Figure 6. HPV Ivag prime/boost immunization induces polyfunctional CD8⁺ T cells with cytotoxic activity.

Depo-Provera–treated mice were immunized with 5 × 10⁷ IU HPV16MM2 or HPV16 control Ivag, and 1 month later, mice were immunized with 5 × 10⁷ IU HPV45MM2 or HPV45 control, respectively. (A) Two weeks after the last immunization, the production of IFN-γ, TNF-α, and IL-2 by CD8⁺ T cells was measured after in vitro stimulation with M2_82–90 peptide in cervicovaginal, spleen, and ILN cell suspensions. The percentage of CD8⁺ T cells in each quadrant is indicated in each plot. (B) Relative proportion of single-, double-, and triple-cytokine-producing CD8⁺ T cells restimulated with M2_82–90 peptide after subtraction of background cytokine production of unstimulated CD8⁺ T cells. (C) In vivo cytotoxic activity was assessed 2 weeks after the last immunization. Target cells consisting of an equal mixture of two splenocyte populations labeled with a high and a low concentration of CFSE were pulsed with M2_82–90 peptide or remained unpulsed, respectively, were injected Ivag and i.v. Twenty-four hours later, cervicovaginal, spleen, and ILN cell suspensions were analyzed by flow cytometry. Representative histograms gated on CFSE-positive cells. Mean percentage ± SD of M2_82–90 specific lysis is indicated.