Reduced T-cell and antibody responses after subcutaneous immunization in the absence of GM-CSF and FL. (A) T-cell proliferation assay. Naive OT-II CD4⁺CD45.1⁺ T cells were sorted and labeled with CSFE before transfer into CD45.2⁺ WT, GM-CSF^−/−, FL^−/−, and DKO mice (6 × 10⁴ cells/recipient). The next day, mice were immunized subcutaneously in the right flank with 2 μg OVA protein and 4 μg MPL. Draining LNs (right axillary and inguinal) and nondraining LNs (shown as representative WT control) were analyzed 3 days later. The proliferation of the transferred OT-II T cells was analyzed by gating on CD45.1⁺CD3⁺CD4⁺ cells. Histograms show the percentage of OT-II T cells having diluted the CFSE label. Results are representative of 2 independent experiments with a total of 3 to 7 mice/group. (B) Production of OVA-specific IgG antibodies. WT, GM-CSF^−/−, FL^−/−, and DKO mice were immunized in the footpads with 2 μg whole OVA protein and 4 μg MPL, and serum was collected at the indicated time points. A second immunization was given at day 21. OVA-specific IgG antibodies were measured by ELISA (n = 7-8 mice/group). ***P < .001.