Figure 9. Repeated Ag stimulation in vivo leads to the generation of functionally impaired effectors. Naive AND TCR Tg GFP CD4 T cells were mixed with PCCF-pulsed DCs and transferred into intact B10.Br mice. Subsequently, empty DCs (acute stimulation) or PCCF-pulsed DCs (RS) were injected every 24 h. On day 5, donor T cells (GFP CD4 ) were (A) enumerated, and assayed for expression of (B) CD69 versus CD62L, and (C) IFN by ICCS with (filled histograms) or without (dotted lines) PMA/ ionomycin restimulation. (D) Total numbers of donor T cells recovered from all organs and total numbers of IFN donor T cells were calculated. (E) On day 5 of in vivo HNT TCR Tg effector generation, host mice were 1108 CD3/anti-CD28 continual stimulation technique. Typically this technique is used to stimulate polyclonal CD4 and CD8 T cells in vitro, which are then examined for effector re- sponsiveness in a variety of model systems. A previous study indicated that mitomycin-c–treated, Ag-pulsed APCs nor- mally disappear from in vitro culture systems between 48–60 h of culture with Ag-specific CD4 T cells, and that the late phases of T cell differentiation are driven by growth-pro- moting cytokines like IL-2 (8). We would suggest that plate- bound anti-CD3 stimulation can only mimic Ag-pulsed APC stimulation conditions if T cells are removed from the plate-bound anti-CD3 stimulation between 48–60 h of cul- ture and transferred, without washing, into uncoated tissue culture containers for the remainder of the culture period. Otherwise, repeatedly stimulated effectors will be generated, which produce lower levels of cytokines upon restimulation, are unable to provide protection against lethal influenza in- fection, and are inhibitory to cognate help. The acutely and repeatedly stimulated effectors that were generated in vitro were able to secrete polarized cytokines in a dose dependent manner in response to plate-bound anti- CD3 restimulation, and were able to proliferate in response to common -chain cytokines and Ag restimulation; this suggested that the repeatedly stimulated effectors were not anergic. The ability of repeatedly stimulated effectors to pro- liferate in response to cytokines like IL-7—known to be an important CD4 T cell survival factor (35)—could help to explain how repeatedly stimulated effectors persisted as memory T cells following adoptive transfer, but died during the in vitro cultures. The availability of common -chain cytokines in vivo could have provided survival or expansion signals to repeatedly stimulated effectors. However, the re- peatedly stimulated effectors remained poorly functional as memory T cells, even though they were allowed to rest from TCR stimulation for 21 d before being restimulated. The implications of a persisting population of poorly functional memory T cells has been discussed by many groups (36–39). As individuals age, thymic output decreases and T cell reper- toire diversity contracts; this makes it even more important that any persisting memory T cells have optimal functional capacities. The persistence of a large population of poorly functional memory T cells could compromise severely the ability to establish and maintain a diverse T cell repertoire of functional effector and memory T cells. The repeatedly stimulated effectors that were generated in vitro inhibited the up-regulation of several surface mole- cules that had been implicated in promoting T cell migration to the spleen and tertiary sites; however, when the repeat- edly stimulated effectors were generated in vivo, they did migrate in greater numbers to the spleen and tertiary sites. lethally infected with influenza virus and survival was monitored. Donor cell frequencies in (A) and (D) are means SD of triplicate mice. FACS analysis data (B and C) are one representative of triplicate mice. Data (E) have a starting population of five mice per group. Data are representative of two to four independent experiments. REPEATED STIMULATION LIMITS CD4 T CELL FUNCTION | Jelley-Gibbs et al.