Figure 2. PAP-specific regulatory cells exist prior to immunization, and are CD8+CTLA-4+.

Panel A. PBMC samples from multiple prostate cancer patients were tested for pre-existing PAP-specific regulatory responses by tvDTH. PBMC were stimulated with the described antigens and antibody treatments, and tvDTH reactivity was measured. Data shown are representative of at least two independent experiments. Differences in mean tvDTH among different antigen/antibody treatment groups (indicated by solid lines) were compared using a student’s t-test, with * indicating p≤ <0.05. Panel B. PAP-specific regulatory responses were evaluated in tvDTH studies using samples from patients identified in Panel A. These studies were conducted using whole PBMC (left section), PBMC samples that had been depleted of CD8+ T-cells (center section), or PBMC samples that had been depleted of CD8+ T-cells and were subsequently supplemented with autologous CD8+ T-cells (right section). Data shown are representative of at least two independent experiments. Statistical differences between group mean DTH values (indicated by solid lines) were analyzed using a student’s t-test, with * indicating p≤0.05. Panel C. PBMC from patients with CD8-dependent regulatory responses identified in Panel B were evaluated for MHC dependency in tvDTH bystander suppression assays using antibodies blocking MHC class I or MHC class II. Statistical differences between group mean DTH values (indicated by solid lines) were analyzed using a student’s t-test, with * indicating p≤0.05. Panel D. PBMC samples from patient ID022 were evaluated for tetanus bystander suppression by tvDTH. PBMC samples were depleted of CD8+ T-cells, and tested alone (left section) or supplemented with magnetic bead-sorted CD8+CTLA-4+ (center section) or CD8+CTLA-4− T-cells (right section) along with antigens described.