A R T I C L E Figure 5. Repeatedly stimulated effectors are responsive to re- stimulation. Four-d AND TCR Tg effectors were CFSE labeled, recultured with common -chain binding cytokines, and analyzed every 24 h for (A) CFSE dye loss (filled histograms 24 h, thick lines 48 h, thin lines 72 h), and (B) were enumerated. (C) TCR expression was determined by anti- V 11 staining. (D) Effectors were restimulated with 10 g/ml plate-bound anti-CD3 and IL-2 and enumerated every 24 h of reculture. Histograms (A and C) are representative of triplicate cultures. Data (B and D) are means of triplicate cultures SD. Data are representative of three independent experiments. lished data) and CXCR3 (25, 26). Overall, repeatedly stimu- lated effectors exhibited extensive up-regulation of activation markers, and an interesting blockade of the modulation of all adhesion markers, except for CD62L (Fig. 6, E and F). Repeated stimulation reduces the ability of Th1 effectors to protect mice from lethal influenza challenge Previous studies showed that IFN production by Th1 and Tc1 effectors is critical to clearance of influenza virus (3, 27– 29). Therefore, we tested the ability of adoptively transferred acutely and repeatedly stimulated influenza-specific Th1 and Th1RS effectors to protect mice that were challenged le- thally with influenza virus. Fig. 7 A shows that all of the mice that were infected with a 2LD50 of influenza virus sur- vived when 5 106 Th1 or Th1RS effectors were trans- ferred. When the number of effectors was reduced to 2.5 106 per mouse, Th1 effectors remained 100% protective (Fig. 7 A-middle graph), but only 25% of the mice that re- ceived Th1CS effectors were protected. Reducing the num- ber of effectors to 0.5 106 per mouse resulted in neither the Th1 nor the Th1RS effector being able to protect against a 2LD50 of influenza virus (Fig. 7 A-bottom graph). JEM VOL. 201, April 4, 2005 The protection against lethal influenza challenge, afforded by sufficient numbers of Th1 polarized effectors, correlated with clearance of live virus from the lungs of the mice (un- published data). These data indicate that it may be the quan- tity of IFN which is produced by the CD4 effectors that determines their ability to protect against lethal influenza challenge. Interestingly, despite the unique phenotypic dif- ferences between Th1 and Th1RS effectors (Fig. 6), the re- peatedly stimulated effectors were recovered from the in- fected mice in numbers similar to, or even slightly greater than, the acutely stimulated effectors (Fig. 7 B). Further- more, host CD4 and CD8 numbers were similarly recruited to the lungs, draining lymph nodes, and spleens of infected mice 4 d after receiving Th1 or Th1RS therapy (Fig. 6 D). Repeated stimulation impairs the ability of Th2 effectors to provide cognate help Following immunization, CD4 T cells provide helper func- tions that are necessary for the generation of B cell–rich ger- minal centers (30). To examine if repeatedly stimulated ef- fectors could provide cognate help to B cells, we adoptively transferred 4-d acutely and repeatedly stimulated Th2 effec- 1105