3a). astrocytes in autoimmune CNS inflammation. Thus, our study defines novel mechanisms of action of IFN- in EAE pathogenesis, and also highlights an opportunity for development Indoximod (NLG-8189) of MS therapies directed at CNS cells. H37Ra (Difco). Two hundred ng of pertussis toxin (List Biological Lab, Epsom, England) was given i.p. on days 0 and 2 post immunization (p.i.). For passive EAE, GFAP-shIFN-R or GFAP-shVec lentivirus injected mice were transferred with 3.0107 polarized MOG35-55-specific Th1 or Th17 cells/mouse 4 hours after sublethal irradiation (550 Rad). To prepare MOG-specific polarized T cell populations, draining lymph nodes and spleen cells were prepared from mice immunized as described above at day 9 p.i. Cells were cultured for 4 days with MOG35-55 at a concentration of 25 g/ml under Th1- (20 ng/ml rmIL-12 [PeproTech], 2 g/ml anti-IL23p19 [eBioscience]) or Th17- (20 ng/ml rmIL-23 [PeproTech]) polarizing conditions (28). Mice were scored daily for appearance of clinical signs of EAE on a scale from 0 to 5 as described previously (29): 0, no clinical signs; 1, fully limp tail; 2, paralysis of one hind limb; 3, paralysis of both hind limbs; 4, paralysis of trunk; 5, moribund or death. Construction of pLenti-GFAP-EGFP-mi-shIFN-R, pLenti-CD11b-EGFP-mi-shIFN-R and control lentiviral vectors Vectors were constructed as previously described (30). Briefly, for pLenti-GFAP-EGFP-mi-shIFN-R vector construction, the shAct1 cassette in pLenti-GFAP-EGFP-mi-shAct1 was replaced by a fragment containing miR-30 based shIFN-R cassette (Open Biosystems, Cat No. RMM4431-98920699). For pLenti-CD11b-EGFP-mi-shIFN-R vector construction, the GFAP promoter in pLenti-GFAP-EGFP-mi-shIFN-R vector was replaced by CD11b promoter sequence, which was composed of bp ?1704- bp +83 of the 5 untranslated region of human CD11b gene amplified from human genomic DNA (31). The constructed vector sequence was verified Indoximod (NLG-8189) by sequencing. The vector without insertion of mi-shIFN-R was used as control. Primers used for vectors construction are listed in Supplemental Table 1. Isolation of primary astrocytes and microglia The whole brain of mice embryos (E16) was harvested and dissociated with Neural Tissue Dissociation Kit (Miltenyi Biotech Inc., Auburn, CA) following the manufacturers instructions. Astrocytes were purified with anti-ASCA-2+ microbeads (Miltenyi Biotech Inc., Auburn, CA) following the manufacturers MACS instructions (30). The purified astrocytes were centrifuged at 300 g for 10 min, and APAF-3 then resuspended with D-MEM/10% FBS for cell culture. Microglia cells were purified with anti-CD11b microbeads (Miltenyi Biotech Inc., Auburn, CA) following the manufacturers MACS instructions. The purified microglia cells were centrifuged at 300 g for 10 min, then resuspended with D-MEM/10% FBS plus 5 ng/ml M-CSF (PeproTech), and seeded on 60-mm dishes at a density of 1106/dish. After 7 days, cultures were trypsinized Indoximod (NLG-8189) and replated in Petri dishes. Cells from cultures that had been passaged once were used as microglia cells. Viral infection of purified astrocytes, microglia and injection For virus infection, purified astrocytes or microglia were rooted Indoximod (NLG-8189) in poly-lysine coated 6-well plates at a concentration of 5105 cells/well. Two days later, culture medium was replaced by fresh complete DMEM medium supplemented with 1106 IU/well of different lentiviruses and 8 g/ml polybrene, and then incubated for 16 hrs at 37C. After incubation, the medium with virus soap was replaced by fresh medium, and cultured for further use. For in vivo injection, mice were anaesthetized and fitted with i.c.v. cannula for virus microinjection. A microsyringe was inserted into 2.0-mm lateral, 1.0-mm caudal to bregma, and 2.5 mm below the skull surface. 1107 IU/mouse GFAP-shIFN-R, CD11b-shIFN-R or their control virus (in 20 l volume) was given to the mice. Injection speed was maintained at 1 l/min to prevent leaking. Astrocyte and microglia treatment stimulation. To isolate CNS cells, spinal cords were mechanically dissociated through a 70 m cell strainer and washed with PBS. Washed cells were fractionated on a 60/30% Percoll gradient by centrifugation at 300g for 20 min. Infiltrating mononuclear cells were collected from the interface and washed with PBS for use. Intracellular staining and flow cytometry Cells isolated Indoximod (NLG-8189) from spleen or spinal cord were stimulated with PMA (50 ng/ml; Sigma-Aldrich), ionomycin (500 ng/ml; Sigma-Aldrich), and.