A R T I C L E Figure 2. Repeated Ag stimulation impairs effector cytokine pro- duction. Viable AND TCR Tg (A) Th1 and (B) Th2 polarized 4 -d effectors were restimulated for 24 h with plate-bound anti-CD3 and supernatants were analyzed by ELISA. Four-d (C) Th1 and (D) Th2 effectors were cultured for 24 h without (open histograms) or with 10 g/ml of plate-bound anti- CD3 restimulation (gray filled histograms) and analyzed by ICCS. Numbers within the histograms are % of cytokine-producing cells in the restimu- lated cultures. Data in (A and B) are means of triplicate cultures SD. Data in (C and D) are representative of triplicate cultures. Data shown are representative of three independent experiments. transferred into AT BM hosts there was no significant dif- ference in the number of memory T cells that were recov- ered (center graph, 25 d memory). However, when we ex- trapolate from the total number of effectors that originally were generated in the primary cultures ( 36 106 Th1 and 19 106 Th1RS), the total number of Th1RS memory cells that are generated as a result of RS would be reduced compared with Th1 memory cells (right graph). Fig. 3 B in- dicates that when equal numbers of viable 4-d Th2 and Th2RS effectors were transferred into AT BM hosts, there was a 25% reduction in the ability of Th2RS effectors that were generated with RS to progress to memory (center graph). Even more striking was the 75% reduction in the number of Th2RS memory cells that were generated as a re- sult of RS that would have resulted if the total number of ef- fectors that were generated in primary culture ( 55 106 Th2 and 25 106 Th2RS) had been transferred into the AT BM hosts (right graph). We then restimulated equal numbers of memory cells with Ag-pulsed APCs for 24 h to JEM VOL. 201, April 4, 2005 Figure 3. Impaired cytokine production persists in memory T cells. AND TCR Tg CD4 T cells stimulated with acute or RS under (A) Th1 or (B) Th2 polarizing conditions were enumerated at days 0 and 4 of in vitro cul- ture. 10 106 effectors were transferred into AT BM hosts and recov- ered from lymph nodes and spleens 21 d later. Twenty-five–d memory T cells were enumerated, and extrapolated memory frequencies were deter- mined by multiplying the number of memory cells recovered by the fraction of 4-d effectors that were generated in vitro. (C) Th1 and (D) Th2 polarized memory cells were restimulated for 24 h with Ag-pulsed APCs and super- natants were analyzed by ELISA. Data (A and B) are means of triplicate cul- tures SD. Data (C and D) are representative of duplicate cultures. Data are representative of three independent experiments. determine if the transition to a memory-like state could re- store the cytokine-producing function of repeatedly stimu- lated T cells. Fig. 3, C and D indicate that the Th1 and Th2 memory cells that were generated from repeatedly stimu- lated effectors still produced lower levels of IFN and IL-4, respectively, compared with memory cells that were gener- ated from acutely stimulated effectors. Repeated T cell receptor stimulation impairs polyclonal CD4 T cell responses The examination of TCR Tg CD4 T cells did not enable us to observe the evolution of a normal polyclonal response, and it was possible that the repeated Ag stimulation of poly- clonal CD4 T cells could inhibit the functions of dominant T cell clones while permitting the expansion of subdominant clones. Therefore, it was important to demonstrate that the effects of repeated Ag stimulation were not confined to the responses of TCR Tg CD4 T cells. Naive polyclonal BALB/c CD4 T cells were cultured under acute and RS conditions with plate-bound anti-CD3 to generate Th1 and 1103