Naïve female C57BL/6 mice were primed (Day 0) and boosted (Day 14) with DCs endogenously expressing male antigens or loaded with male antigens using various methods as outlined in materials and methods. (a) Representative contour plots showing the frequency of Uty tetramer-binding T cells in the spleens of immunized animals on day 28. (b) Antigen loading of bone marrow-derived DCs results in expansion of Uty tetramer-binding cells. N=7/group. **-p<0.05, ***p<0.01 compared to apoptotic cell-loaded DCs. (c) The number of splenocytes producing interferon-γ in response to peptide-specific stimulation is significantly increased following immunization with apoptotic cell-loaded DCs when compared to all other methods. Uty represents the dominant CD8 HY epitope, Smcy the subdominant CD8 epitope and Dby the dominant CD4 epitope.

(a) Purified sca1+/lin− cells expand 50-fold when cultured with Flt-3 ligand (25 ng/mL), IL3 (20 ng/mL) and stem cell factor (100 ng/mL), GM-CSF (1000U/mL) and IL-4 (1000 U/mL). This is a 5-fold increase in yield over cells cultured in GM-CSF and IL-4 alone (dotted line). (b) Analysis surface phenotype on Days 1, 5, and 8 (post-activation) of culture indicates that these conditions expand a population of stem cells without a committed lineage that later differentiate into dendritic cells expressing high levels of CD11c, CD40, CD80 and CD86 compared to isotype controls. DCs that are activated (solid) on Day 7 with aCD40 monoclonal antibody (20 ug/mL) and CpG ODN 1555 (5 ug/mL) show an upregulation of CD40, CD80 and CD86 (solid histogram) compared to nonactivated DCs (- -) and isotype controls (—).(c) DCs on Day 7 retain an immature phenotype, as indicated by their ability to endocytose FITC-conjugated albumin at 37° (solid histogram), a phenomenon that disappears at 0° (open histogram). Following activation, DCs lose the ability to take up FITC protein at 37. (d) Morphology of cultured DCs on day 5(pre-activation) and day 8 (post-activation) showing the acquisition of dendrites. (e) Activated DCs (solid histogram) produce significantly higher levels of intracellular IL12 than nonactivated DCs (light grey line). Isotype control staining of permeabilized cells is represented by the black line.

(a) Tumor-free survival in C57BL/6 female mice given a DC vaccine intraperitoneally on days 0 and 14 followed by subcutaneous challenge with 3 × 10⁶ HY-expressing MB49 cells on day 28. All DC loading methods were statistically superior to female DCs (p<0.02). Immunization with male DCs resulted in protection of 80% percent of the mice. However, loading DCs with apoptotic cells resulted in statistically improved protection when compared to all other methods (p<0.001) except male DC vaccination and 100% survival. (b) With higher dose tumor challenge (5 × 10⁶ HY-expressing MB49 cells on day 28), immunization with DCs loaded with apoptotic cells still resulted in complete protection whereas 100% tumor-induced lethality occurred in all other vaccinated groups (p<0.002).