Early removal of the DLNs alters memory CD8⁺ T cell phenotype but not secondary responses. (A,C) B6 mice were immunized with 10⁷ pfu of rHuAd5-SIINFEKL-Luc intramuscularly and their DLNs were surgically removed 7 or 20 days later. Spleens were harvested from these mice approximately 100 days after surgery and SIINFEKL-specific CD8⁺ T cells were identified using tetramer staining. Expression of various phenotypic markers on tetramer-positive CD8⁺ T cells was examined after lymphadenectomy shown at day 7 (A-B) or day 20 (C). (B) Frequency of T_EFF (CD127⁻CD62L⁻), T_EM (CD127⁺CD62L⁻), and T_CM (CD127⁺CD62L⁺) within the tetramer-positive population after LN removal at day 7. Data points represent a total (± SEM) of 8 to12 mice. (D-E) B6 mice were immunized with 10⁷ pfu of rHuAd5-SIINFEKL-Luc intramuscularly and their DLNs were surgically removed 7 days later. Mice were challenged intraperitoneally with 10⁷ pfu of rVV-ESOVA approximately 100 days after surgery. (D) Total number of SIINFEKL-specific IFN-γ⁺ CD8⁺ T cells in the spleen before and 7 days after rVV challenge. Data points represent a total (± SEM) of 5 mice. (E) Virus titers in the ovaries 7 days after rVV challenge. Each point represents a single mouse. *P < .05, **P < .01.