strain (K 318) containing the gB498–505 epitope. Mice containing CFSE-labeled gBT-I.1 CD8 T cells were in- fected cutaneously with the mutant viruses or the wild- type KOS strain and analyzed for proliferating cells. The absence of dividing cells after infection with the K 5C strain, but not the K 318 mutant, indicates that de novo synthesis of the viral peptides is required in order to stimu- late a response by CD8 T cells (Fig. 2). This data rules out activation of the T cells via antigen obtained from the injected virions. There Is a 6-h Lag between Infection and T Cell Activation in the PLNs. It has been shown that CD8 T cells require a 24-h lag between TCR stimulation and the first division, possibly to recruit the various cellular machinery required for division (16). To determine whether this is the case for gBT-I T cells, mice were immunized intravenously with 1 g gB498–505 peptide after the transfer of CFSE-labeled transgenic T cells. The use of peptide should provide im- mediate TCR engagement. As shown in Fig. 3, prolifera- tion was first observed in the spleen 24 h after immuniza- tion. This data agrees with the results of Ohen et al. using LCMV transgenic T cells and peptide stimulation (16). More importantly, given the observation that cutaneous in- fection results in appearance of the first cell division within PLNs at 30 h, we calculate that the HSV-1–specific T cells are most likely activated 6 h after virus inoculation. In Vivo Antigen Presentation Correlates with CD8 T Cell Activation. To directly demonstrate this early activation, transgenic CD8 T cells were analyzed for expression of the early activation marker CD69, which is expressed rap- idly after TCR engagement (18). Specific up-regulation of this marker was observed 6 h after infection and further in- creased after 8 h (Fig. 4 A). This matches the timing of events predicted from the CFSE proliferation experiments described above. The arrival of APCs presenting gB498–505, was deter- mined using a lacZ-inducible hybridoma cell line, HSV- 2.3.2E2 expressing the same receptor as the gBT-I.1 trans- genic mice (9). Suspensions of cells from the PLNs of in- fected mice were treated with collagenase to isolate DCs and other APCs, and graded numbers of these cells were incubated with the LacZ-inducible hybridoma cells. Pre- sentation of gB498–505 was first detected 4–6 h after cutane- ous infection (Fig. 4 B) and continued to increase over the next 48 h. The combined data shows that synthesis of gB, transport to the draining lymph nodes, and presentation occurs within hours of infection. The actual identity of the APCs involved remains unknown, although recent experiments using bone marrow chimeric mice has shown that these are of haemopoietic origin (data not shown). Therefore these are likely to be professional APCs probably of DC origin (1). It remains unclear whether presentation involves the migration of antigen-bearing APCs or the simple transfer of cell-independent antigen to the draining lymph nodes. Tracking studies have demonstrated the migration of DCs from the skin to lymph nodes as early as 30 min after con- tact sensitization (2). In addition, gB has been shown to be expressed within 2–3 h of infection (19–21). Taken to- gether, there is sufficient time for viral infection and pro- tein synthesis in the epithelium, followed by transport of antigen via APCs to the PLNs, consistent with the timing of events described above. Antigen Presentation and T Cell Activation Does Not Require the Presence of Virus in the Draining Lymph Nodes. Inocula- tion of mice with HSV-1 via the footpad involves a subcu- taneous injection that may drain to the PLNs via the lym- phatics. It was formally possible that direct infection of APCs in the lymph nodes may have accounted for the rapid antigen presentation and swift T cell activation after infection. We attempted to amplify viral DNA from the PLNs by PCR using HSV-1–specific oligonucleotides (13). Specific bands were amplified from the PLNs, indi- cating the presence of significant amounts of viral DNA Figure 2. De novo synthesis of viral peptides is required to elicit a gB-specific T cell re- sponse. Mice receiving CFSE- labeled gBT-I.1 CD8 T cells were infected with wild-type HSV-1 KOS or the gB mutant strains K 318 or K 5C. 42 h af- ter infection, CD8 T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms rep- resent 5–10,000 live events. 654 Early Antigen Presentation and Activation of Anti-HSV CTL Figure 3. Naive T cells require a minimum 24-h lag between activa- tion and commencement of prolifer- ation. Mice receiving CFSE-labeled gBT-I.1 cells were immunized intra- venously with gB498–505 peptide and the transgenic CD8 T cells in the spleen analyzed 18, 24, and 30 h later for the presence of dividing cells. Histograms represent 5–10,000 live events.