The cell line EL-4 (C57BL/6, H-2b, Thymoma) was maintained in

RPMI complete media (CM) supplemented with 10% heat-inactivated FBS, 50 U/mL of penicillin–streptomycin Everolimus molecular weight and 50 μg/mL gentamycin. The synthetic peptide corresponding to the CTL epitopes of chicken ovalbuman (SIINFEKL) was purchased from American Peptide (Sunnyvale, CA, USA), dissolved in dimethyl sulfoxide, DMSO (Sigma, St. Louis, MO, USA) and diluted in 1× PBS at a final concentration of 1 mg/mL for cell culture studies. The OVA protein was purchased from Sigma. α-GalCer was purchased from Diagnocine LLC (Hackensack, NJ, USA) and dissolved in DMSO (Sigma) at a concentration of 1 mg/mL. Mice were immunized by the intranasal or intravenous routes 1–2 times at 0 and 5 or 23 days with a

mixture of the OVA protein at 100 μg/mouse/dose and the synthetic glycolipid α-GalCer at 2 μg/mouse/dose. For intranasal immunizations, mice were anaesthetized by intraperitoneal (i.p.) injection of ketamine–xylaxine mixture, and 10 μl of the adjuvant–antigen mixture in 1× PBS was introduced into each nostril as reported earlier 7, 27. For intravenous immunizations, 200 μl of the adjuvant–antigen mixture in 1× PBS was injected into the tail vein of the mouse. At various time point post-immunization, mice were sacrificed and perfused and cell suspensions were prepared from the spleen, lung, liver, and lymph nodes by homogenization or enzymatic EPZ015666 mw dissociation using collagenase type IV (Sigma). Lymphocytes from liver were further isolated through a percoll (Sigma) gradient of 44 and 67%. The CTL responses in single-cell suspension from from spleens of immunized mice were assayed as described

previously 28. Briefly, spleen cells were re-stimulated for 5 days with the OVA peptide (SIINFEKL). These effector cells were tested for cytolytic activity against 51Cr-labeled syngeneic EL-4 target cells that were pre-incubated with either medium alone or OVA peptide. The percentage (%) of specific lysis was calculated using the following formula: % specific lysis=(experimental release−spontaneous release)/(maximum release−spontaneous release)×100, where the spontaneous release represents the radioactivity obtained when the target cells were incubated in culture medium without effectors and maximum release represents the radioactivity obtained when the target cells were lysed with 5% Triton X-100. Cells isolated from the lung and MdLN of immunized mice were subjected to ELISpot assay for enumerating the numbers of antigen-specific IFN-γ-producing cells as described earlier 29 using the reagent kit from BD Biosciences (San Jose, CA, USA). The spots, representing individual IFN-γ-producing cells as spot forming cells (SFC), on the membrane were enumerated by Zellnet Consulting, New York, NY using the KS-ELISPOT automatic system (Carl Zeisis, Thornwood, NY, USA).