primed T cells to mount a secondary response. Recent at- tempts at vaccination of mice with Leishmania major parasites that only survived for a short time revealed that the T cells that were generated with this vaccination strategy were par- tially primed T cells and were protective to an extent. How- ever, the most efficient protection against these pathogens requires the maintenance of large numbers of highly acti- vated Th1 effectors, and that resting memory T cells were not present in sufficient numbers to afford a high level of protection against rechallenge (49, 50). The findings regard- ing chronic pathogen infections all have a common theme; CD4 T cells that were generated in response to chronic pathogen infections had qualitative deficits in their func- tional capacities during the chronic phase of the infection. Our study revealed that chronic Ag stimulation by Ag-bear- ing APCs was too much of a good thing and may be the critical factor that is involved in inducing a functionally non- responsive state in Ag-specific T cells. These findings have important implications for vaccine design and may lead to a better understanding of how to combat persistent infections. MATERIALS AND METHODS Animals. AND (B10.Br) TCR Tg, AND TCR Tg GFP, Thy1.1 HNT (BALB/c ByJ) TCR Tg, OT-II (C57BL/6) TCR Tg, and BALB/c ByJ mice were used as sources of naive CD4 T cells at 4–6 wk of age, and were bred in the animal breeding facilities at Trudeau Institute. B10.Br, BALB/c ByJ, and C57BL/6 CD4 KO mice were used at 2–4 mo of age, and were bred in the animal facilities at Trudeau Institute or purchased from Jackson ImmunoRe- search Laboratories. Experimental procedures were approved by Trudeau In- stitute’s Institutional Animal Care and Use Committee. Preparation of APCs. All cells were cultured in cell culture medium (RPMI 1640 supplemented with penicillin [200 g/ml; Sigma-Aldrich], streptomycin [200 g/ml; Sigma-Aldrich], glutamine [4 mM; Sigma- Aldrich], 2-ME [50 M; Sigma-Aldrich], Hepes [10 mM; Sigma-Aldrich], and 8% FBS [Intergen]). T-depleted B cells were prepared by isolating lym- phocytes from spleens of intact mice in cell wash buffer [RPMI 1640 sup- plemented with Hepes and 1% FBS], and depleting T cells using antibody and complement as described previously (8). The T-depleted spleen cells were stimulated for 2–3 d with LPS and dextran sulfate (Sigma-Aldrich) as described previously (8). Bone marrow–derived DCs were prepared by iso- lating cells from bone marrow of B10.Br or BALB/c ByJ mice, and cultur- ing at 4 105 cells/ml with 20 ng/ml GM-CSF (Preprotech) and 20 ng/ml IL-4 for 6–8 d, with 25 ng/ml LPS (Sigma-Aldrich) added for the last 18– 24 h. The DCs were loaded with 5 M pigeon cytochrome c peptide frag- ment (PCCF) or HNT peptide. B cells were treated with 100 g/ml of mi- tomycin-c (Sigma-Aldrich) before use, whereas DCs that were used in vivo were not treated. Naive CD4 T cell isolation. The enrichment of naive CD4 T cells from pooled spleen and lymph node lymphocytes was performed as described previously (8). In brief, lymphocytes were passed through nylon wool, treated with MHC class II and CD8 depletion antibodies followed by com- plement and DNase I (Sigma-Aldrich) treatment, and discontinuous Percoll (Sigma-Aldrich) gradient separation. Cells at the 63%/80% Percoll interface were used for T cell assays. The purified cell populations were 85% CD4 cells, 90–95% of which had a naive phenotype (CD45RBhi, CD62Lhi, CD44low, CD25low). In vitro culture of CD4 T cells and memory generation. Naive TCR Tg CD4 T cells (3 105/ml) were labeled with CFSE (Molecular Probes Inc.) as previously described (8), and cultured with T-depleted splenic APC blasts (3 105/ml) pulsed with 5 M peptide Ag (AND TCR Tg T cells were stimulated with B10.Br APC PCCF peptide; HNT TCR Tg T cells were stimulated with BALB/c APC HNT peptide; and OT-II TCR Tg T cells were stimulated with C57BL/6 APC OVA pep- tide). All peptides were purchased from New England Peptide. Th1 effec- tors were generated by culture with IL-2, IL-12, and anti–IL-4, and Th2 ef- fectors were generated by culture with IL-2, IL-4, and anti-IFN as described previously (8). Acutely stimulated CD4 effectors (Th1 and Th2) were generated in vitro by adding peptide-pulsed APC at day 0, followed by the addition of fresh nonpulsed APCs every 24 h. Repeatedly stimulated CD4 effectors (Th1RS and Th2RS) were generated by adding peptide-pulsed APCs at day 0, followed by addition of fresh peptide-pulsed APCs every 24 h. Acutely stimulated polyclonal effectors were generated by culturing naive polyclonal BALB/c ByJ CD4 T cells for 48 h on plate-bound anti-CD3 (10 g/ml) with soluble anti-CD28 (5 g/ml), and then removing the T cells without washing to uncoated flasks for the last 48 h of culture (8). Repeat- edly stimulated polyclonal effectors were generated by culturing on the plate-bound anti-CD3 for the entire 96 h of culture. 10 106 4-d effector T cells were transferred i.v. into AT BM mice as previously described (9), and allowed to return to a resting state for 21 d. 25-d memory T cells were harvested from spleens and peripheral lymph nodes, enumerated, and assayed by ELISA for cytokine production. Analysis of cytokine production. T cells (5 105/ml) were restimu- lated for 24 h with 10 g/ml plate-bound anti-CD3, unless indicated oth- erwise, and culture supernatants were analyzed by ELISA (8). Alternatively, T cells (5 105/ml) were restimulated for 24 h with T-depleted, spleen- derived APC blasts (5 105/ml) that were pulsed with peptide Ag (10 g/ ml), followed by ELISA or ICCS as described previously (8). Flow cytometry. Lymphocytes were stained with saturating concentra- tions of fluorochrome-labeled mAbs as described previously (8). All surface- staining reagents were purchased from BD Biosciences. PE-labeled anti- V 3 (AND TCR Tg), anti-V 8.3 (HNT TCR Tg), anti-V 5 (OT-II TCR Tg), allophycocyanin or peridium chlorophyll protein–labeled anti- CD4 and peridium chlorophyll protein–labeled anti-Thy1.1 were used to identify donor CD4 T cells in all FACS analysis. Flow cytometry was per- formed using FACScalibur flow cytometers (BD Biosciences), and the data were analyzed with FlowJo software (Tree Star, Inc.). Restimulation of T cells with common -chain–binding cytokines. 4-d effector T cells were washed extensively, labeled with CFSE, and cul- tured at 2 105/ml with the indicated concentration of common -chain binding cytokines (Preprotech). Virus infections. A/PR/8/34 (PR8) strain of influenza A virus was grown in the allantoic fluid of 10-d-old embryonated chicken eggs, from an aliquot that originally was obtained from D. Morgan (The Scripps Research Institute, La Jolla, CA). Mice were inoculated intranasally during light iso- flurane anesthesia with 50 103 PFU/ml (2LD50) of virus in 100 l PBS. 5 106, 2.5 106, or 0.5 106 4-d effectors that were generated in vitro from Thy1.1 HNT TCR Tg CD4 T cells, or 200 l of PBS alone, were transferred i.v. into infected mice. Donor and host T cells were isolated for analysis 4 d following transfer from draining lymph nodes (parathymic and mediastinal), spleens, and perfused lungs as previously described (3). Analysis of B cell help. C57BL/6 CD4 KO mice were immunized by i.p. injection of 200 g 4-hydroxy-3-nitrophenyl acetyl (Sigma-Aldrich) conjugated OVA (NP-OVA) or PBS in 100 l alum (Sigma-Aldrich). 107, 106, or 105 4-d effectors that were generated in vitro from OT-II TCR Tg CD4 T cells, or 200 l of PBS alone, were transferred i.v. into immunized mice. Host B cells were isolated from spleens 16 d following transfer, and serum was harvested from blood. The total number of host NP-specific, germinal center phenotype (CD38lo/PNAhi) B cells were calculated by as- 1110 REPEATED STIMULATION LIMITS CD4 T CELL FUNCTION | Jelley-Gibbs et al.