During a primary response, generation of large numbers of CD4 effectors able to secrete high titers of cytokines is critical in order to quickly clear the immunogen. Using a TCR-Tg mouse model, we have found several profound alterations in CD4 effector cell generation that are associated with aging, and our results suggest that the low production of IL-2 by aged naive cells is largely (or totally) responsible for these deficiencies. Aged CD4 T cell defects included lower production of IL-2, loss of late phase expansion, and failure to differentiate into effectors that express phenotypic markers associated with that stage and that secrete high levels of cytokines. Importantly, provision of exogenous IL-2 both supports the expansion and differentiation of aged naive CD4 cells and leads to generation of effectors that themselves are capable of optimum IL-2 production. Furthermore, we show here that whereas both IL-2 and IL-15 as well as IL-4 and IL-7 can enhance the expansion of aged CD4 T cells, only IL-2 can support the efficient generation of polarized Th2 effectors, indicating a unique role for IL-2 signaling through the high affinity IL-2R in effector development.
Aged Tg+ CD4 cells expand less than young cells during the 5-d period of in vitro effector generation (). The early stage of expansion (days 1–3) is quite similar in both the young and aged cultures, whereas later expansion (days 4 and 5) is three to five times greater in the cultures of young cells. This is most dramatically observed by flow cytometric analysis of CFSE-labeled young and aged Tg+ CD4 cells ( C), in which we can actually visualize that the aged cells do not divide after day 3, whereas the young cells continue dividing. This effect is mirrored in the expansion of cells, which continues after day 3 in young but not aged populations. Moreover, the aged CD4 effectors expressed decreased levels of CD25 (IL-2Rα). We suggest that the early stage of proliferation is more dependent upon Ag-induced TCR stimulation, and the latter stage is more dependent upon IL-2, which is present in greater amounts in the young cultures. The phenotype of the day 4 effectors generated from aged naive cells was between that of a naive cell and a day 4 effector from young mice, i.e., the aged effector cells had a less differentiated phenotype that had high levels of CD44 but did not substantially downregulate CD62L and did not upregulate IL-2R (CD25) expression. This suggests that cytokines (IL-2 and perhaps others) acting midway (days 2–3) in the primary response (when IL-2 disappears from cultures of aged cells) are required for full differentiation into effectors. Effectors generated from these Tg+ CD4 cells from aged mice produced less IL-2, and the frequency of effector cells secreting IL-2, as detected by intracellular staining, was also significantly reduced in the aged, supporting dependence on the continued presence of IL-2 for efficient effector generation.
These results thus support a large impact of aging on CD4 effector development but also suggest that the spectrum of deficiencies found in effector generation of aged CD4 T cells may be attributable largely or totally to the decreased secretion of IL-2 upon initial TCR stimulation. In vitro, IL-2 supports the progression of activated T cells through the cell cycle, the regulation of the differentiation of T cell effectors, and the differentiation of CD4 T cells so they become susceptible to Fas/FasL-induced activation-induced cell death 303132
. Although other cytokines that signal through the γc chain may substitute for IL-2, IL-2 is the only γc chain–binding cytokine produced when naive CD4 T cells are stimulated in vitro and is thus solely responsible under these circumstances where no other T cells are present and other cell populations are limited. Therefore, the finding that IL-2 secretion by CD4 Tg+
cells upon TCR stimulation is markedly reduced in the aged is predicted to have profound effects on the generation of effector cells and the ensuing effector response. Moreover, as IL-2 regulates its own receptor expression 333435
, the effects of any reduction in IL-2 production in the early stages of an immune response may be amplified enormously. It then becomes important to know whether these deficiencies may be overcome simply by the addition of IL-2 or other cytokines or if other intrinsic defects in the effector cells' ability to respond to IL-2 are present.
When aged CD4+
effectors are generated in the presence of IL-2 (IL-2 effectors, Th1 and Th2 effectors), they become phenotypically () and functionally () similar to effectors derived from younger mice. In addition to upregulating CD44, they upregulate CD25 expression and downregulate CD62L. They also expand at the same rate and to the same extent as seen in cultures of young Tg+
cells (). When aged effectors generated in the presence of IL-2 are restimulated with Ag/APC, they secrete levels of cytokines that are the same as young effectors (). Additionally, similar proportions of IL-2 effectors in both the young and aged populations are actively secreting IL-2 (), and similar amounts of cytokines are secreted on a per-cell basis. These findings suggest that the major defect in effector cell generation in the aged is the inability to secrete sufficient IL-2 levels to sustain cell expansion and induce further differentiation to fully functional effector cells. However, the addition of sufficient levels of IL-2 during effector cell generation abrogates these age-related differences. Once adequately differentiated effectors are generated, it is not surprising that the effector progeny are capable of high levels of cytokine production, based on recent publications reporting the heritability of differential gene methylation patterns (epigenetic remodeling) that occurs during effector generation 363738
. This model proposes that activation and effector generation of T cells involves demethylation and increased accessibility of the cytokine gene locus (IL-2 in this case) to transcription factors as a consequence of signaling from the appropriate cytokine receptor. This remodeling is permanent and heritable, thus explaining our finding that aged IL-2 effectors generated in the presence of high levels of IL-2 can produce high levels of IL-2 upon restimulation. Experiments to test this hypothesis on young and aged effectors are planned.
When IL-2 remains low during effector generation in cultures of aged CD4 T cells, only a fraction of the potential effector generation is realized. It is not clear if this partially activated population can later be recruited into the responding population, as a lack of IL-2 during effector cell generation has been reported to lead to anergy in some models 3940
. The presence of a significant population of nonresponding or poorly responding T cells in aged individuals is well documented 121941424344
, and these cells have been shown to display a phenotype similar to the aged no cytokine effectors generated in our model. Furthermore, in IL-2−/−
, and IL-2β−/−
, many CD4 T cells express an Ag-experienced phenotype but appear to be nonresponsive to TCR stimulation. There is also impaired in vivo peripheral deletion of these Ag-experienced cells in both models. This has lead to the hypothesis that in the absence of IL-2 induced signals, stimulation of T cells fails to activate the death pathway, which leads to the accumulation of Ag-experienced anergic cells in the periphery, and we suggest that this is what may be occurring in aged individuals.
During an in vivo response, other cytokines that signal through the γc chain may be present and could possibly replace IL-2 and restore function of aged naive cells. The experiments shown in demonstrate that other cytokines that signal through the γc chain (IL-15, IL-4, and IL-7) can indeed increase the proliferative capacity of naive aged Tg+ CD4 cells. It seems likely that these effects are via an IL-2–independent mechanism that does not involve increased IL-2 production or upregulation of IL-2Rα expression. Most interesting, from a therapeutic viewpoint, is the effect of IL-15. IL-15 was able to support not only enhanced expansion of aged CD4 T cells without IL-2Rα expression but also development of Th1 polarized effectors. However, although IL-2 addition allows aged CD4 T cells to develop into polarized effectors under the influence of polarizing cytokines, IL-15 is unable to support optimum development of Th2 effectors. These data support the hypothesis that IL-2, acting through the high affinity IL-2R, plays a unique role in Th2 effector generation and that lower IL-2 production by aged CD4 T cells results in a defect in both expansion of responding naive cells and efficient generation of Th2-polarized effectors.
Recent reports have shown that adjuvants such as LPS, poly I:C, and nonvertebrate DNA can induce type 1 IFN, which then induces APCs to secrete IL-15 4849
. If these adjuvants can induce adequate IL-15 levels in vivo, they may be able to stimulate enhanced proliferation of activated T cells in aged individuals, which could lead to the development of more efficacious vaccines. Even though aged effectors generated in the presence of IL-15 in vitro are not as differentiated as those generated in the presence of IL-2, the increased expansion of these aged “IL-15 effectors” may boost the in vivo aged immune response sufficiently to gain an advantage over an invading pathogen. However, based on our results, one might expect defects in generation of Th2 polarized effectors and in response to pathogens cleared by Th2-dependent mechanisms. Experiments are currently underway in our laboratory to examine the in vivo potential of this approach.
In conclusion, it is clear that the decline in IL-2 production by naive CD4 T cells that occurs with aging has a dramatic negative impact in the generation of T cell effectors in vitro. Assuming that such a decrease also occurs in vivo, this would lead to decreased and ineffective responses, thus helping to explain the increased incidence of mortality and infection with aging. In fact, preliminary studies suggest that aged naive CD4 T cells also respond poorly in vivo (Haynes, L., and S.L. Swain, unpublished data). Moreover, it is conceivable that any cell that partially undergoes a limited response and receives subthreshold signals from IL-2 may become anergic, and these anergic cells (which do not undergo apoptosis) could potentially accumulate with aging and fill the peripheral immune system with nonresponsive lymphocytes. However, the ability of exogenous IL-2 and other γc-binding cytokines to reverse the proliferative defect of aged CD4 cells in vitro raises the possibility that adjuvants that work to induce inflammatory cytokines (IL-15 or other γc reacting cytokines) might boost the primary response and restore effector generation in the elderly.