Mouse Lymphoid Cells Research Articles

Collaborative Study of Thresholds for Mutagens: Adaptive Responses in the Micronucleus Test and Gene Induction by Mutagenic Treatments.

We have been conducting a collaborative study on the thresholds of mutagens. In our previous examinations of cell activity and cell proliferation as endpoints, both displayed hormesis. This time, we conducted experiments to determine thresholds using the micronucleus test as an endpoint. The micronucleus test was conducted using Chinese hamster CHL/IU cells and mouse lymphoid L5178Y cells. Additionally, we conducted preliminary investigations into the gene expression using human TK6 cells. When adhesive CHL/IU cells were treated with mitomycin C (MMC), and the hormetic response was examined, hormesis was not observed clearly. When L5178Y cells were treated with methyl methanesulfonate (EMS), AF-2, MMC, and colchicine, all of them exhibited an adaptive response. Additionally, cross-adaptive responses using AF-2 and MMC or EMS and MMC were conducted, both combinations showed a cross-adaptive response. When the gene expression patterns of six genes were investigated by RT-PCR after treatment with MMC, EMS, and H2O2 using TK6 cells, two genes, GADD45A and P21, were induced in a dose- and time-dependent manner. Adaptive responses arise from preconditioning. As hormesis is inherently linked to preconditioning, adaptive responses observed in this study strongly suggest that hormesis was induced, hence existence of thresholds.

Fermented rice bran supplementation attenuates colonic injury through modulating intestinal aryl hydrocarbon receptor and innate lymphoid cells in mice with dextran sodium sulfate-induced acute colitis

This study investigated the effects of fermented rice bran (FRB) on modulating intestinal aryl hydrocarbon receptor (AhR) expression, innate lymphoid cell (ILC)3 populations, the fecal microbiota distribution, and their associations with dextran sodium sulfate (DSS)-induced acute colitis. C57BL/6 mice were assigned to four groups: 1) NC group, normal mice fed the AIN-93M diet; 2) FRB group, normal mice fed a diet supplemented with 5% FRB; 3) NCD group, DSS-treated mice fed AIN-93M; 4) FRBD group, DSS-treated mice fed a 5% FRB-supplemented diet. DSS was administered for 5 d and followed by 5 d for recovery. At the end of the experiment, mice were sacrificed. Their blood and intestinal tissues were collected. Results showed that there were no differences in colonic biological parameters and function between the NC and FRB groups. Similar fecal microbiota diversity was noted between these two groups. Compared to the non-DSS-treated groups, DSS administration led to increased intestinal permeability, enhanced inflammatory cytokine production and disease severity, whereas tight junctions and AhR, interleukin (IL)-22 expressions were downregulated, and the ILC3 population had decreased. Also, gut microbiota diversity differs from the non-DSS-treated groups and more detrimental bacterial populations exist when compared to the FRBD group. FRB supplementation in DSS-treated mice attenuated fecal microbial dysbiosis, decreased intestinal permeability, improved the barrier integrity, upregulated AhR and IL-22 expression, maintained the ILC3 population, and pathologically mitigated colonic injury. These findings suggest enhanced ILC3- and AhR-mediated functions may be partly responsible for the anti-colitis effects of FRB supplementation in DSS-induced colitis.

Chenopodium quinoa's Ingredients Improve Control of the Hepatic Lipid Disturbances Derived from a High-Fat Diet.

This study explored the effects of Chenopodium quinoa's ingredients on the major lipids' hepatic profile and the functional selective differentiation of monocyte-derived macrophages and innate lymphoid cells in mice on a high-fat diet. Six-week-old Rag2-/- and Rag2-/-Il2-/- mice received (12 days) a low-molecular-weight protein fraction (LWPF) or the lipid fraction (qLF) obtained from the cold pressing of C. quinoa's germen. At the end of the experiment, mouse serum and liver tissue were collected. The differences in triglycerides, phospholipids, and the major lipids profile were analyzed. Infiltrated monocyte-derived macrophages and innate lymphoid cells (ILCs) and the expression of liver metabolic stress-related mRNA were measured. In the Rag2-/- mice, feeding them LWPF appeared to improve, to a larger extent, their hepatic capacity to utilize fatty acids in comparison to the qLF by preventing the overwhelming of triglycerides (TGs), despite both reducing the hepatic lipid accumulation. An analysis of the hepatic major lipids profile revealed significant increased variations in the PUFAs and phospholipid composition in the Rag2-/- mice fed with the LWPF or LF. The Rag2-/-Il2-/- mice, lacking innate and adaptive lymphocytes, seemed resistant to mobilizing hepatic TGs and unresponsive to lipid accumulation when fed with the LF. Notably, only the Rag2-/- mice fed with the LWPF showed an increased proportion of hepatic CD68+F4/80+ cells population, with a better controlled expression of the innate immune 'Toll-like' receptor (TLR)-4. These changes were associated with an oriented expansion of pluripotential CD117+ cells towards ILC2s (CD117+KLRG1+). Thus, C. quinoa's ingredients resulted in being advantageous for improving the mechanisms for controlling the hepatic lipotoxicity derived from a high-fat diet, promoting liver macrophage and ILCs expansion to a selective functional differentiation for the control of HFD-driven immune and metabolic disturbances.

Immunomodulatory Properties of Citrus limon Extracts on BALB/c Mouse Lymphoid and Myeloid Lineage Cells

Immunomodulatory Properties of Citrus limon Extracts on BALB/c Mouse Lymphoid and Myeloid Lineage Cells

Alcohol-mediated susceptibility to lung fibrosis is associated with group 2 innate lymphoid cells in mice.

Chronic alcohol ingestion promotes acute lung injury and impairs immune function. However, the mechanisms involved are incompletely understood. Here, we show that alcohol feeding enhances bleomycin-induced lung fibrosis and inflammation via the regulation of type 2 innate immune responses, especially by group 2 innate lymphoid cells (ILC2s). Neuroimmune interactions have emerged as critical modulators of lung inflammation. We found alcohol consumption induced the accumulation of ILC2 and reduced the production of the neuropeptide calcitonin gene-related peptide (CGRP), primarily released from sensory nerves and pulmonary neuroendocrine cells (PNECs). CGRP potently suppressed alcohol-driven type 2 cytokine signals in vivo. Vagal ganglia TRPV1+ afferents mediated immunosuppression occurs through the release of CGRP. Inactivation of the TRPV1 receptor enhanced bleomycin-induced fibrosis. In addition, mice lacking the CGRP receptor had the increased lung inflammation and fibrosis and type 2 cytokine production as well as exaggerated responses to alcohol feeding. Together, these data indicate that alcohol consumption regulates the interaction of CGRP and ILC2, which is a critical contributor of lung inflammation and fibrosis.

The Ca2+ concentration impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages in vitro.

Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αβ T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca2+ concentrations. This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca2+ concentrations. Furthermore, elevated Ca2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ levels can boost cytokine production by both mouse and human lymphoid cells.

Nuclear IL-33 in Fibroblasts Promotes Skin Fibrosis

Systemic sclerosis (SSc) is a complex and poorly understood connective tissue disease characterized by diffuse fibrosis in several organs, especially the skin ( Asano, 2017 Asano Y. Recent advances in the treatment of skin involvement in systemic sclerosis. Inflamm Regen. 2017; 37: 12 Crossref Scopus (6) Google Scholar , Bhattacharyya et al., 2012 Bhattacharyya S. Wei J. Tourtellotte W.G. Hinchcliff M. Gottardi C.G. Varga J. Fibrosis in systemic sclerosis: common and unique pathobiology. Fibrogenesis Tissue Repair. 2012; 5: S18 Crossref Scopus (30) Google Scholar , Denton et al., 2006 Denton C.P. Black C.M. Abraham D.J. Mechanisms and consequences of fibrosis in systemic sclerosis. Nat Clin Pract Rheumatol. 2006; 2: 134-144 Crossref Scopus (156) Google Scholar ). Interleukin (IL)-33 is found to regulate tissue fibrosis in multiple organs ( Kotsiou et al., 2018 Kotsiou O.S. Gourgoulianis K.I. Zarogiannis S.G. IL-33/ST2 Axis in Organ Fibrosis. Frontiers in immunology. 2018; 9: 2432 Crossref PubMed Scopus (96) Google Scholar , Xu et al., 2016 Xu J. Zheng J. Song P. Zhou Y. Guan S. IL33/ST2 pathway in a bleomycininduced pulmonary fibrosis model. Mol Med Rep. 2016; 14: 1704-1708 Crossref Scopus (13) Google Scholar ). However, the exact role of IL-33 in fibrosis remains controversial ( Kotsiou et al., 2018 Kotsiou O.S. Gourgoulianis K.I. Zarogiannis S.G. IL-33/ST2 Axis in Organ Fibrosis. Frontiers in immunology. 2018; 9: 2432 Crossref PubMed Scopus (96) Google Scholar ). Previous studies have shown a pro-fibrotic role for IL-33 cytokine through its receptor, interleukin-1 receptor-like 1(IL1RL1 or ST2), regulating the secretion of pro-fibrotic cytokine, IL-13, from eosinophils, M2 macrophages and type 2 innate lymphoid cells (ILC2) ( Li et al., 2014 Li D. Guabiraba R. Besnard A.G. Komai-Koma M. Jabir M.S. Zhang L. et al. IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice. The Journal of allergy and clinical immunology. 2014; 134: 1422-1432 e11 Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar , Rankin et al., 2010 Rankin A.L. Mumm J.B. Murphy E. Turner S. Yu N. McClanahan T.K. et al. IL-33 induces IL-13-dependent cutaneous fibrosis. J Immunol. 2010; 184: 1526-1535 Crossref PubMed Scopus (188) Google Scholar , Wu et al., 2018 Wu L. Luo Z. Zheng J. Yao P. Yuan Z. Lv X. et al. IL-33 Can Promote the Process of Pulmonary Fibrosis by Inducing the Imbalance Between MMP-9 and TIMP-1. Inflammation. 2018; 41: 878-885 Crossref PubMed Scopus (30) Google Scholar ). However, IL-33 can also play an anti-fibrotic role through the induction of ILC2 cells and regulatory T cells in several organs including the skin ( Cheon et al., 2022 Cheon S.Y. Park J.H. Ameri A.H. Lee R.T. Nazarian R.M. Demehri S. IL-33/Regulatory T-Cell Axis Suppresses Skin Fibrosis. The Journal of investigative dermatology. 2022; Google Scholar , Garbern et al., 2019 Garbern J.C. Williams J. Kristl A.C. Malick A. Rachmin I. Gaeta B. et al. Dysregulation of IL-33/ST2 signaling and myocardial periarteriolar fibrosis. J Mol Cell Cardiol. 2019; 128: 179-186 Abstract Full Text Full Text PDF Scopus (12) Google Scholar , Riedel et al., 2017 Riedel J.H. Becker M. Kopp K. Duster M. Brix S.R. Meyer-Schwesinger C. et al. IL-33-Mediated Expansion of Type 2 Innate Lymphoid Cells Protects from Progressive Glomerulosclerosis. J Am Soc Nephrol. 2017; 28: 2068-2080 Crossref Scopus (71) Google Scholar ). Thus, it is critical to determine the full spectrum of IL-33 effect in fibrosis to develop optimal therapies for its utilization in the treatment of SSc and other fibrosis-associated diseases.

IL-33-induced keratoconjunctivitis is mediated by group 2 innate lymphoid cells in mice

BackgroundInterleukin-33 (IL-33) is involved in type 2 innate immunity by inducing type 2 cytokines, such as IL-5 and IL-13, through the activation of group 2 innate lymphoid cells (ILC2s) or T helper 2 (Th2) cells. We previously reported that mice overexpressing IL-33 (IL-33Tg) in the cornea and conjunctiva spontaneously develop atopic keratoconjunctivitis-like inflammation. Despite previous studies, it is not fully understood what types of immune cells contribute to the disease process of IL-33-induced keratoconjunctivitis. MethodsTo defect Th2 cells, IL-33Tg mice were crossed with Rag2KO mice. To defect ILC2s, IL-33Tg mice received bone marrow transplantations from B6.C3(Cg)-Rorasg/J mice that lacked ILC2. Immunostaining techniques were used to determine where ILC2 is distributed in the cornea and conjunctiva. We analyzed the transcriptomes of ILC2 from the conjunctiva by using single-cell RNA-seq analysis. To investigate whether tacrolimus reduces type 2 cytokine production by ILC2, ILC2 was cultured with tacrolimus, and the percentage of cytokine-producing ILC2 was examined. To investigate whether tacrolimus can inhibit IL-33-induced keratoconjunctivitis in vivo, IL-33Tg mice were treated with tacrolimus eye drops. ResultsILC2 infiltrated the conjunctival epithelium and subepithelial tissue. Keratoconjunctivitis developed spontaneously in Rag2KO/IL-33Tg mice, but keratoconjunctivitis was abolished in IL-33Tg mice lacking ILC2. ILC2 was not a uniform cluster but a heterogeneous cluster. Tacrolimus inhibited cytokine production from ILC2s in vitro, and tacrolimus eye drops inhibited keratoconjunctivitis in IL-33Tg mice in vivo. ConclusionsILC2 plays a pivotal role in IL-33-induced keratoconjunctivitis in mice.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

Stem Cell Factor Neutralization Protects From Severe Anaphylaxis in a Murine Model of Food Allergy.

Food allergy is a growing public health problem with ~15 million people affected in the United States. In allergic food disease, IgE on mast cells bind to ingested antigens leading to the activation and degranulation of mast cells. Stem cell factor (SCF) is mast cell growth and activation factor that is required for peripheral tissue mast cells. We targeted a specific isoform of SCF, the larger 248 amino acid form, that drives peripheral tissue mast cell differentiation using a specific monoclonal antibody in a model of food allergy. Ovalbumin sensitized and intragastrically challenged mice were monitored for symptoms of anaphylaxis including respiratory distress, diarrhea, and a reduction in body temperature. During the second week of challenges, allergic mice were injected with an antibody to block SCF248 or given IgG control. Mice treated with α-SCF248 had a decreased incidence of diarrhea and no reduction in body temperature suggesting a reduction in anaphylaxis compared to IgG control treated animals. Re-stimulated mesenteric lymph nodes indicated that α-SCF248 treated mice had decreased OVA-specific Th2 cytokine production compared to IgG control treated allergic animals. The reduction of food induced anaphylaxis was accompanied by a significant reduction in gut leak. The mesenteric lymph node cells were analyzed by flow cytometry and showed a decrease in the number of type 2 innate lymphoid cells in mice injected with α-SCF248. Morphometric enumeration of esterase+ mast cells demonstrated a significant reduction throughout the small intestine. Using a more chronic model of persistent food-induced anaphylaxis, short term therapeutic treatment with α-SCF248 during established disease effectively blocked food induced anaphylaxis. Together, these data suggest that therapeutically blocking SCF248 in food allergic animals can reduce the severity of food allergy by reducing mast cell mediated disease activation.

Multifaceted effects of soluble human CD6 in experimental cancer models

BackgroundCD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation...

Mahuang Xixin Fuzi decoction protects against ovalbumin-induced allergic rhinitis by inhibiting type 2 innate lymphoid cells in mice

Abstract Objective As a traditional herbal formula, Mahuang Xixin Fuzi decoction (MXFD) has been used to treat allergic rhinitis (AR). It regulates the transcription factor GATA3 in T cells, blocks Th2 skewing and rebalances the Th1/Th2 immunity. Type 2 innate lymphoid cells (ILC2s) are closely associated with GATA3. However, it remains unknown whether ILC2s could be one mechanism through which MXFD acts. We sought to elucidate the efficacy and mechanism of action of this decoction in AR treatment. Methods Forty C57BL/6J female mice were equally divided into control, model, loratadine- and MXFD-treated groups in random. AR was induced by ovalbumin (OVA), and then treatment with loratadine or MXFD was administered. AR scores were evaluated and compared before and after treatment. Pathological changes in the nasal mucosa and lung were observed using hematoxylin-eosin staining of tissue samples. Activation of ILC2 in nasal mucosa was assessed by immunofluorescence and quantitative polymerase chain reaction analysis. ILC2 cell count in lungs was measured by flow cytometry and levels of interleukin- (IL) 4, IL-5 and IL-13 in serum were detected by ELISA. Results The decoction alleviated the symptoms of AR in mice, improved vascular congestion and expansion, glandular hyperplasia and inflammatory cell infiltration in mouse nasal mucosa and slowly improved the interstitial pneumonia and lesions. Meanwhile, MXFD reduced the number and percentage of ILC2s in the nasal mucosa and lungs, downregulated the expression of GATA3 mRNA and RORα mRNA, and reduced the related inflammatory cytokine levels, including IL-4, IL-5 and IL-13. Conclusion These data suggest that inhibition of ILC2s by MXFD may be an important means by which to treat AR.

Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.

Effect of targeting CD40 for DC vaccination in pancreatic adenocarcinoma.

e15783 Background: Although immunotherapy yields striking results in various malignancies, results in pancreatic cancer have been disappointing. Both a highly immunosuppressive tumor microenvironment and a dense desmoplastic stroma have been found to prohibit proper T-cell infiltration in these tumors, thereby preventing immunotherapy efficacy. We hypothesize that a rational and translational multistep approach is needed to sensitize pancreatic cancer to immunotherapy. In an aggressive murine pancreatic ductal adenocarcinoma model, we assessed the effectiveness of dendritic cell (DC) vaccination in combination with αCD40 treatment, as these treatments are known to induce effector T cells and degrade stroma, respectively. Methods: Immune competent C57BL/6 mice were inoculated subcutaneously with pancreatic tumor cells (KPC3). Mice with established tumors were vaccinated with tumor-loaded monocyte derived DCs and consequently treated with αCD40 agonistic antibodies. Tumor sizes were monitored over time. Immune responses were determined by flow cytometry of cells in peripheral blood, spleen and tumor. NanoString Technologies were applied on tumor samples. Results: A significant delay in tumor growth was found in the combination therapy arm compared to untreated mice and mice treated with DCs or αCD40 alone. Monotherapy had no effect on tumor growth. Survival of mice treated with the combination therapy was also improved compared to untreated mice or mice treated with monotherapy (P < 0.001). Interim blood analysis showed significant increases in frequencies of activated and proliferating T cells in treated animals and those cells also displayed an effector memory phenotype. This was more pronounced for CD4 T cells in mice treated with DCs while αCD40 therapy induced a confined response in CD8 T cells. Increased frequencies of tumor infiltrating lymphocytes were found in all treated mice compared to untreated mice. mRNA expression analysis indicated less exhausted phenotype of intratumoral lymphoid cells in mice treated with DCs and αCD40 compared to monotherapy DCs or αCD40. Conclusions: These results demonstrate the potency of this novel form of combination immunotherapy and reveals a mechanistic insight into the requirements of effective immunotherapy in pancreatic cancer.

Ribosomal Lesions Promote Oncogenic Mutagenesis.

Ribosomopathies are congenital disorders caused by mutations in ribosomal proteins (RP) or assembly factors and are characterized by cellular hypoproliferation at an early stage. Paradoxically, many of these disorders have an elevated risk to progress to hyperproliferative cancer at a later stage. In addition, somatic RP mutations have recently been identified in various cancer types, for example, the recurrent RPL10-R98S mutation in T-cell acute lymphoblastic leukemia (T-ALL) and RPS15 mutations in chronic lymphocytic leukemia (CLL). We previously showed that RPL10-R98S promotes expression of oncogenes, but also induces a proliferative defect due to elevated oxidative stress. In this study, we demonstrate that this proliferation defect is eventually rescued by RPL10-R98S mouse lymphoid cells that acquire 5-fold more secondary mutations than RPL10-WT cells. The presence of RPL10-R98S and other RP mutations also correlated with a higher mutational load in patients with T-ALL, with an enrichment in NOTCH1-activating lesions. RPL10-R98S-associated cellular oxidative stress promoted DNA damage and impaired cell growth. Expression of NOTCH1 eliminated these phenotypes in RPL10-R98S cells, in part via downregulation of PKC-θ, with no effect on RPL10-WT cells. Patients with RP-mutant CLL also demonstrated a higher mutational burden, enriched for mutations that may diminish oxidative stress. We propose that oxidative stress due to ribosome dysfunction causes hypoproliferation and cellular insufficiency in ribosomopathies and RP-mutant cancer. This drives surviving cells, potentiated by genomic instability, to acquire rescuing mutations, which ultimately promote transition to hyperproliferation. SIGNIFICANCE: Ribosomal lesions cause oxidative stress and increase mutagenesis, promoting acquisition of rescuing mutations that stimulate proliferation.

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OBJECTIVES/SPECIFIC AIMS: Trichuris trichiura, is a leading cause of chronic colitis worldwide, resulting in growth stunting, anemia, and cognitive deficits, predominately in children. Our long-term goal is to develop a vaccine against T. trichiura. Both T. trichiura and the closely related Trichuris muris release excretory/secretory (ES) macromolecules from the stichosome organ, which facilitates intracellular invasion into the cecum. We exploited the high degree of genetic sequence homology between T. trichiura and T. muris to evaluate T. muris stichosome proteins as vaccine candidates. METHODS/STUDY POPULATION: Through immunological screening of the T. muris stichosome cDNA library and T. muris ES macromolecules generated in culture, we identified, cloned, and expressed several vaccine candidates. In ranking these antigens, we selected the most promising recombinant proteins, WAP and CAP-1, and vaccinated AKR mice to evaluate the immunogenicity and efficacy of our candidate. In addition, we collected 240 serum samples in the T. trichiura endemic region of Honduras to measure the recognition of WAP and CAP-1 in naturally infected human. RESULTS/ANTICIPATED RESULTS: We measured a statistically significant reduction in larval worm burden in WAP and ES vaccinated mice, but not CAP-1, by microscopy and real-time PCR of T. muris DNA. We found a significant relationship between antigen-specific IgG1:IgG2a ratio in the mouse serum and degree of protection. Mouse splenocytes, vaccine-sensitized draining lymph nodes, and mesenteric lymph nodes were antigen-stimulated and their secreted Th1, Th2, and Th17 cytokines were measured by Luminex®. Stimulated mouse lymphoid cells were further analysed for T helper, T cytotoxic, and central/effector memory T cells by Flow Cytometry. Human IgG1, IgG2, and IgE antibody titers against WAP and CAP-1 were measured by ELISA. DISCUSSION/SIGNIFICANCE OF IMPACT: In our study, we describe the T cell dependent mechanism of humoral immunity of 2 promising ES-derived vaccines recombinant proteins, WAP and CAP-1. We evaluated the immune response, indicating a driving a Th2-induced humoral response necessary for protection. We further predict protection and allergenicity of WAP in humans using serum from a cohort in an T. trichiura endemic region.

Expression of IL-33 in ocular surface epithelium induces atopic keratoconjunctivitis with activation of group 2 innate lymphoid cells in mice

In a transgenic mouse line hK14mIL33tg, with the expression of interleukin-33 (IL-33) driven by a keratin 14 promoter, keratoconjunctivitis developed spontaneously between 18 and 22 weeks of age under specific-pathogen-free conditions. These mice showed blepharitis and corneal impairments, and the histology revealed epithelial thickening in the conjunctiva and the cornea with infiltration of eosinophils, mast cells and basophils. IL-5, IL-13 and CCL11 were abundant in lacrimal fluid in the mice, and the gene expressions of IL-4, IL-5, IL-13, IL-33, Prg2 and Mmcp8 were significantly increased in the cornea. Furthermore, group 2 innate lymphoid cells (ILC2) producing IL-5 and IL-13 were markedly increased in the cornea. These phenotypes closely resemble human atopic keratoconjunctivitis (AKC). The characteristic ocular phenotype in these mice strongly suggests that IL-33 is crucial for the development of AKC. The mouse line may be useful as a novel model for research and development of therapeutic strategies for AKC.

The T-cell leukemia-associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling.

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10-R98S) found in T-ALL. The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10-R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10-R98S mice and T-ALL xenograft samples. RPL10-R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10-R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10-R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10-R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10-R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.

Abstract A83: Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily

Abstract Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells highlighting their importance in multiple locations and phases of the anti-tumor immune response. Whereas agonistic stimulation of TRAIL-Receptor-1 or -2 can directly induce apoptosis in tumor cells, signaling through many TNFRSF members, such as CD40, CD27, OX40, HVEM, GITR and 4-1BB, results in co-stimulation of immune cells. Therefore, agonistic stimulation of certain members of the TNFRSF is considered to have a positive impact on immune-based therapeutic concepts in clinical oncology. Apogenix has developed a proprietary technology platform for the construction of novel hexavalent TNFRSF agonists (HERA) for the treatment of cancer. HERA fusion proteins are based on a perfect molecular mimic of the TNFSF cytokine structure. The HERA core unit consists of one single polypeptide chain comprising the three receptor-binding domain-forming subsequences (TNFSF-protomers). These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) preserve the structural organization of the trimeric natural TNFSF cytokine and can be utilized to engineer fully human fusion-proteins in a modular manner. Fusing an IgG1 Fc-domain as a dimerization scaffold to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist from two trivalent scTNFSF-RBDs. As a result of this molecular design, each molecule is capable of clustering six receptors in a spatially well-defined manner in close proximity to each other. Therefore, signaling following treatment with the Apogenix HERA scTNFSF-RBD-Fc fusion proteins in vivo is independent of secondary clustering through Fc-γ receptors that is required for many agonistic anti-TNFRSF antibodies (e.g., anti-TRAIL-R2 or anti-CD40). The described HERA engineering concept has been successfully translated to TRAIL, CD40L, LIGHT and CD27L resulting in hexavalent agonists suitable for further development. CD27-Ligand is a potent co-stimulatory molecule that drives T-cell activation and survival through interaction with its receptor (CD27). Here we show in vitro and in vivo data for APG1293 (scCD27L-RBD-Fc), a hexavalent CD27 agonist. APG1293 was expressed in CHO suspension cells followed by a lab-scale purification process including affinity chromatography and SEC-based polishing, resulting in homogenous aggregate-free protein lots. The purified protein binds its respective target-receptor with high affinity. In vivo stability/PK studies have been performed in addition to in vitro experiments with primary human and mouse lymphoid and myeloid cell populations. Specifically, APG1293 was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells and importantly, binding significantly increased T-cell expansion following activation. In vivo the efficacy of APG1293 was evaluated in the colorectal syngeneic murine tumor models MC38-CEA and CT26. In both models APG1293 treatment resulted in a dose dependent tumor growth inhibition. In summary, the data on the hexavalent APG1293 indicate a potent immune cell driven anti tumor efficacy. Therefore, APG1293 could be applied as a single agent or in combination with check-point inhibitors. Citation Format: Christian Gieffers, Christian Merz, David Richards, Mauricio Redondo, Viola Marschall, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A83.

Back to the drawing board: Understanding the complexity of hepatic innate lymphoid cells.

Recent studies of immune populations in nonlymphoid organs have highlighted the great diversity of the innate lymphoid system. It has also become apparent that mouse and human innate lymphoid cells (ILCs) have distinct phenotypes and properties. In this issue of the European Journal of Immunology, Harmon etal. [Eur. J. Immunol. 2016. 46: 2111-2120] characterized human hepatic NK-cell subsets. The authors report that hepatic CD56(bright) NK cells resemble mouse liver ILC1s in that they express CXCR6 and have an immature phenotype. However, unlike mouse ILC1s, they express high levels of Eomes and low levels of T-bet, and upon stimulation with tumor cells, secrete low amounts of cytokines. These unexpected findings further support the differences between human and mouse immune populations and prompt the study of the role of hepatic ILC subsets in immune responses.