Infectious-Colitis, Inflammation-Associated Colon Cancer or Airway Hypersensitivity.

The murine interleukin-4 treated macrophage (MIL4) exerts anti-inflammatory and pro-healing effects and has been shown to reduce the severity of chemical-induced colitis. Positing M(IL4) transfer as an anti-inflammatory therapy, the possibility of side-effects must be considered. Consequently, bone marrow-derived M(IL4)s were administered via intraperitoneal injection to mice concomitant with Citrobacter rodentium infection (infections colitis), azoxymethane/dextran sodium sulphate (AOM/DSS) treatment [a model of colorectal cancer (CRC)], or ovalbumin sensitization (airway inflammation). The impact of M(IL4) treatment on C. rodentium infectivity, colon histopathology, tumor number and size and tissue-specific inflammation was examined in these models. The anti-colitic effect of the M(IL4)s were confirmed in the di-nitrobenzene sulphonic acid model of colitis and the lumen-to-blood movement of 4kDa FITC-dextran and bacterial translocation to the spleen and liver was also improved by M(IL4) treatment. Analysis of the other models of disease, that represent comorbidities that can occur in human inflammatory bowel disease (IBD), revealed that M(IL4) treatment did not exaggerate the severity of any of the conditions. Rather, there was reduction in the size (but not number) of polyps in the colon of AOM/DSS-mice and reduced infectivity and inflammation in C. rodentium-infected mice in M(IL4)-treated mice. Thus, while any new therapy can have unforeseen side effects, our data confirm and extend the anti-colitic capacity of murine M(IL4)s and indicate that systemic delivery of one million M(IL4)s did not exaggerate disease in models of colonic or airways inflammation or colonic tumorigenesis.

Oral vaccination with invasive Lactobacillus plantarum delivered nucleic acid vaccine co-expressing SS1 and murine interleukin-4 elicits protective immunity against Trichinella spiralis in BALB/c mice

Trichinellosis is a foodborne zoonosis caused by Trichinella spiralis (T. spiralis) that not only causes considerable economic losses for the global pig breeding and food industries, but also seriously threats the health of human. Therefore, it is very necessary to develop an effective vaccine to prevent trichinellosis. In this study, the invasive Lactobacillus plantarum (L. plantarum) expressing fibronectin-binding protein A (FnBPA) was served as a live bacterial vector to deliver DNA to the host to produce a novel oral DNA vaccine. Co-expressing T. spiralis SS1 and murine interleukin-4 (mIL-4) of DNA vaccine were constructed and subsequently delivered to intestinal epithelial cells via invasive L. plantarum. At 10 days after the third immunization, the experimental mice were challenged with 350 T. spiralis infective larvae. The results found that the mice orally vaccinated with invasive L. plantarum harboring pValac-SS1/pSIP409-FnBPA not only stimulated the production of anti-SS1-specific IgG, Th1/Th2 cell cytokines, and secreted(s) IgA but also decreased worm burden and intestinal damage. However, the mice inoculated with invasive L. plantarum co-expressing SS1 and mIL-4 (pValac-SS1-IL-4/pSIP409-FnBPA) induced the highest protective immune response against T. spiralis infection. The DNA vaccine delivered by invasive L. plantarum provides a novel idea for the prevention of T. spiralis infection.

Foxo3a tempers excessive glutaminolysis in activated T cells to prevent fatal gut inflammation in the murine IL-10-/- model of colitis.

Mutations in susceptibility alleles correlate with gut-inflammatory diseases, such as Crohn's disease; however, this does not often impact the disease progression indicating the existence of compensatory genes. We show that a reduction in Foxo3a expression in IL-10-deficient mice results in a spontaneous and aggressive Crohn's- like disease with 100% penetrance, which is rescued by deletion of myeloid cells, T cells and inhibition of mTORC1. In Foxo3a-/- IL-10-/- mice, there is poor cell death of myeloid cells in the gut, leading to increased accumulation of myeloid and T cells in the gut. Myeloid cells express high levels of inflammatory cytokines, and regulatory T cells are dysfunctional despite increased abundance. Foxo3a signaling represses the transcription of glutaminase (GLS/GLS2) to prevent over-consumption of glutamine by activated T cells and its conversion to glutamate that contributes to the TCA cycle and mTORC1 activation. Finally, we show that Foxo3a restricts the abundance of colitogenic microbiota in IL-10-deficient mice. Thus, by suppressing glutaminolysis in activated T cells Foxo3a mediates a critical checkpoint that prevents the development of fulminant gut inflammatory disease.