within 2 h of infection (Fig. 5 A). However, the intensity of bands amplified 6 and 24 h after infection demonstrate a rapid decrease in the amount of HSV-1 in the lymph nodes, and by 48 h we were unable to detect viral DNA within the PLNs. Moreover, this lack of viral DNA at 48 h Figure 5. Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled ax- illary and inguinal lymph nodes, respectively. 100 ng of DNA was ampli- fied by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control ( ). A no DNA control was included to rule out contamination ( ). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after in- fection. Histograms represent 5–10,000 live events. Figure 4. Activation of CD8 T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 CFSE cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ-inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted mi- croscopically. Numbers presented represent the total number of lacZ cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD (n 8–12). did not correlate with detectable antigen presentation, which continued to increase over this time period, suggest- ing that direct APC infection was not necessary for gB- specific T cell activation. To verify that the virus did not need to be present in lymph nodes to initiate a response we infected mice via scarification of the flank (11). Unlike footpad infection, scarification removes only the top layer of skin thereby confining the infection to the upper epithelial layers and restricting direct viral entry into the dermal lymphatics (22). Therefore, only antigen transported via APCs should reach the draining lymph nodes. As predicted, mice in- fected in the flank displayed no detectable viral DNA in the draining axillary and inguinal lymph nodes at any time over the 48 h period after infection (Fig. 5 B). Nonethe- less, flank inoculation resulted in effective T cell activa- tion and the same kinetics of proliferation as found using footpad injection. This was shown by comparing CD8 T cell proliferation 48 h after cutaneous footpad infection or flank scarification. The number of CFSE peaks in the draining lymph nodes indicated that the gB-specific T cells were activated at the same time after both routes of cutaneous infection (Fig. 5 C). Thus, despite the discrep- ancy in the presence of viral DNA between the two pe- ripheral infection routes, both showed the same relatively short lag between infection and activation of the gB-spe- cific T cells. Overall, our results suggest that the presence of viral DNA in the draining PLNs is largely immunologically ir- relevant, as activation of the gB-specific T cells appears to occur independent of detectable viral DNA. While HSV-1 can infect DCs, this has been associated with downmodula- tion of costimulatory molecule expression as well as severe reduction in the T cell–stimulatory capacity of these cells (23). Therefore direct DC presentation of HSV-1 antigens may prove inefficient at T cell priming. Cross-priming, which involves the transfer of antigen from infected cells to professional APCs (24), represents an effective means of 655 Mueller et al. Brief Definitive Report