repeated TCR stimulation (RS) as that which results from the addition of Ag-pulsed APCs on the day of culture initia- tion and every 24 h for the duration of the 4 d culture. Here, we further investigate the consequences of acute versus re- peated exposure to Ag-pulsed APCs on the generation and function of CD4 effector and memory T cells. The results of this study reveal that highly activated, and well-polarized effectors can be generated under conditions of RS. However, RS leads to the generation of effectors that produce lower levels of cytokines in response to restimula- tion than acutely stimulated effectors. Moreover, repeatedly stimulated Th1 effectors fail to provide protection in mice that were lethally challenged with influenza virus. Addition- ally, Th2 polarized effectors that were generated with RS fail to provide cognate help to B cells. We also have investigated the effects of acute and RS in vivo, and have determined that RS profoundly affects the migration and function of the resulting effectors. We suggest that an initial 48–60 h of TCR stimulation during T cell priming results in an ex- panded population of optimally functional effectors that traf- fic efficiently through lymphoid organs, and are able to par- ticipate in antiviral responses and provide B cell help. We also suggest that longer durations of Ag stimulation result in T cells that exhibit dramatically reduced effector function, and fail to provide other lymphocytes with the cognate help that is necessary to stimulate vigorous immune responses. RESULTS Repeated T cell receptor stimulation results in poor effector expansion Naive and TCR transgenic (Tg) CD4 T cells were cultured with acute or repeated stimulation to generate Th1 and Th2 polarized effectors. By day 4, cell recovery in Th1 and Th2 cultures that were exposed to RS was 50% reduced, com- pared with acute stimulation (Fig. 1, A and B, respectively). Interestingly, carboxyfluorescein succinimidyl ester (CFSE) profile analysis showed that acutely and repeatedly stimulated effectors underwent equivalent rounds of division through- out the culture (Fig. 1 C). Analysis of cell viability indicated that the repeatedly stimulated effectors had fewer viable cells; this suggested that the decrease in effector recovery could be attributed to increased cell death compared with effectors that were generated with acute stimulation (Fig. 1 D). Repeated stimulation impairs effector cytokine production Because the protective immune functions of T cells are me- diated, in large part, by the effector cytokines that they pro- duce (15, 16), we examined the effects of acute versus RS on the cytokine production by Th1 and Th2 effectors. Acutely and repeatedly stimulated Th1 and Th2 effectors had the ca- pacity to secrete polarized cytokines in response to plate- bound anti-CD3 in a dose dependent manner (Fig. 2, A and B, respectively). However, repeatedly stimulated effectors produced significantly reduced amounts of IFN (Th1) and IL-4 (Th2) when compared with effectors that were gener- ated with acute stimulation (Fig. 2, A and B). To determine 1102 Figure 1. Repeated Ag stimulation impairs effector T cell expansion. Naive AND TCR Tg CD4 T cells, stimulated with acute or repeated Ag-pulsed APC stimulation under (A) Th1 or (B) Th2 polarizing conditions were enu- merated, (C) analyzed for CFSE dye loss, and (D) cell viability. Data are means of triplicate cultures SD. Data shown in (C) is one representative of triplicate cultures. Data are representative of three to six independent experiments. if the reduction in cytokine production by repeatedly stimu- lated effectors was a result of fewer effectors that were capa- ble of producing cytokines, or if the effectors were produc- ing less cytokine on a per cell basis, intracellular cytokine staining (ICCS) was performed on viable effectors that were restimulated for 24 h with optimal concentrations of Ag- pulsed APCs. Fig. 2, C and D show that fewer effectors that were generated with RS were capable of producing cyto- kines, and those that were producing cytokines were making less cytokine on a per cell basis. Impaired cytokine production persists in memory T cells Several groups have reported that, following primary stimu- lation, T cells can enter a state of transient unresponsiveness (17, 18). This nonresponsive state is of limited duration, and lasts only 6 d for CD4 T cells and 2 weeks for CD8 T cells (17, 18). Additionally, the repeatedly stimulated effectors were tested for cytokine production immediately following their last encounter with Ag-pulsed APCs, whereas the acutely stimulated effectors were tested for cytokine produc- tion following 2 d of “rest” without Ag-pulsed APC stimu- lation. Therefore, to determine if RS during CD4 effector generation is inducing a temporary state of nonresponsive- ness, we adoptively transferred 4 d Th1 and Th2 polarized effectors, generated with acute or RS, into adult thy- mectomized, lethally irradiated, bone marrow reconstituted (AT BM) host mice and waited for 3 wk for the progres- sion to resting memory (9). Fig. 3 A indicates that when equal numbers of viable 4-d Th1 and Th1RS effectors were REPEATED STIMULATION LIMITS CD4 T CELL FUNCTION | Jelley-Gibbs et al.