Lymphocytes were isolated from nasal-associated lymphoid tissues (NALT), nasal passages (NPs), head and neck lymph nodes (HNLNs), submaxillary glands (SMGs), spleens, small intestinal lamina propria (iLP), Peyer’s patches (PPs), lumbar lymph Selleck GDC-0199 nodes (LLNs), sciatic lymph nodes (SLNs), and popliteal lymph nodes (PopLNs). HNLN, splenic, PP, LLN, SLN, and PopLN mononuclear cells were isolated by conventional methods using Dounce homogenization [26] and [27]. To isolate the mononuclear cells from NALT, NPs, SMGs, and iLP, the tissues were minced and digested using 300 units/ml of Clostridium histolyticum

Type IV collagenase (Worthington, Freehold, NJ) for 30 min at 37 °C in spinner flasks [26]. After incubation, the digestion mixtures were passed through Nitex mesh (FairviewFabrics, Hercules, CA) to remove undigested tissues. Mononuclear cells were separated by Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation with cells interfacing between 40% and 60% Percoll. Greater than 95% viability was obtained for all lymphocytes isolated from

each tissue, as determined by trypan blue exclusion. On wk 14, sets of studies were terminated to collect NALT, NP, HNLN, SMG, splenic, iLP, PP, LLN, and PopLN mononuclear cells from the immunized mice. NALT, NP, HNLN, SMG, splenic, iLP, and PP mononuclear cells were used from i.n.-immunized mice, and NP, HNLN, splenic, iLP, LLN, and PopLN mononuclear cells were used from i.m.-immunized mice. Ag-specific Ab-forming cell (AFC) responses by the ELISPOT method were detected, using mixed Selleck PLX4032 cellulose ester membrane-bottom microtiter plates (MultiScreen-HA; Millipore, Bedford, MA) by coating with 5 μg/ml F1- or V-Ag in sterile PBS, as previously described [27]. For total IgA or IgG AFC responses, wells were coated with 5 μg/ml goat anti-mouse IgA or IgG Abs (Southern Biotechnology Associates) in sterile PBS. On wk 7 or 14, groups of i.n.- or i.m.-immunized mice, respectively, were evaluated for cytokine responses to F1- and V-Ags. I.m.-immunized mice were boosted nasally with F1-Ag protein at 8 and 9 wks and with both DNA and nasally dosed with F1-Ag protein at 12 wks. From i.n.-immunized mice, HNLN,

splenic, and PP mononuclear cells were obtained, and HNLN, splenic, and peripheral lymph nodes (PLNs), containing second LLN, SLN, and PopLN mononuclear cells, were obtained from i.m.-immunized mice. Total mononuclear cells from each lymph tissue were resuspended in CM. Mononuclear cells were restimulated with 10 μg of recombinant F1-Ag, V-Ag, or with media as control in the presence of 10 U/ml human IL-2 (PeproTech) for 2 days at 37 °C in a humidified 5% CO2 incubator. Cells were washed and resuspended in CM, and then these stimulated lymphocytes were evaluated by IFN-γ-, IL-4-, IL-5-, IL-10-, and IL-13-specific ELISPOT assays, as described previously [24], [25] and [27]. To determine cytokine responses to F1- and V-Ags, on wk 7 or 14, groups of immunized i.n. or i.m. mice were used, respectively.