Mice Lacking Expression of Secondary Lymphoid Organ Chemokine Have Defects in Lymphocyte Homing and Dendritic Cell Localization

Gunn, Michael D.; Kyuwa, Shigeru; Tam, Carmen; Kakiuchi, Terutaka; Matsuzawa, Akio; Williams, Lewis T.; Nakano, Hideki

Skin DC Migration and Staining. Skin explant culture, preparation of dermal and epidermal whole mounts, and staining of DCs were performed as described previously (31) . In brief, ears were rinsed with 70% ethanol and split with forceps into dorsal and ventral halves. Dorsal ear halves were floated directly on 2 ml of complete medium in 24-well plates for 3 d. Epidermal and dermal sheets were prepared by floating ear halves (fresh or cultured) or freshly prepared abdominal skin dermal side down on 0.5 M ammonium thiocyanate for 20 min at 37 Њ C. Epidermis was separated from dermis with fine forceps and stained immediately. Epidermal and dermal sheets were cut into 3 ϫ 3-mm sections, fixed in acetone for 30 min, and rehydrated in PBS. They were incubated in 1:20 biotinylated anti-I-Ad overnight at 4 Њ C, washed in PBS three times, and incubated with SA-FITC at 1:100 for 90 min at room temperature. After three washes, sheets were mounted on microscope slides and evaluated by UV microscopy. Nonadherent migratory cells were recovered from the bottom of tissue culture wells after 72 h by gentle rinsing. Aliquots were counted and cells were cytospun onto microscope slides for staining with 1:100 biotinylated anti-I-Ad using a Vectastain ABC kit (Vector Labs) and DAB and counterstained with hematoxylin. The proportion of I-A ϩ cells was determined by examining 10 fields/slide over 3 slides. Viral Infection. Mouse hepatitis virus, strain A59 (MHV-A59), was propagated and plaque assayed on DBT cells as described previously (32, 33) . A single pool of virus was divided into aliquots and stored at Ϫ 80 Њ C until use. Mice were infected by intraperitoneal injection of 0.2-2 ϫ 10 6 PFU of MHV-A59 in a 0.2 ml vol. Mortality was assessed at 14 d after infection. LD 50 were calculated by the PROBIT method.