1 in 105–106 (6). Taken together, this suggests that a mas- sive expansion of the antigen-specific population must oc- cur relatively soon after infection in order to achieve the production of such a large CTL population at the height of the response. Recently, efforts have been made to quantitate specific antigen-presenting cells (7), and to follow the development of effector functions by CD8 T cells (8) after infection. Despite this, the kinetics of initial antigen presentation in combination with the activation and generation of a func- tional CTL population have not yet been fully elucidated. To overcome the limitation of low naive CTL precursor frequencies and effectively analyze the events that occur immediately after cutaneous infection of C57BL/6 mice with HSV-1, we have made use of an MHC class I–restricted TCR transgenic mouse line specific for the gB498–505 deter- minant (9). Here we describe the rapid activation of the transgenic CD8 T cells coinciding with the earliest ap- pearance of specific APCs in the draining PLNs within hours of cutaneous infection with HSV-1. Materials and Methods Mice, Viruses, and Peptide. C57BL/6 and the gB-specific, TCR transgenic MHC class I–restricted (gBT-I.1) mice were obtained from the Department of Microbiology and Immunology, Uni- versity of Melbourne, animal house and kept in specific pathogen-free conditions. The gBT-I.1 transgenic mice express a TCR from a CTL clone (HSV-2.3) that recognizes the HSV-1 gB498–505 determinant complexed with H-2Kb and is described in detail elsewhere (9). The KOS strain of HSV-1 was propagated and titered using VERO cells grown in Minimal Essential Media plus 10% FCS (MEM-10). The mutant K 318 and K 5C viruses were propagated and titered on D6•VERO cells grown in MEM- 10 (10). The gB498–505 peptide with the sequence SSIEFARL was obtained from Auspep. Monoclonal Antibodies and 5-Carboxyfluorescein Diacetate Succini- midyl Ester Labeling. Cells were labeled with 5-carboxyfluores- cein diacetate succinimidyl ester (CFSE; Molecular Probes) as described previously (5). Single cell suspensions prepared from lymph nodes or spleen were stained with anti-CD8 -allophy- cocyanin (APC; 53–6.7) and anti-CD69-PE (H1.2F3) ob- tained from BD PharMingen. 2 106 cells were stained for 20 min on ice and washed with PBS-azide containing 1% BSA. Dead cells were excluded using propidium iodide and 5–10,000 live CFSE events collected on a Becton Dickinson FACSort™. Adoptive Transfers, Virus Infections, and Peptide Immunizations. Lymph node cells obtained from gBT-I.1 mice containing 106 CD8 T cells as determined by flow cytometry were either la- beled with CFSE or left untreated and transferred into C57BL/6 mice by intravenous injection. 24 h after transfer, mice were in- jected in each hind footpad with 4 105 PFU HSV-1 KOS, K 318, or K 5C in PBS, or infected with 106 pfu HSV-1 by flank scarification (11). For peptide injection, mice were immu- nized intravenously with 1 g of gB498–505 peptide in PBS. The draining PLN (footpad infection), pooled axillary and inguinal lymph nodes (flank infection), or spleen (intravenous peptide in- jection) were removed and single cell suspensions produced and counted by trypan blue exclusion before antibody staining for FACS®. In Vivo CTL Assay. Splenocyte target cell suspensions from C57BL/6 were evenly split into two populations. One was pulsed with 10 6 M gB498–505 peptide for 45 min at 37 C and then labeled with a high concentration (2.5 M) of CFSE (CF- SEhi population). The other was incubated for 45 min at 37 C without peptide and labeled with a low concentration (0.25 M) of CFSE (CFSElo population). An equal number of cells from each population (107) were mixed together and adoptively trans- ferred into HSV-1–infected mice which were then killed 4 h later. Cell suspensions were analyzed by FACS® and each popula- tion distinguished by their different fluorescent intensities. Per- cent specific lysis was determined by loss of the peptide-pulsed CFSEhi population compared with control CFSElo population us- ing the formula described previously (5). LacZ-inducible Hybridomas, APC Assays, and X-gal Assays. The CTL clone HSV-2.3 was fused with the BWZ.36 cell line containing the NFAT-lacZ construct (12) to produce the T cell hybridoma HSV-2.3.2E2. For detection of APCs, PLNs were re- moved from mice infected with HSV-1 in the footpad at various times and separately teased into tubes containing 0.5 ml collage- nase/DNase solution (1 mg/ml collagenase type II [Worthington Biochemicals] and 0.1% grade II bovine pancreatic DNase I [Boehringer] in Dulbeccos modified eagles medium plus 10% FCS). The suspension was then pipetted for 20 min and 50 l 0.1 M EDTA added before a further 5 min of pipetting. Twofold serial dilutions of the cells were made in flat-bottom, 96-well plates starting at 106 cells/well. Hybridoma cells (105) were added to each well before overnight culture. Background lacZ expression was determined using hybridoma cells alone or with EL4 cells minus peptide, while positive controls consisted of peptide or peptide-pulsed EL4 cells. X-Gal assays were performed on the cultures to identify the responding hybridomas as described pre- viously (7). Cultures were examined microscopically for the pres- ence of blue cells after 8–12 h incubation at 37 C. PCR Detection of HSV DNA. The draining PLNs from HSV-1 footpad infected mice were removed at various times after in- fection and placed into DNAsol™ (Molecular Research Center) supplemented with proteinase K (50 g) at room temperature overnight. A footpad from a mouse primed 24 h earlier was also removed and digested as above to act as a positive control. Ge- nomic DNA was isolated as described by the manufacturer’s in- structions. The HSV-1 DNA was amplified by PCR by using 100 ng of genomic DNA, 50 pmol of primers, 0.2 mM deoxynucleo- side triphosphate, and 1.5 U of Taq polymerase (GIBCO BRL). The primers used for PCR amplification were HSV-1a (5 - CCCTGTCTCGCGCGACGGAC-3 ) and HSV-1b (5 - TCACCGACCCATACGCGTAA-3 ) (13). Amplification in- volved 35 cycles of 93 C for 30 s, 55 C for 1 min, and 72 C for 1 min followed by one cycle of 93 C 30 s, 55 C for 1 min, and 72 C for 7 min with a DNA Thermal Cycler (PerkinElmer). For each specimen, 100 ng of genomic DNA was evaluated for ampli- fication competence by 25 PCR cycles using insulin primers (5 - CGAGCTCGAGCCTGCCTATCTTTCAGGTC-3 and 5 - CGGGATCCTAGTTGCAGTAGTTCTCCAG-3 ). A 20- l aliquot of each PCR product was analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide. Results and Discussion Rapid Recruitment of gB-specific T Cells into the Dividing Pool and Concurrent Differentiation into Effector Cells. To examine the early events that give rise to the large CTL 652 Early Antigen Presentation and Activation of Anti-HSV CTL