Figure 4. Repeated Ag stimulation impairs polyclonal CD4 T cell responses. Naive BALB/c polyclonal CD4 T cells that were cultured with acute or repeated plate-bound anti-CD3 stimulation under (A) Th1 or (B) Th2 polarizing conditions were enumerated. Four-d (C) Th1 and (D) Th2 effectors were restimulated for 24 h with 10 g/ml plate-bound anti-CD3 and supernatants were analyzed by ELISA. Data (A and B) are means of triplicate cultures SD. Data (C and D) are one representative of triplicate cultures. Data are representative of two independent experiments. Th2 polarized effectors. Cell recovery in 4-d Th1 and Th2 cultures that were exposed to RS was reduced by at least 50%, compared with acute stimulation (Fig. 4, A and B, re- spectively). We next examined the effects of acute versus RS on the cytokine-producing potential of Th1 and Th2 effec- tors. As observed with the TCR Tg CD4 effectors, repeat- edly stimulated polyclonal effectors produced significantly reduced amounts of IFN (Th1) and IL-4 (Th2) when com- pared with effectors that were generated with acute stimula- tion (Fig. 4, C and D), although they maintained their re- spective polarized phenotypes. Repeated Ag stimulation does not result in cytokine unresponsiveness Strong TCR stimulation or Ag persistence can induce a state of permanent unresponsiveness, or anergy (1, 19–21). This hypo-responsive state often is defined as the maintenance of T cells with a reduced ability to proliferate to IL-2 and/or produce effector cytokines. To explore further the func- tional potential of acutely versus repeatedly stimulated effec- tors, we determined whether effectors that were generated with RS were able to proliferate in response to IL-2 or other cytokines that stimulate T cells through the common -chain (19). Therefore, we CFSE-labeled 4-d Th1, Th1RS, Th2, and Th2RS effectors and recultured them in fresh T cell medium using optimal concentrations of common -chain binding cytokines. The acutely and repeatedly stim- ulated effectors divided in response to IL-2, IL-4, IL-7, and 1104 IL-15 (Fig. 5 A). Interestingly, the repeatedly stimulated ef- fectors expanded when recultured with IL-2 and IL-7, whereas the acutely stimulated effectors simply were main- tained during this reculture period (Fig. 5 B). The 4-d primary effectors that were generated with RS had the potential to have down-regulated TCR expression as a result of excessive TCR stimulation during the primary culture. Lower levels of TCR expression could have a dra- matic impact on the ability to restimulate the effectors for cytokine production. Despite the fact that 4-d Th2 effectors that were stimulated repeatedly (Fig. 5 C-Th2RS) had a slight down-regulation of their TCR (V 11 shown, V 3- unpublished observation), these were the effectors that were most capable of expanding in response to an excess of TCR and IL-2 stimulation (Fig. 5 D). Repeatedly stimulated effectors have unique phenotypic characteristics CD69 is one of the most prominently cited markers of re- cent Ag stimulation, and CD62L is one of the only mole- cules that has been clearly established as playing an important role in lymphoid homing and T cell trafficking in vivo; therefore, we focused on the expression of these two mole- cules on acutely and repeatedly stimulated effectors. Under Th1 and Th2 polarizing conditions, RS resulted in a more pronounced elevated level of expression of CD69 by 4-d ef- fectors (Fig. 6, A and B). This indicated that RS is capable of maintaining a high level of TCR signaling throughout the 4-d culture period. Additionally, repeatedly stimulated effec- tors exhibited more extensive down-modulation of CD62L under Th1 and Th2 polarizing conditions when compared with effectors that were generated with acute stimulation. This pattern of expression indicated that repeatedly stimu- lated effectors underwent higher levels of activation and dif- ferentiation compared with T cells that were generated with acute stimulation. Because expression of these markers has been shown to be transient, a static view of 4 d-effectors did not allow us to determine if there were differences in the kinetics of expres- sion of these markers during effector generation. Therefore, we labeled naive CD4 T cells with CSFE and analyzed the T cells every 24 h during effector generation. These data are presented as flow cytometry dot plot overlays of days 1 (red), 2 (blue), 3 (green), and 4 (orange). Th1 (Fig. 6 C) and Th2 (Fig. 6 D) effectors exhibited a progressive differentiation to a CD62Llo phenotype, which is more dramatic with RS. Tran- sient expression of CD69 was observed when effectors were generated with acute stimulation; up-regulated expression lasted only for the first 48 h of culture, whereas CD69 ex- pression was maintained throughout the entire 4 d of effector generation when stimulated with RS (Fig. 6, C and D). We also analyzed effectors that were stimulated acutely and re- peatedly for expression of a variety of other activation mark- ers (CD25, and CD44, unpublished data and references 3, 22); adhesion markers (CD43, CD49d, and CD162; refer- ences 3, 22–24); and chemokine receptors CCR5 (unpub- REPEATED STIMULATION LIMITS CD4 T CELL FUNCTION | Jelley-Gibbs et al.