e n i c i d e M l a t n e m i r e p x E f o l a n r u o J e h T CORRESPONDENCE Dawn M. Jelley-Gibbs: djgibbs@trudeauinstitute.org Abbreviations used: Ag, antigen; AT BM, adult thymectomized, lethally irradiated, bone marrow reconstituted; CFSE, carboxy- fluorescein succinimidyl ester; ICCS, intracellular cytokine staining; NP, 4-hydroxy- 3-nitrophenyl acetate; PCCF, pigeon cytochrome c peptide fragment; RS, repeated stimula- tion; Tg, TCR transgenic. JEM © The Rockefeller University Press $8.00 Vol. 201, No. 7, April 4, 2005 1101–1112 www.jem.org/cgi/doi/10.1084/jem.20041852 A R T I C L E Repeated stimulation of CD4 effector T cells can limit their protective function Dawn M. Jelley-Gibbs, John P. Dibble, Svetlana Filipson, Laura Haynes, Roslyn A. Kemp, and Susan L. Swain Trudeau Institute Inc., Saranac Lake, NY 12983 Chronic infections often result in CD8 T-cell deletion or functional nonresponsiveness. However, to date no definitive studies have attempted to determine the impact of repeated T cell receptor stimulation on CD4 effector T cell generation. We have determined that when antigen presentation is limited to 2 d, optimum in vitro CD4 effector generation is achieved. Alternatively, repeated stimulation results in decreased CD4 effector expansion, decreased cytokine production, and altered migration. Similarly, functionally impaired effectors develop in vivo when antigen-pulsed antigen-presenting cells are replenished every 24 h during a primary immune response. CD4 effectors that are generated with repeated stimulation provide no protection during influenza infection, and have an impaired ability to provide cognate help to B cells. These results suggest that duration of antigen presentation dictates CD4 effector function, and repeated T cell receptor stimulation in vitro and in vivo that exceeds an optimal threshold results in effectors with impaired function. Efficient priming of naive T cells is essential for the generation of effective immunity to pathogens. The initiation of a protective adap- tive immune response depends on successful interactions between T cells and APCs. When naive CD4 T cells successfully recognize anti- gen (Ag) that is presented by APCs, they ex- pand into highly activated effectors that exhibit immediate cytokine-secreting function (1–3). We hypothesize that reaching an optimal threshold of TCR stimulation in the initial phases of a T cell response determines if an effector survives and acquires the functions that are necessary to provide cytokine or cognate help to other lymphocytes. The progressive differentiation model that was proposed by Lanzavecchia and colleagues (4, 5) suggests that the level of TCR signal that is accumulated determines if T cells reach hier- archical thresholds for induction of prolifera- tion and differentiation. In this model, random encounters of variable duration with APCs and cytokines result in the generation of effectors with different fates (6). The concept that TCR signal accumulation results in progressive differ- entiation of effectors is supported by the finding that the commitment of naive T cells to prolif- erate is reached in 6–12 h if they are stimulated by a high dose of Ag plus costimulation (7), but 40 h is required to generate an expanded effector population (8). Moreover, it has been demonstrated that multiple rounds of prolifer- ation, and 2–4 d of culture, are required for naive T cells to expand into effectors that are capable of rapidly producing large amounts of cytokines (9, 10). Less is known about the impact of repeated TCR stimulation on effector development, or the ability of the effectors that are generated to protect against infectious agents and provide help to other lymphocytes (11). The current body of literature primarily has focused on determining the minimal stimulation that is necessary to drive multiple rounds of T cell division (7, 12–14), rather than the optimal stimulation that is required for the generation of functional effectors. The generation of effectors in vitro typi- cally involves stimulating naive CD4 T cells with Ag-pulsed APCs, often with exogenous IL-2 and polarizing cytokines. Our previous studies demonstrated that under these condi- tions, the Ag-pulsed APCs disappear from cul- ture within 48–60 h of culture initiation, and that cell division in the final 2–4 d of a 4-d culture is driven by cytokine (IL-2) stimula- tion. We define this set of conditions, where Ag-pulsed APCs are added only at the initia- tion of culture, as acute Ag stimulation. Alter- natively, prolonging Ag stimulation beyond the first 2 d of culture resulted in reduced numbers of effectors (8). Therefore, we define 1101