B6 cg tg tcratcrb 425cbn j

To characterize MOG-specific T cell interactions, DCs from naïve C57BL/6J mice were isolated, as above, and treated with CpG (1 μg) and either soluble MOG-R₃ (60 μg), GpG (10 μg), or CTRL (10 μg), or iPEM formulations (10 μg). After 16 h of culture, CD4⁺ T cells were isolated from the spleens of transgenic 2D2 mice (C57BL/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J, The Jackson Laboratory) with a magnetic isolation kit (StemCell Technologies), according to the manufacturer’s instructions. Isolated CD4⁺ T cells were then incubated with a cell proliferation dye (eFluor 670, eBioscience), washed, and 2.5 × 10⁵ labelled cells were added to DC cultures. After 72 h of co-culture, cells were collected, washed and blocked, as above. Cells were then stained with anti-CD4 (BD Biosciences) for 20 min at room temperature, washed to remove unbound antibody, and resuspended in DAPI for analysis by flow cytometry. In separate studies, CD4⁺ T cells with receptors specific for OVA_323–339 were isolated from the spleens of transgenic OT-II mice (B6.Cg-Tg(TcraTcrb)425Cbn/J, The Jackson Laboratory), labeled with proliferation dye, and added to DC cultures, as above. In these experiments, control wells were treated with CpG (1 μg) and soluble OVA_323–339 (60 μg).

AB wild-type, lck:gfp and rag1-mutant strains were obtained from the Zebrafish International Resource Center, bred and maintained at the aquatics facility at Brigham and Women’s Hospital. rag2-mutant strains were donated by L. Zon (Boston Children’s Hospital). The collection of embryos and maintenance of larvae were carried out as described previously^{5 (link),8 (link),51 (link)}. Eight-week-old male C57BL/6J, B6(Cg)-Zbtb46tm1(HBEGF) Mnz/J, FVB.Cg-Tg(HIV-EGFP,luc)8Tsb/J, B6.D2N-Ahrd/J, B6.129S7-Rag1tm1Mom/J and B6.Cg-Tg(TcraTcrb)425Cbn/J mice were obtained from the Jackson Laboratory, and Cebpb^+/+ and Cebpb^−/− (Cebpbtm1Vpo/J) mice were obtained from A. Mildner. B6.129S4-Il17atm1.1Lky/J mice were a gift from T. Korn. Animals were kept in a pathogen-free facility at Hale Building for Transformative Medicine, Brigham and Women’s Hospital. Germ-free mice were bred in-house in tightly controlled and monitored isolators (Class Biologically Clean company) in an animal facility specifically dedicated to housing germ-free mice in the Massachusetts Host-Microbiome Center at the Brigham and Women’s Hospital. All of the experimental protocols were approved by the Institutional Animal Care and Use Committee of Brigham and Women’s Hospital.

Male C57BL/6 mice were purchased from Jackson Laboratories. IL17A^GFP/+ mice (Jax:C57BL/6-Il17atm1Bcgen/J) were a gift from Dr. Jun Huh. OT-II TCR-transgenic mice were obtained from Jackson Laboratories (B6.Cg-Tg(TcraTcrb)425Cbn/J). H2-Ab1-deficient mice were previously described^{36 (link)}. To construct bone marrow chimeras, bone marrow cells were harvested from both femurs and tibias, and treated with ACK buffer (Lonza) to remove red blood cells. CD45.1⁺/CD45.2⁺ mice were irradiated (10 Gy) and reconstituted with equal proportions (~5 million cells each) of CD45.1 and CD45.2 (Ab KO) bone marrow cells. All mice were bred and maintained in our specific-pathogen-free facilities at Harvard Medical School (IACUC protocols IS1257, IS187-3, IS2221).

Six- to ten-week-old C57BL/6, B6.SJL-ptprc^a (CD45.1⁺), B6.129S2-Cd4^tm1Mak/J (CD4 KO), B6.129S2-H2^dlAb1-Ea/J (MHC II KO), C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I), and B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Thymectomized animals underwent adult thymectomy at the Jackson Laboratory (Bar Harbor, ME). E1/E3 deleted Ad5-SIINFEKL-Luc, Ad5-OVA, and Ad5HVR48(1–7)-Gag have been previously described (35 (link)–38 (link)). Mice were immunized intramuscularly with 10⁹ viral particles of the indicated vector in the quadriceps using 100 µl divided equally between the two legs. NYVAC-Env has been previously described (39 (link)) and was used at 10⁶ pfu per mouse. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines of Beth Israel Deaconess Medical Center.