TC Mice Research Articles

Targeting T Cell Activation and Lupus Autoimmune Phenotypes by Inhibiting Glucose Transporters.

CD4+ T cells have numerous features of over-activated cellular metabolism in lupus patients and mouse models of the disease. This includes a higher glycolysis than in healthy controls. Glucose transporters play an essential role in glucose metabolism by controlling glucose import into the cell from the extracellular environment. We have previously shown that treatment of lupus-prone mice with 2-deoxy-D-glucose, which inhibits the first step of glycolysis was sufficient to prevent autoimmune activation. However, direct targeting of glucose transporters has never been tested in a mouse model of lupus. Here, we show that CG-5, a novel glucose transporter inhibitor, ameliorated autoimmune phenotypes in a spontaneous lupus-prone mouse model, B6.NZM2410.Sle1.Sle2.Sle3 (Triple-congenic, TC), and in a chronic graft- vs. host-disease (cGVHD) model of induced lupus. In vitro, CG-5 blocked glycolysis in CD4+ T cells, and limited the expansion of CD4+ T cells induced by alloreactive stimulation. CG-5 also modulated CD4+ T cell polarization by inhibiting Th1 and Th17 differentiation and promoting regulatory T (Treg) induction. Moreover, CG-5 treatment reduced lupus phenotypes including the expansion of germinal center B (GC B) cells, as well as the production of autoantibodies in both TC mice and cGVHD models. Finally, CG-5 blocked glycolysis in human T cells. Overall, our data suggest that blocking glucose uptake with a small molecule inhibitor ameliorates autoimmune activation, at least partially due to its inhibition of glycolysis in CD4+ T cells.

SAT-575 Radiolabeled Somatostatin Receptor Analog 86Y-EB-TATE is Characterized by Superior Tumor Uptake Compared to 68Ga-DOTA-TATE and 68Ga-DOTA-JR11 in Thyroid Cancer Mice Models

Background: Differentiated thyroid cancer (DTC) and medullary thyroid cancer (MTC) are characterized by the overexpression of somatostatin receptor type 2 (SSTR2). There are limited data on the use of SSTR2 as a molecular target for imaging and treatment of DTC and MTC. The goal of our study was to compare the diagnostic accuracy of three different radiolabeled SSTR2 analogs in DTC and MTC mouse models: JR11, an antagonist; DOTATATE, an agonist; and EB-TATE, a novel modified agonist characterized by a longer half-life in the blood. Methods: SSTR2 expression was analyzed in two DTC cell lines (BCPAP and FTC133) and one MTC cell line (TT) by RT-PCR and immunoblotting. A rat pancreatic cell line (AR42J), characterized by intrinsically high SSTR2 expression, was used as a positive control. A metastatic (MT) mouse model was generated using immunocompromised mice which develop lung and liver metastases after tail vein injection of FTC133 and BCPAP cells. Subcutaneous (SC) xenograft mice models were generated by injecting FTC133, BCPAP, TT, and AR42J cells. The fast-growing FTC133 and AR42J cells formed soft, sizable tumors in 2 weeks, while BCPAP and TT cells formed very solid tumors after 4 and 8 weeks, respectively. Maximum standard uptake value of 68Ga-DOTATATE, 86Y-EB-TATE, and 68Ga-DOTA-JR11 (IPSEN®) was compared in each mouse in MT and SC models by PET/CT. Results: Among thyroid cancer cell lines, TT cells were characterized by the highest SSTR2 expression at the mRNA level compared with BCPAP (p<0.001) and FTC133 (p<0.001). At the protein level, BCPAP and TT cells were characterized by similar SSTR2 expression and FTC133 cells had the lowest expression. Among examined radiolabeled SSTR2 analogs, 86Y-EB-TATE showed the highest tumor uptake in all MT and SC mouse models in comparison to 68Ga-DOTATATE (p< 0.001) and 68Ga-DOTA-JR11, (p<0.001). 68Ga-DOTA-JR11 was characterized by higher tumor uptake than 68Ga-DOTATATE in only the SC FTC133 mouse model (p=0.01). The AR42J mouse model (positive control) had the highest tumor uptake of all three radioactive analogs in comparison to DTC and MTC mice models (p<0.001). Among thyroid cancer mice models, aggressively growing FTC133 tumors were characterized by the highest uptake of 68Ga-DOTA-JR11 and 86Y-EB-TATE compared with slow-growing BCPAP (p<0.01 & p<0.001, respectively) and TT tumors (p<0.05 & p<0.001, respectively) in SC mice models. There was no significant difference in the 68Ga-DOTATATE uptake between FTC133 and BCPAP mice models (p= 0.97), which were characterized by significantly higher uptake than the TT mouse model (p= 0.01 & 0.03, respectively). Conclusion: We show for the first time that a novel SSTR2 analog 86Y-EB-TATE is characterized by superior tumor uptake compared to 68Ga-DOTATATE and 68Ga-DOTA-JR11 in TC mice models. Uptake of the radiolabeled analogs not only depends on SSTR2 expression levels, but also on the tumor aggressiveness.

FcmR Shapes BCR Signaling in IgM-Positive Leukemia

Background: The Fc receptor for IgM (FcmR/ TOSO) is significantly overexpressed on chronic lymphocytic leukemia (CLL) cells from peripheral blood, but becomes down-regulated in the tumor microenvironment by e.g. CD40:CD40L interaction. Since the functional role of FcmR on lymphomagenesis is still not understood, we developed a conditional knockout mouse with B cell-specific FcmR-depletion. These mice were crossbred with the Eµ-TCL1 murine model, which develops a CLL-like phenotype.Results: The depletion of FcmR/TOSO in TCL1 mice (Eµ-Tcl1tg/wt FcmRfl/fl CD19cre/wt; further on called TCT) revealed a significantly shorter overall survival (296 days; n=40) compared to the TOSO expressing control mice (Eµ-Tcl1tg/wt FcmRwt/wt CD19cre/wt; TC; 344 days; n=106; Log-rank p<0.0001). In addition, these mice show a significantly higher blood leukocyte count and lower platelet and erythrocyte count. Leukocytes could be identified as CLL-characteristic leukemic CD19+/CD5+ B cells. Altogether TCT exhibited a faster progress of disease. Spleen immunohistochemistry revealed the transformation of most TCT (14/17 transformed) into an even more aggressive phenotype with increased splenomegaly and change in tissue and cell morphology compared to TC (9/9 not transformed). While characterizing these cells by flow cytometry, we identified a significantly higher expression of IgM on malignant B cells from TCT in comparison to TC mice. This finding indicates that the BCR itself might have a different contribution to lymphomagenesis in FcmR knock-out settings.Therefore, to validate the functional role of FcmR in the process of lymphomagenesis, we performed transcriptome profiling by RNA-Seq using splenic leukemic cells (CD19+ CD5+) from 36-week old TC (n=4) and TCT (n=4) mice. 2089 genes were found to be significantly modulated in the malignant cells of TCT mice, from which 1221 were downregulated and 868 showed an upregulation (significant change in mean expression; p<0.05). To investigate the role of IgM on TCT mice, purified malignant B cells were incubated for two hours with F(ab')2 goat anti-mouse IgM. Strikingly, TCT mice showed 3941 genes (2054 downregulated, 1887 upregulated) with significant difference in expression compared to TC (p<0.05). The gene expression profiles of the anti-IgM treated mice revealed a stronger regulation of BCR signalling in TCT mice, suggesting that FcmR represents an important factor in these processes. We examined the gene expression profiles, using Ingenuity Pathway Analysis Software. Analysis revealed that the most deregulated functions include interferon-signalling, recruitment of leukocytes, infection of cells and cellular movement.Conclusion: Here we present functional evidence that loss of FcmR results in increased IgM/BCR on the surface of non-switched leukemia. Moreover, malignant cells with loss of FcmR are more susceptible to BCR stimulation and show a signature of signalling pathways, which contribute to inflammation in B cell malignancies. DisclosuresFingerle-Rowson:MorphoSys: Employment. Pallasch:Gilead: Research Funding. Wendtner:Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding.

Protection from lethal Flu infections: lessons from a systemic lupus mouse model

Abstract Patients with systemic lupus erythematosus (SLE) are more susceptible to infections than healthy subjects but it is unknown whether this is due to disease or treatment. Using mice expressing three SLE susceptibility loci (triple congenics hereafter called TC mice) we unexpectedly found that these mice are resistant to an otherwise lethal respiratory Flu PR8 (H1N1) infection. At the naïve state, TC mice had not only more activated plasmacytoid dendritic cells, inflammatory monocytes and natural killer cells but also more activated CD8+ and CD4+ T cells with higher TNF and IFNg-producing capacity than C57BL/6J controls. At the primary effector phase following intranasal Flu PR8 infection, TC mice mounted poor antibody responses but their Flu-specific CD8+ T cell responses were greater effectors at the membrane level (higher frequencies of KLRG1+IL7R− and KLRG1+IL7R+) and at the transcriptional level (higher T-bet expression) than CD8+ T cells from C57BL/6J counterparts. PR8-immune TC mice also developed more functional Flu-specific CD8+ T cell responses than C57BL/6J mice following to a heterosubtypic Flu X-31 (H3N2) challenge. While immunosuppressive treatments of SLE in patients might be responsible of mounting poor anti-microbial immune responses, lessons from this SLE mouse model could be taken to prevent and protect against severe Flu infections.

Dietary Tryptophan Modulates Autoimmunity in a Mouse Model of Lupus

Abstract Both kynurenine and serotonin are synthesized from dietary tryptophan via indolamine-2,3-dioxygenase (IDO) and tryptophan hydroxylase (tph1) respectively. SLE patients have altered serum and PBMC tryptophan metabolites relative to healthy controls such that there is a depletion of both tryptophan and serotonin and an accumulation of kynurenine. Mass spectrometry analyses showed that SLE B6.Sle1.Sle2.Sle3 congenic (TC) mice on a standard diet that were positive for serum autoantibodies have the same serum tryptophan metabolite alterations reported in SLE patients as compared to B6 controls. Additionally, TC mice have an altered fecal tryptophan metabolite profile relative to B6 controls, and antibiotic treatment restores this profile, suggesting a microbial involvement. Based on these findings, we sought to understand how altering dietary tryptophan would impact autoimmunity in lupus-prone TC mice. Congenic mice consuming low (0.1%) or no (0%) tryptophan for 6 months were protected from autoimmunity whereas those consuming supplemented tryptophan (1%) had exacerbated autoimmunity, including anti-dsDNA IgG production that was correlated with serum kynurenine levels. Furthermore, enzyme expression within the tryptophan catabolic pathway was altered in total CD4+ T cells from SLE congenic mice relative to B6 controls. These data show an altered tryptophan metabolism in lupus prone mice associated with the gut microbiome and with changes in enzyme expression within the tryptophan catabolic pathway in immune cells. Further we showed that manipulation of tryptophan metabolites through diet modulates autoimmunity in these lupus-prone mice.

Autoreactive and immunization-induced follicular helper T cells have opposite glucose and glutamine requirements

Abstract Both glycolysis and mitochondrial oxidative metabolism are elevated in CD4+ T cells from lupus-prone mice, and metabolic inhibitors targeting both of these pathways normalized lupus T cell functions in vitro and reverted disease in mice. Here, we showed that inhibiting glucose metabolism with 2-deoxy-D-glucose (2DG) results in a drastic reduction of follicular helper T (TFH) cell frequency in lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice. However, treatment of TC mice with 2DG had little effect on the production of antibodies or the expansion of TFH cells following immunization with a nominal antigen of T-cell-dependent (TD) humoral responses, and no effect on the frequency of influenza virus-specific TFH cells induced by immunization with PR8 influenza virus. Thus, TFH cells supporting the production of autoantibodies are different from TFH cells providing protective humoral immunity against pathogens. The specific metabolic signature from autoimmune TFH cell is significantly different compared with exogenous antigen-specific TFH cells. Further results obtained with gene expression analysis and treatment with metabolic inhibitors indicated that autoreactive TFH cells depend on glucose metabolism, while exogenous antigen-specific TFH cells are more glutamine-demanding. Overall, our results predict that targeting TFH cellular metabolism provides an effective and safe therapeutic approach for systemic autoimmunity by eliminating autoreactive TFH cells.

Fermented Red Ginseng Alleviates Cyclophosphamide-Induced Immunosuppression and 2,4,6-Trinitrobenzenesulfonic Acid-Induced Colitis in Mice by Regulating Macrophage Activation and T Cell Differentiation.

A variety of products have been developed with red ginseng (RG, the steamed roots of Panax ginseng Meyer). To clarify the immunomodulating effects of water-extracted RG (wRG), 50% ethanol-extracted RG (eRG), enzyme-treated eRG (ERG) and probiotic-fermented eRG (FRG), we examined their immunopotentiating and immunosuppressive effects in mice with cyclophosphamide (CP)-induced immunosuppression (CI) or 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis (TC). Oral administration of RG in CI mice significantly increased blood IFN- levels. Treatment with RG also increased the tumoricidal effects of CI mouse splenic cytotoxic T (Tc) and NK cells against YAC-1 cells. Treatment with RGs, in particular FRG and wRG, significantly increased Th1 cell differentiation. Treatment with RG except wRG increased Treg cell differentiation. However, wRG alone increased IL-6 and IL-17 expression in the colon of CI mice. Furthermore, RG alleviated colitis in TC mice. FRG most potently suppressed TNBS-induced colon shortening, NF- B activation and TNF- and IL-17 expression and increased IL-10 expression. RGs inhibited TNF- expression and increased IL-10 expression in lipopolysaccharide-stimulated primary macrophages in vitro while the differentiation of splenic T cells into type 1 T (Th1) and regulatory T (Treg) cells was increased by FRG in vitro. In conclusion, FRG can alleviate immunosuppression and inflammation by inhibiting macrophage activation and regulating Th1 and Treg cell differentiation.

Modification of single-nucleotide polymorphism in a fully humanized CYP3A mouse by genome editing technology

Cytochrome P450, family 3, subfamily A (CYP3A) enzymes metabolize approximately 50% of commercially available drugs. Recently, we developed fully humanized transchromosomic (Tc) CYP3A mice with the CYP3A cluster including CYP3A4, CYP3A5, CYP3A7, and CYP3A43. Our humanized CYP3A mice have the CYP3A5*3 (g.6986G) allele, resulting in the almost absence of CYP3A5 protein expression in the liver and intestine. To produce model mice for predicting CYP3A5′s contribution to pharmacokinetics, we performed a single-nucleotide polymorphism (SNP) modification of CYP3A5 (g.6986G to A, *3 to *1) on the CYP3A cluster using genome editing in both mouse ES cells and fertilized eggs, and produced humanized CYP3A5*1 mice recapitulating the CYP3A5*1 carrier phenotype in humans. The humanized CYP3A mouse with CYP3A5*1 is the first Tc mouse for predicting the SNP effect on pharmacokinetics in humans. The combination of Tc technology and genome editing enables the production of useful humanized models that reflect humans with different SNPs.

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

BackgroundThree-dimensional (3D) spheroids are frequently used in toxicological study because their morphology and function closely resemble those of tissue. As these properties are maintained over a long term, repeated treatment of the spheroids with a test object is possible. Generally, in the repeated treatment test to assess cytotoxicity in the spheroids, ATP assay, colorimetric measurement using pigments or high-content imaging analysis is performed. However, continuous assessment of cytotoxicity in the same spheroids using the above assays or analysis is impossible because the spheroids must be disrupted or killed. To overcome this technical limitation, we constructed a simple monitoring system in which cytotoxicity in the spheroids can be continuously monitored by nondestructive bioluminescence measurement.ResultsMouse primary hepatocytes were isolated from transchromosomic (Tc) mice harboring a mouse artificial chromosome (MAC) vector expressing beetle luciferase Emerald Luc (ELuc) under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter/rabbit β-globin intron II (CAG) promoter, and used in 3D cultures. We confirmed that both luminescence and albumin secretion from the spheroids seeded in the 96-well format Cell-ableTM were maintained for approximately 1 month. Finally, we repetitively treated the luminescent 3D spheroids with representative hepatotoxicants for approximately 1 month, and continuously and nondestructively measured bioluminescence every day. We successfully obtained daily changes of the dose-response bioluminescence curves for the respective toxicants.ConclusionsIn this study, we constructed a monitoring system in which cytotoxicity in the same 3D spheroids was continuously and sensitively monitored over a long term. Because this system can be easily applied to other cells, such as human primary cells or stem cells, it is expected to serve as the preferred platform for simple and cost-effective long-term monitoring of cellular events, including cytotoxicity.

Gut dysbiosis contributes to autoimmune pathogenesis in lupus-prone mice

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease mediated by a combination of genetic and environmental factors. We have used a congenic model of the disease, the B6.Sle1.Sle2.Sle3 (TC) mice, which share 95% of their genome with B6 controls. TC and B6 mice have a different distribution of gut bacteria based on 16S rDNA sequencing. Fecal transfers into germ free (GF) B6 mice showed that microbiota from aged TC mice was sufficient to induce the production of anti-dsDNA IgG. TC microbiota expanded germinal center (GC)-like organization in Peyer’s patches and TFH cells in the mesenteric lymph node. Co-housing TC and B6 mice for 6 months starting at weaning revealed significant interactions between disease status and the microbiota compared with independently housed mice from both strains. Remarkably, the frequency of TFH cells was decreased in co-housed TC mice, corresponding to a drastic reduction of autoantibodies and amelioration of renal disease. Co-housed TC and B6 mice showed a similar fecal metabolome profile, which was significantly different from those of independently housed mice. Studies to characterize the role of specific metabolites related to disease pathogenesis are ongoing. Finally, treatment of TC mice with a combination of antibiotics significantly delayed anti-dsDNA IgG induction and expanded Treg and TFR cells. Overall, our results suggest that the dysregulated gut microbiota in lupus contributes to disease pathogenesis either directly or indirectly, through the production of critical metabolites.

Loss of TOSO Promotes Richter's Transformation of TCL1A Driven CLL

Background: The immunoglobulin-like protein TOSO, which has been found to serve as Fc receptor for IgM (FcµR), was shown by us and others to be overexpressed on CLL cells and only weakly expressed on more aggressive B-NHL. However the functional role of TOSO on lymphomagenesis has not been explored so far.Methods: To determine the role of TOSO on lymphoma development, we took advantage of the Eµ-TCL1 transgenic mice, which usually end up with an aggressive (IgVH unmutated) CLL-like phenotype. We generated a novel B cell-specific conditional knockout (KO) mouse model in which EµTCL1 mice (TC or control in the following) were crossbred with TOSO-floxed mice, expressing Cre recombinase under the control of the CD19 promoter (EµTCL1;Tosofl/fl;Cd19cre/wt or TCT in the following). TCT mice were further compared with p53 conditional knockout (EµTCL1;Tp53fl/fl;Cd19cre/wt or TCP).Results: In this study, we compared kinetics, overall survival and phenotype of lymphoma/CLL in TC, TCT and TCP mice. Interestingly, TCTmice developed a very aggressive phenotype and resulted in significantly shorter overall survival compared to TC mice (TCT 274 days vs. TC 346 days; p<0.0001). As expected, mice lacking p53 (TCP) died even more rapidly than TCT mice (median survival: TCP 233 days). Initially, all three genotypes (TC, TCT, TCP) developed a CLL phenotype, exhibiting a CD19 and CD5 positive malignant clone. In the TCT mice, shorter overall survival is accompanied by a stronger increase of blood leukocytes. Flow cytometry analysis confirmed a strong increase of leukemic CD19/CD5-positive B cells in the blood of TCT mice. With only 20 weeks of age, leukemic cells already made up 37.5 % (SD ± 15.47; n=14) of lymphocytes (TC: 14.3 % SD ± 9.81; n=31). At the age of 36 weeks, TCT mice showed even a 3.6-fold elevated malignant cell count compared to control mice (n=35 TC, n=14 TCT; p=0.006). All TCT mice developed a splenomegaly, with spleen weight (p=0.01) and size (p=0.018) significantly increased in 36 week old TCT mice (n=7) compared to TC mice (n=7) and comparable to those from TCP mice. Interestingly, between week 28 and 36, we could observe that most of the TCT mice start losing CD19+ cells in the blood in contrast to TC and TCP mice. Immunohistochemistry revealed the expansion of malignant cells with pleomorphic nuclei and abundant cytoplasm in the spleen and bone marrow, as we know it from Richter‘s transformation. To understand the rapid development of leukemia in TCT mice, we first determined the role of the BCR in this model. Interestingly, flow cytometry revealed a higher surface IgM expression (MFI: TCT 9,27; TC 2,05). In addition, in vitro assays revealed a significantly higher resistance of TCT cells towards PI3K inhibition (Idelalisib and Duvelisib) compared to TC cells.To further rule out the role of TOSO under "germinal-center conditions", we stimulated primary human CLL cells with CD40L expressing feeder cells and IL-4. Interestingly, both stimuli (either alone or in combination) resulted in almost complete loss of TOSO on CLL cells. Moreover, we uncovered, that the TOSO promoter is counteractively regulated by NF-κB and BCL6. Furthermore, our data illustrate that DNA hypomethylation of the TOSO promoter is a discriminating characteristic in CLL patients compared to healthy donors, thus explaining the significantly enhanced expression levels. Thus, both, epigenetic regulation and altered NF-κB/ BCL6 expression are critical pathogenetic steps in the development of CLL and aggressive B-NHL by regulating TOSO expression.Conclusion: The transformation of CLL into more aggressive malignancies is still not fully understood. Our data reveal that the loss of TOSO might play a major role in Richter's transformation by upregulation of the BCR and by mimicking the germinal-center phenotype. DisclosuresFingerle-Rowson:Roche: Employment. Wendtner:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann-La Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hallek:GSK: Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Gilead: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria.

Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation

The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10−6). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50 % in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-015-9884-6) contains supplementary material, which is available to authorized users.

Genetic and cellular dissection of the activation of AM14 rheumatoid factor B cells in a mouse model of lupus.

The RF-specific AM14 tg BCR has been used as a model to dissect the mechanisms of B cell tolerance to ICs containing nucleic acids. We have shown previously that AM14 RF B cells break tolerance in the TC mouse model of lupus through the dual engagement of the AM14 BCR and TLR9. In this study, we showed that neither the expression of Sle1 or Sle2 susceptibility loci alone was sufficient to activate AM14 RF B cells, suggesting that the production of antichromatin IgG2a(a) autoAg mediated by Sle1 and an intrinsically higher B cell activation mediated by Sle2 were required. We also showed that the B6 genetic background enhanced the selection of AM14 RF B cells to the MZB cell compartment regardless of the expression of the Sle loci and therefore, of their activation into AFCs. Furthermore, some AM14 RF B cells were selected into the B-1a compartment, where they did not differentiate into AFCs. Therefore, it is unlikely that the selection of AM14 RF B cells to the MZB or B-1a cell compartments in TC.AM14(a) mice is responsible for their breach of tolerance. Finally, we showed that the presence of expression of Sle1 in non-tg cells, most likely T cells, is necessary for the activation of AM14 RF B cells into AFCs. Overall, these results suggest a threshold model of activation of AM14 RF B cells on the B6 background with additive genetic and cellular contribution of multiple sources.

Dysregulated cytokine production by dendritic cells modulates B cell responses in the NZM2410 mouse model of lupus.

The breakdown in tolerance of autoreactive B cells in the lupus-prone NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) mice results in the secretion of autoantibodies. TC dendritic cells (DCs) enhance B cell proliferation and antibody secretion in a cytokine-dependent manner. However, the specific cytokine milieu by which TC DCs activate B cells was not known. In this study, we compared TC and C57BL/6 (B6) control for the distribution of DC subsets and for their production of cytokines affecting B cell responses. We show that TC DCs enhanced B cell proliferation through the production of IL-6 and IFN-γ, while antibody secretion was only dependent on IL-6. Pre-disease TC mice showed an expanded PDCA1+ cells prior to disease onset that was localized to the marginal zone and further expanded with age. The presence of PDCA1+ cells in the marginal zone correlated with a Type I Interferon (IFN) signature in marginal zone B cells, and this response was higher in TC than B6 mice. In vivo administration of anti-chromatin immune complexes upregulated IL-6 and IFN-γ production by splenic DCs from TC but not B6 mice. The production of BAFF and APRIL was decreased upon TC DC stimulation both in vitro and in vivo, indicating that these B cell survival factors do not play a role in B cell modulation by TC DCs. Finally, TC B cells were defective at downregulating IL-6 expression in response to anti-inflammatory apoptotic cell exposure. Overall, these results show that the TC autoimmune genetic background induces the production of B cell-modulating inflammatory cytokines by DCs, which are regulated by the microenvironment as well as the interplay between DC.

A novel transchromosomic system: stable maintenance of an engineered Mb-sized human genomic fragment translocated to a mouse chromosome terminal region

Transchromosomic (Tc) technology using human chromosome fragments (hCFs), or human artificial chromosomes (HACs), has been used for generating mice containing Mb-sized segments of the human genome. The most significant problem with freely segregating chromosomes with human centromeres has been mosaicism, possibly due to the instability of hCFs or HACs in mice. We report a system for the stable maintenance of Mb-sized human chromosomal fragments following translocation to mouse chromosome 10 (mChr.10). The approach utilizes microcell-mediated chromosome transfer and a combination of site-specific loxP insertion, telomere-directed chromosome truncation, and precise reciprocal translocation for the generation of Tc mice. Human chromosome 21 (hChr.21) was modified with a loxP site and truncated in homologous recombination-proficient chicken DT40 cells. Following transfer to mouse embryonic stem cells harboring a loxP site at the distal region of mChr.10, a ~4Mb segment of hChr.21 was translocated to the distal region of mChr.10 by transient expression of Cre recombinase. The residual hChr.21/mChr.10ter fragment was reduced by antibiotic negative selection. Tc mice harboring the translocated ~4Mb fragment were generated by chimera formation and germ line transmission. The hChr.21-derived Mb fragment was maintained stably in tissues in vivo and expression profiles of genes on hChr.21 were consistent with those seen in humans. Thus, Tc technology that enables translocation of human chromosomal regions onto host mouse chromosomes will be useful for studying in vivo functions of the human genome, and generating humanized model mice.

Abstract 2729: A genetically engineered mouse model of de novo pancreatic carcinogenesis .

Abstract Multistep models of PDAC development converge on both abnormalities in KRAS and E2F activation, which are part of the Rb/E2F pathway, and dysfunction of p53, which is associated with the MDM2/p53 pathway. Recent progress in the understanding of the protective roles of the Rb/E2F and MDM/p53 pathways against carcinogenesis showed that cross talk and complementary actions of these pathways are present in many types of cancers. In this study, we addressed directly questions regarding the collaborative roles of the Rb/E2F and MDM2/p53 pathways in suppressing the progression of multistep PDAC carcinogenesis by generating TKC (loxP-βgeo-STOP-loxPtemperature-sensitive SV40 T antigen (tsTAg)), loxP-STOP-loxP-KrasG12D, and Pdx1-Cre) mice which exhibit impairment of both pathways and K-rasG12D expression in pancreatic epithelial cells. The PDAC induced in TKC mice exhibited rapid development and absence of inflammation or PanIN formation, which occurred by de novo carcinogenesis. The tumors resembled human PDAC at the histological and molecular level. In in vitro study, we established PDAC cell lines from TKC mice and immortalized pancreatic ductal cell lines from TC mice, Cytokeratin 19 expression with which was applied to select cells as pancreatic ductal cell differentiation. In collagen gel 3D culture, PDAC cells formed atypical gland structures the same as in subcutaneous transplanted tumor in nude mice, while the Cytokeratin 19 positive immortalized ductal cell lines from TC mice formed ductal structures similar to either intralobular or interloblar pancreatic ducts. The results of the analysis of the highlighted molecular mechanisms suggest that the synthetic interaction among mutated Kras, uncontrolled E2f activation, and dysfunction of p53 results in de novo pancreatic carcinogenesis. This model represents an important tool to explore molecular targets for diagnostics and cancer therapy for PDAC via de novo pancreatic carcinogenesis.This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and New Energy and Industrial Technology Development Organization (NEDO) of Japan, and supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Citation Format: Takashi Yamaguchi, Sanae K. Ikehara, Yuzuru Ikehara. A genetically engineered mouse model of de novo pancreatic carcinogenesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2013-2729

Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes.

A Novel and Stable Mouse Artificial Chromosome Vector

Human chromosome fragments (hCFs) and human artificial chromosomes (HACs) can be transferred into mouse ES cells to produce trans-chromosomic (Tc) mice. Although hCFs and HACs containing large genomic DNAs can be autonomously maintained in Tc mice, their retention rate is variable in mouse ES cell lines and Tc mouse tissues, possibly because of centromere differences between the species. To improve the retention rate of artificial chromosomes in mouse cells, we constructed novel mouse artificial chromosome (MAC) vectors by truncating a natural mouse chromosome at a site adjacent to the centromeric region. We obtained cell clones containing the MAC vectors that were stably maintained in mouse ES cells and various tissues in Tc mice. The MACs possess acceptor sites into which a desired gene or genes can be inserted. Thus, Tc mice harboring the MAC vectors may be valuable tools for functional analyses of desired genes, producing humanized model mice, and synthetic biology.

Mechanisms for non-antigen specific rheumatoid factor B cell activation in the NZM2410 mouse model of lupus (159.9)

Abstract Systemic lupus erythematosus is characterized by the presence of autoantibodies, including rheumatoid factor (RF), due to a breakdown in B cell tolerance. In the AM14 model, the AM14 transgenic heavy chain combined to Vκ8 produces a RF specific for IgG2aa. Contrary to the MRL/lpr lupus model, RF B cell activation required the presence of chromatin-IgG immune complexes (ICs), but could occur in the absence of the IgG2aa autoantigen in the B6.NZM2410.Sle1.Sle2.Sle3 (TC) strain, which contains the three major NZM2410-derived lupus susceptibility loci (Sle1, Sle2, Sle3). Treatment of young TC.AM14 and control B6.AM14 mice with a TLR7/9 antagonist did not decrease RF B cell activation after immunization with antigen non-specific anti-chromatin ICs. Splenic DCs from mice injected with anti-chromatin hybridomas showed elevated expression of IL-6 and IFN-γ mRNA in TC mice in comparison to B6 controls. B cell co-cultures with TC-derived BMDCs induced greater B cell proliferation than B6-derived BMDCs. Finally, B cell activation of aged AM14 mice expressing Sle1 or Sle2 showed that while Sle1 promoted antigen specific activation, Sle2 induced B-cell activation is polyclonal in nature. Therefore, our results show that while cognate-antigen independent RF B cell activation is not affected by inhibition of TLR7/9 signaling, it is enhanced by DC-derived cytokines and the expression of the Sle loci.

Abstract 4434: Development of pancreatic ductal adenocarcinoma (PDAC) by the expression of temperature-sensitive SV40 large T antigen (tsTAg) and K-ras G12D on pancreatic epithelial linage cells

Abstract Based on studies using genetically engineered mouse (GEM) models producing pancreatic ductal adenocarcinoma (PDAC), pancreatic intraepithelial neoplasia (PanIN) - PDAC linear pathway has been proposed. Whereas the positive association between the carcinogenesis and the engineered genetic abnormalities K-ras, Ink4a/Arf, Trp53, and Smad4, it has been pointed out the oversimplification on the postulated carcinogenesis steps. Indeed, there should be various routes leading to PDAC on the etiological heterogeneity, and it remains controversial whether PanIN-1 lesions are required for development of PDAC in view of the pathogenesis. In order to increase knowledge about the routes leading to PDAC, we generated GEM harboring loxP-μgeo-stop-loxP-tsTAg (LbsL-tsTAg); loxP-stop-loxP-K-rasG12D; Pdx1-Cre (TKC) alleles that express temperature-sensitive SV40 T antigen (tsTAg) and K-rasG12D on epithelial cells in pancreas. The tsTAg has been widely used to generate immortalized cells by inhibiting tumor suppressor molecules, P53 and Rbs (Rb1, Rbl1, Rbl2), and we previously reported the generation of GEM with LbsL-tsTAg and the success to establish the immortalized lymphatic endothelial cells using this mice (Yamaguchi T. et al, FEBS J. Vol.275: p1988-1998, 2008). In TKC mice, PDAC foci appeared at 5th days after birth without PanIN-1 lesions, and showed rapid progression and invasion into duodenum, resulting in death within 3 weeks. On the contrary, though tsTAg-expressing TC mice develop neither PDAC nor PanIN, dysplastic feature appeared on acinar cells at 15-20 weeks after birth. In in vitro assay, the characteristics of the established PDAC cell lines from TKC mice was compared with the immortalized cell lines expressing Cytokeratin 19 (Ck19, pancreatic ductal cell marker) from TC mice. PDAC cells were positive for Ck19 and formed atypical gland structures in collagen gel 3D culture the same as in subcutaneous transplanted tumor in nude mice, while the Ck19 positive immortalized cell lines from TC mice formed ductal structures in collagen gel similar to either intralobular or interloblar pancreatic ducts. The expression of Tacstd2, SerpinB2, and microRNA-200 group genes were upregulated in PDAC cells more than the Ck19 positive immortalized cell lines, which were commonly in human PDAC. These results suggest that PDAC can develop without PanIN-1 lesion under the given conditions as TKC mice, and that constitutive active mutation of K-ras plays an important role for lineage addiction to show ductal phenotype. Our newly established mice models and in vitro assay systems are feasible not only to establish molecular taxonomy of PDAC, but also to develop biomarkers for early detection, and help to discover the therapeutic target molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4434. doi:1538-7445.AM2012-4434