Figure 7. Repeatedly stimulated Th1 effectors fail to protect against lethal influenza infection. 5 106, 2.5 106, and 0.5 106 4-d HNT TCR Tg Th1 or Th1RS effectors were transferred into mice that were lethally infected with influenza virus. (A) Host survival was monitored, and (B) donor CD4 and host CD4 and CD8 T cells were enumerated 4 d follow- ing transfer. Data (A) have a starting population of five mice per group. Data (B) are means of three mice per group SD. Data are representative of two to three independent experiments. tors with the capacity to produce high levels of IFN (Fig. 9, C and D). In contrast, effectors that were generated in vivo with RS failed to expand into a large population of IFN – producing effectors (Fig. 9 D). This is despite the fact that RS in vivo resulted in only a modest increase in total num- bers of effectors (Fig. 9 D). Repeatedly stimulated effectors that were generated in vivo also produced less IL-2 when compared with the acutely generated effectors; neither pop- ulation produced any detectable IL-4 (unpublished data). Because RS of T cells in vivo generated effectors of simi- lar functional deficiencies as those that were generated in vitro, we determined if the repeatedly stimulated effectors that JEM VOL. 201, April 4, 2005 A R T I C L E Figure 8. Th2RS effectors provide minimal cognate B cell help in vivo. Four-d Th2 or Th2RS effectors, or PBS alone, were transferred into CD4 KO hosts that were immunized with NP-OVA in alum. Sixteen d follow- ing immunization (A) NP B cells were harvested from spleens and analyzed for germinal center (GC ) phenotype (CD38 /PNA ). (B) total numbers of NP GC B cells were determined (*P 0.006, **P 0.007), and (C) serum was analyzed for NP-specific IgG1 (‡P 0.00006). Data (A) is one repre- sentative of triplicate mice. Data (B and C) are means SD of triplicate mice. Data are representative of three independent experiments. were generated in vivo could protect against lethal influenza challenge. However, we needed to optimize the conditions of in vivo stimulation of adoptively transferred HNT TCR Tg CD4 T cells because it was demonstrated that large numbers if IFN –producing T cells would be necessary to protect mice against lethal challenge. Careful titration of naive HNT TCR Tg CD4 T cells and HNT-pulsed DCs enabled us to generate enough acutely stimulated effectors to protect 80% of the mice from lethal challenge (Fig. 9 E), whereas only 40% of the mice survived when the T cells were stimulated with re- peated injections of HNT-pulsed DCs. DISCUSSION Repeated stimulation of CD4 T cells during primary culture led to the generation of effectors that produced strikingly low levels of polarized cytokines, without changing their overall polarization profiles. Repeated stimulation did not induce anergy, as defined by the ability of effectors to prolif- erate to -chain binding cytokines, restimulation with Ag, or their ability to persist in vivo. The repeatedly stimulated effectors also were inefficient in protection against influenza virus and were inhibitory to cognate help. We have determined that RS in vitro, whether achieved by adding Ag-pulsed APCs every 24 h of culture or contin- ual culture on plate-bound anti-CD3 (8) compromises pri- mary CD4 T cell expansion and differentiation into cyto- kine-producing effectors. This finding has significant implications regarding the quality of the effectors that are generated with the widely used 4–7 d plate-bound anti- 1107