These findings seemed to be inconsistent, until we reconsid- ered the modulation of CD62L to be not only a measure of the level of activation of T cells, but also a potent modulator of CD4 T cell migration between lymph nodes and tertiary sites. Of the adhesion markers that we analyzed, CD62L is the most thoroughly published as being able to dictate the migration of T cells in and out of lymphoid compartments (22), and it is with respect to CD62L expression that our in vitro phenotype data and our in vivo migration data are most consistent. Unfortunately, although the modulation of adhe- sion molecules and chemokine receptors has been established as being relevant for the trafficking and tissue localization of activated T cells during immune responses (23), no study has revealed conclusively the definitive combination of surface molecule expression that is required to promote optimal CD4 T cell migration between the lymph nodes, spleen, and tertiary sites. The only conclusion that we have drawn from this study is that repeated Ag stimulation in vivo led to the generation of effectors that preferentially migrate to the spleen and tertiary sites, rather than the lymph nodes, and that repeated exposure to Ag stimulation has a dramatic im- pact on the migratory potential of primary effectors. Further studies are necessary to assess the role of individual surface molecules—particularly those that failed to become up-reg- ulated in response to RS—on the migration and interaction of CD4 effectors with other lymphocytes in vivo. The finding that large numbers of repeatedly stimulated effectors failed to provide, or directly impaired, efficient cognate help to B cells also had interesting implications, par- ticularly with regards to immunization strategies. The poor helper function was not surprising, considering that repeat- edly stimulated Th2 effectors had a marked reduction in their ability to produce IL-2 and IL-4. In fact, Grabstein et al. (40) demonstrated that CD4 T cell–derived cytokines were essential for contact-dependent T cell help for Ag-spe- cific primary antibody responses. Specifically, the induction of polyclonal Ig secretion required IL-4 and IL-5, and opti- mal Ag-specific antibody formation required IL-2. Because repeatedly stimulated Th2 polarized effectors produced low levels of IL-2 and IL-4, it was not surprising that they failed to provide optimum levels of cognate help to B cells. How- ever, because the deficit in T cell help was most pronounced when a high number of effectors were transferred, these re- sults also would be consistent with the induction of an in- hibitory mechanism at high T cell numbers. In fact, Eaton et al. (31) recently reported that the transfer of too few or too many naive CD4 T cells resulted in reduced cognate helper function. Additionally, Erickson et al. (41) reported that al- though CD40L ligation of CD40 on B cells has been used as a potent immune adjuvant for short-lived humoral-mediated immunity, increasing the magnitude of T cell help ablated germinal center formation and prematurely terminated the humoral immune response. In fact, the highly activated phe- notype that was characteristic of repeatedly stimulated effec- tors included a dramatic up-regulation of CD40L surface ex- pression (unpublished data). JEM VOL. 201, April 4, 2005 A R T I C L E Several studies have focused on the impact of varied dura- tions of Ag stimulation on CD4 T cell activation and devel- opment of effector functions. However, the central question of the studies that have published to date was to determine the minimal duration of Ag stimulation that was necessary to promote CD4 T cell activation, division, and development of a cytokine-producing phenotype. The general consensus of these studies was that longer durations of TCR stimulation— from 8 to 20 h—were required to induce IL-2 and IFN production when Ag dose or affinity was low, and that high Ag dose or affinity could drive more progressive differentia- tion of proliferating T cells with as little as 4–6 h of TCR stimulation (12, 42, 43). These studies suggested that the quantity of TCR stimulation augmented T cell activation, at least in part, by reducing the time that is required for cyto- kine secretion. However, in most of the protocols, TCR stimulation and/or T cell culture was terminated by 48 h, and hence, did not determine the consequences of prolonged Ag stimulation on the peak of effector responses, which typically is determined at 4–7 d of a primary in vitro immune re- sponse. Even studies that had attempted to establish if cell di- vision could determine the development of cytokine produc- tion by effectors used in vitro APC populations, which have been established to be eliminated from in vitro cultures within the first 48–60 h, and hence, would be unable to stim- ulate repeated Ag stimulation (8, 10, 44). This study is the first to determine the direct impact of repeated Ag presenta- tion on primary CD4 effector responses. Generally, it had been accepted that different pathogens pose unique challenges to the immune system, and variations in pathogen load and Ag persistence may affect primary T cell responses dramatically. Recent studies revealed that Ag-bear- ing DCs were highly efficient at recruiting and stably captur- ing T cells with high avidity interactions for long periods of time (6). The ability to introduce Ag-bearing DCs repeatedly to induce effectors with lower IFN production suggested that persistent infections might induce poor CD4 T cell re- sponses as a result of repeated Ag stimulation, particularly when CTLs are not able to eliminate Ag-bearing APCs. Studies of CD4 T cell responses to chronic or persistent pathogens like HIV, Mycobacterium tuberculosis, EBV would support the hypothesis that Ag persistence leads to T cell death and functional nonresponsiveness of surviving CD4 T cells. Following infection with HIV, M. tuberculosis, and EBV, the frequency of Ag-specific CD4 T cells that is re- covered from individuals during the latent phase of the in- fection is significantly lower than that detected during pri- mary infection, rather than maintaining high frequencies of Ag-specific CD4 T cells throughout the infection (45–48). Alternatively, failed attempts to vaccinate against diseases, like leishmaniasis and malaria, suggested that the mainte- nance of protective CD4 T cell–mediated immunity would require the continued presence of Ag stimulation (49, 50). These infections induce strong CD4 effector T cell re- sponses, and because of their persistent nature, may drive all of the T cells to terminal exhaustion; this leaves no partially 1109