pool detectable at the peak of the response to HSV-1 infec- tion, gBT-I.1 CD8 T cells were labeled with CFSE (14) and transferred into normal mice before footpad infection with HSV-1. The first appearance of a dividing cohort of CD8 T cells appeared in the PLNs at 30 h after infec- tion (Fig. 1 A). Progressive analysis indicated that these cells then proceeded to double consistently every 5–6 h thereaf- ter, having undergone four divisions after 48 h and 7 di- visions by 72 h. Initially, very few cells were present in the dividing pool, as indicated by the relatively small peak of divided cells at 30 h. However, as the response proceeded the number of cells that were recruited into the dividing pool continued to increase, thereby reducing the size of the undivided population. The relatively small pool of dividing cells observed early in the response may reflect either the small number of gB- specific precursor cells present within the PLNs at the time of infection, or a limited level of initial antigen presentation in the PLNs, or both. Due to the limited size of the PLNs, the number of gB-specific CTL precursors present here in a naive mouse would be very small, yet these cells would be stimulated first due to their proximity to the site of in- fection. A 15–20-fold increase in total cellularity within the PLNs is observed over the first 24 h after infection (Fig. 1 B), most of which is due to an influx of resting lympho- cytes (3). The multiple peaks of proliferating cells indicate that the time for recruitment into the dividing pool varies as more naive T cells enter the PLNs, yet once activated all cells divide at a regular rate (15). While we show that CD8 T cells first divide at 30 h af- ter infection, significant CTL activity is not detectable in the PLNs in normal mice until 2–3 d after infection with HSV-1 (5), even though CTLs are thought to be armed af- ter one cell division (16). One possible reason for this delay is the low precursor frequency in C57BL/6 mice which prevents detection earlier in the response when cell division begins. To overcome this limitation we used an in vivo CTL assay method (17) in combination with the adoptive transfer of naive gBT-I.1 CD8 T cells in numbers (106) that would greatly exceed the natural precursor frequency of host gB-specific cells. This enabled the direct detection of significant CTL activity in the PLNs as early as 30 h after infection, which continued to increase, more than doubling over the next 18 h (Fig. 1 C). This data corresponds closely to the increase in proliferating cells over this same period (Fig. 1 A), and provides in vivo evidence suggesting that the first round of division and the development of effector functions by CD8 T cells occur simultaneously. De Novo Antigen Synthesis Is Required for Initiation of the gB-specific Response. Given the very rapid activation of specific T cells in the PLNs, it was possible that the anti- gen was derived from the injected virions rather than syn- thesized de novo upon infection. To exclude this, we made use of mutant strains of the HSV-1 KOS virus (K 318 and K 5C) that express defective forms of the gB protein (10). While both mutants are packaged with a cell line that supplies the wild-type gB protein, they express distinct truncated forms of this protein, with only one Figure 1. Concurrent in vivo proliferation and CTL activity by gB-specific CD8 T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 106 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSEhi) while unpulsed control targets were labeled with a low concentration of CFSE (CFSElo). 4 h after target cell transfer, mice were killed and PLN cells an- alyzed for relative elimination of the CFSEhi versus CFSElo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD. 653 Mueller et al. Brief Definitive Report