Figure 6. Repeated Ag stimulation generates effectors with a more activated phenotype. CD62L and CD69 surface expression on 4 d (A) Th1 and (B) Th2 effectors (acute stimulation filled histograms, RS solid lines) were compared with naive (dotted line) AND TCR Tg CD4 T cells. (C) Th1 and (D) Th2 polarized T cells were analyzed every 24 h for surface CD62L or CD69 expression versus CFSE loss. (E) Th1 and (F) Th2 polarized T tors [OT-II TCR Tg, specific for a peptide of ovalbumin (OVA)] into CD4 KO hosts (31). Analysis of host 4-hydroxy- 3-nitrophenyl acetate (NP)-binding B cells 16 d following immunization revealed that almost three times as many NP- specific B cells differentiated to a germinal center phenotype (CD38loPNAhi) in the presence of 107 Th2 effectors that were generated with acute stimulation, compared with Th2 effectors that were generated with RS (Fig. 8 A). Fig. 8 B indicates that the transfer of 106 or 107 Th2 effectors gener- ated a significant expansion of NP-specific, germinal center phenotype B cells, whereas Th2RS were unable to generate an expanded population of NP-specific, germinal center phenotype B cells. Additionally, significantly more NP-spe- cific IgG1 was detected in the serum of mice when 107 Th2 effectors were transferred into NP-OVA immunized hosts, compared with the transfer of 107 Th2RS effectors (Fig. 8 C). We also assayed for the production of NP-specific IgM, IgG2a, and IgG2b and found no significant production of these antibodies (unpublished data). Effectors generated with repeated stimulation in vivo have profound functional deficiencies To reproduce conditions of RS in vivo we used an adoptive transfer model that involves the cotransfer of Ag-pulsed DCs with naive Ag-specific CD4 T cells into intact hosts. The cotransfer of Ag-pulsed DCs with Ag-specific CD4 T cells was necessary to optimize the primary stimulation of naive CD4 T cells in vivo, and enabled us to visualize the trans- ferred T cells in sufficient numbers to perform phenotypic and functional analysis. Previous studies suggested that Ag- bearing APCs would be deleted rapidly by CTLs (13, 32– 1106 cells were analyzed every 24 h for surface CD43, CD162, CXCR3, or CD49d expression versus CFSE loss. Red day 1, blue day 2, green day 3, and orange day 4. Numbers represent percentage of day 4 T cells in the upper left quadrant. Data are representative of three to five indepen- dent experiments. 34), and, in fact, Ag-pulsed DCs were undetectable within 2 d of transfer (R.A. Kemp, unpublished data). To over- come this, we cotransferred Ag-pulsed DCs with naive T cells at day 0, followed by transfer of Ag-pulsed DCs on days 1, 2, 3, and 4 of a 5-d in vivo immune response to model RS. We modeled acute stimulation by cotransferring Ag- pulsed DCs with naive T cells at day 0, followed by transfer of nonpulsed DCs on days 1, 2, 3, and 4. On day 5, following naive T cell transfer, we recovered and analyzed donor T cells from peripheral lymph nodes, spleens, lungs, and livers of host mice. Effectors that were generated in vivo with acute stimulation migrated primarily to the peripheral lymph nodes and spleens; few cells mi- grated to tertiary sites (e.g., lungs, liver; Fig. 9 A). Con- versely, we found increased migration of effectors that were generated in vivo with RS to the spleen, lung, and liver (Fig. 9 A). This was not unexpected because phenotypic analysis of the in vitro generated effectors revealed that repeatedly stimulated effectors had a largely CD62Llo phenotype, which has been shown to promote T cell migration out of lymph nodes (22). In fact, effectors that were generated in vivo with acute stimulation had down-regulated CD62L to a lower extent when compared with the highly activated phe- notype of repeatedly stimulated Th1 effectors (Fig. 9 B). This was true particularly for those effectors that had mi- grated to the lymph nodes and spleen, because the effectors that migrated to the lungs and liver exhibited a more highly activated phenotype, regardless of the conditions under which they were stimulated (Fig. 9 B). Effectors that were generated in vivo with acute stimula- tion resulted in an expanded population of Th1-like effec- REPEATED STIMULATION LIMITS CD4 T CELL FUNCTION | Jelley-Gibbs et al.