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Figure S1 B A C D +Tam.

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Presentation on theme: "Figure S1 B A C D +Tam."— Presentation transcript:

1 Figure S1 B A C D +Tam

2 Figure S1 Development of obesity in OvaxCreERT2 male mice upon high fat diet feeding Depiction of the cassette design in OvaxCreERT2 transgenic mice: Mice carrying the Ova antigen cassette under the control of ROSA26 promoter flanked with inversely oriented loxP sites were crossed to CreERT2 mice carrying the CreERT2 gene cassette under the control of the liver specific Albumin promoter (OvaxCreERT2). Liver specific antigen expression occurs by Cre recombinase mediated inversion of the cassette upon a single administration of Tam (weight adjusted). In presence of Tam, Cre mediated inversion of Ova is reversible but becomes fixed once the Tam is cleared. The mRNA levels in induced (+Tam) mice are 5 fold higher than from non-induced (-Tam) mice which refer to as high and low antigen load, respectively. Body weight of HFD fed male OvaxCreERT2 (n= 7-19 per group **p≤0.01) Liver sections stained with haematoxylin/eosin (H&E) from 16 week SD/HFD OvaxCreERT2 mice. Characterization of OvaxCreERT2 mice with respect to the metabolic cholesterol, glucose, triglyceride (TG), AST concentration in blood plasma. (n=5-12 per group ** p≤0.01)

3 Figure S2 A

4 Figure S2

5 Figure S2 C d3 d13 + + c + + c + + c + + c + - + + - + + - + + - +
OvaxCreERT2 Tam c c OvaxCreERT2 Tam SD HFD SD HFD

6 Figure S2 D

7 Figure S2 Efficiency and functionality of T cell response (A) The number of intrahepatic Ova specific Thy1.1 OT-I cells in SD/HFD OvaxCreERT2 mice was determined at d3 and d13 post adoptive transfer of OT-I cells. (B) CD69, Lag 3 and (C) CD44 and CD62Lhigh expression on intrahepatic Ova specific OT-I cells in SD/HFD OvaxCreERT2 mice was analyzed by flow cytometry at d3 and d13 post adoptive transfer of T cells. (D) CD25 expression in the CD8 T cell population in SD/HFD OvaxCreERT2 mice on the indicated days after T cell transfer.

8 Figure S3 A

9 Figure S3 B

10 Figure S3 C

11 Figure S3 Phenotypic characterization of T cell response upon HFD in non-induced OVA x CRERT2 x OT-I mice (A) Time kinetics of hepatitis induction measured by ALT concentration in blood plasma of non-induced (-Tam) SD/HFD OvaxCreERT2xOT-I mice. The indicated days refer to time points after Tam induction of mice as described in Figure 2 (n= 5-9 per group). (B) CD69, CD44 and CD62L expression on intrahepatic Ova specific OT-I cells SD (d33) and HFD (d44) OvaxCreERT2xOT-I mice (-Tam) was analyzed by flow cytometry. (C) At the respective days, the expression of the exhaustion markers PD1 and Lag3 on intrahepatic OT-I cells from non-induced (-Tam) SD and HFD OvaxCreERT2xOT-I mice was analyzed by flow cytometry.

12 Figure S4 SD HFD MAC2 CD11c +Tam Tam Tam Tam

13 Figure S4 Histology analysis of liver tissue sections in induced/non-induced OvaxCreERT2xOT-I mice SD and HFD OvaxCreERT2xOT-I mice were sacrificed on day 33 and 44, respectively, according to the procedure described in Figure 2. Sections were stained for macrophages (anti-MAC2, brown), Dendritic and NK cells (anti-CD11c, brown). The figure is a representative picture of liver samples taken from 1 of 3 mice.

14 Supplementary methods
Measurement of metabolic parameters Blood was collected by retro-orbital puncture from mice fed for 19 weeks on both SD or HFD diets and mixed 1:4 with Heparin 1,25I.E. (Ratiopharm, Ulm, Germany). Serum Glucose, Cholesterol, Triglycerides, Aspartate transaminase (AST) and Alanine transaminase (ALT) concentrations were measured using a Reflovet®Plus reader (Roche Diagnostics, Mannheim, Germany).  Histological examination Hematoxylin-eosin staining of liver sections was performed as described previously (Cebula et al, 2013). Liver samples were fixed in 4% formalin (pH 7,4), dehydrated and embedded in paraffin. 3 µm thick sections were stained with anti-MAC2 antibody (Cedarlane , Cl8942AP, Biozol Diagnostica Vertrieb GmbH, Germany), or anti CD11c (Synaptic Systems GmbH, Goettingen ,Germany, ). Biotinylated Goat anti rat IgG antibody ( ,KPL Inc, Maryland, USA), Biotinylated Goat anti rabbit antibody ( ,KPL Inc, Maryland, USA) was used as secondary antibody, 3,3'-Diaminobenzidine (DAB) was used as chromogen. Flow cytometry Isolated lymphocytes were aliquoted in 96 well plates and stained with the fluorescently labelled monoclonal anti-mouse antibodies such as anti-CD8PerCPCy5.5 (Bioscience/Biolegend), anti-CD69clone H1.2F3 (eBioscience/ Biolegend), anti-PD-1-FITC clone: J43, anti-LAG-3-PE eBioL9B7W (eBioscience) and anti-Thy1.1-PE (e- Bioscience, San Diego, CA) anti-CD44 APC; clone: IM7 ( eBioscience) anti-CD4 eFlour 450, clone: GK 1.5 ( eBioscience), anti- CD62L PE-Cy7, clone: MEL-14 (eBioscienc). eCells were subjected to FACS LSRII (Becton Dickinson, Heidelberg, Germany) and analysis was done on FlowJo-software (TriStar Inc, Oregon, USA).;


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