Antigen dose–dependent regulation of polyclonal CD4⁺ and CD8⁺ T cell responses. (A) Mice were immunized in the footpad with indicated doses of recombinant GP along with CpG; 8 d later, spleens were examined for presence of H2-Db GP.Tetramer⁺ (GP.TET) CD8⁺ T cells and I-Ab GP.TET⁺ CD4⁺ T cells by flow cytometry. Tetramer⁺ cells were also examined for Vb2-14 staining (Vb8.1/2 demonstrates representative staining). (B) Enumeration of the total number of I-Ab GP.TET⁺ CD4⁺ and H2-Db GP.TET⁺ CD8⁺ T cells after immunization with the indicated doses of GP and CpG. Error bars are mean ± range. (C) Analysis of the Vb repertoire of the tetramer⁺ T cells. Bars indicate individual animals. (D) Enumeration of the total number of Vb families contributing to the CD4⁺ (I-Ab TET⁺) and CD8⁺ (H2-Db TET⁺) T cell responses to GP. Error bars are mean ± range. Paired t test was performed comparing the familial representation within the CD4⁺ versus CD8⁺ T cell response within the same mice. GP immunization data represent two independent experiments. (E) Mice were immunized with low (0.25 µg) or high (25 µg) doses of OVA protein along with CpG, and 7 d later spleens were examined for presence of H2-Kb OVA.TET⁺ CD8⁺ T cells. (F) Enumeration of the mean number of Vb families contributing to the total OVA-specific CD8⁺ T cell response. Mice lacking detectable responses (0.25 µg dose group) were excluded from analysis. Error bars are mean ± SD; n = 5. OVA immunization data represent at least three independent experiments. (G) Correlation analysis comparing the total number of Vb families participating in a given response and the total number of antigen-specific CD8⁺ T cells within that response. Responses against GP and OVA antigens were combined for analysis. For analyses of relative familial composition (D, F, and G), control nonimmunized samples were first used to determine the maximum potential contribution of nonspecific events in the Tetramer⁺ gates, and this value was used as a cutoff to enumerate the number of true responder families. Numbers in flow plots represent frequencies. **, P ≤ 0.01.