In this paper, we investigated the potential of IL-7 to function in the homeostasis of memory CD4 cells in vivo. Our results suggest that IL-7 is a key regulator of memory CD4 cell survival not only long term, but also during earlier stages when resting memory cells develop from primary cells that have undergone response to antigen. The notion that IL-7 might be a cytokine candidate for control of memory CD4 cell homeostasis was initially revealed by our in vitro assessment of the effects of rIL-7 on the survival of resting memory CD4 cells compared with naive CD4 cells (). By using highly purified resting TCR transgenic CD4 cells, we ensured greater uniformity of memory populations than in previous analyses of memory phenotype cells from normal animals. Importantly, we observed naive and memory CD4 cells express equivalent amounts of IL-7Rα and Bcl-2 and up-regulate Bcl-2 to comparable levels after exposure to IL-7, supporting the possibility that both populations have the potential to be similarly regulated by this cytokine. IL-7–mediated survival by memory CD4 cells was not accompanied by division, as shown previously for naive CD4 cells (32
). The data suggest that survival and homeostatic division can be separately regulated in both naive and memory CD4 cells.
In vivo, IL-7 deprivation resulted in the disappearance of transferred memory CD4 cells in both intact and immunodeficient recipients (). Although deficiency of IL-7 also disrupts both T and B cell lymphopoeisis (37
), it is striking that IL-7– and IL-7R–deficient animals, which display a similar phenotype with regard to a lack of mature lymphocytes, show a profound difference in their ability to maintain resting memory CD4 cells. Few memory CD4 cells were recoverable from either the lymphoid or nonlymphoid compartments in the absence of IL-7. The potential for IL-7 to regulate memory CD4 cell survival in vivo is further supported by the results of IL-7 blocking studies in normal recipients, where both donor and host memory CD4 cells decayed in recipients rendered deficient in IL-7 (). These data provide strong support for a conclusion that IL-7 is at least one factor that contributes to maintaining memory CD4 cells in vivo.
Although several previous studies have not detected a requirement for IL-7 in regulating the homeostasis of memory CD4 cells, it is important to bear in mind that a primary focus has been proliferative capacity in lymphopenic recipients (13
). Our data suggest that IL-7 may not affect memory CD4 cell expansion. In addition, we find that under lymphopenic conditions where heterogeneous populations of normal memory CD4 cells undergo homeostatic division, reduced recovery is observed in the absence of IL-7 (unpublished data), in line with our current results from IL-7 blocking studies in normal recipients. The use of irradiated hosts for analysis of cytokine dependence in some studies may further complicate analysis of requirements for individual cytokines due to the rapid induction of multiple cytokines, including TGF-β (38
) and IL-6 (39
), that have the potential to mediate T cell survival.
Analyses of γc cytokine receptor–deficient mice indicate that some T cells can be generated in the absence of a capacity to respond to this cytokine family (21
). In addition, when TCR transgenic mice are crossed to γc receptor knockout mice, CD4 cells with an activated phenotype that are highly susceptible to apoptosis arise. These findings suggest that, whereas other mediators can support expansion of the few naive CD4 cells that are generated in the absence of a γc cytokine response, most of γc-deficient naive CD4 cells fail to survive. However, TCR transgenic CD4 cells from γc receptor–deficient mice can survive upon activation and differentiation into memory CD4 cells in γc receptor–deficient recipients (18
). Although this finding was interpreted to indicate that CD4 memory cell homeostasis is regulated independently of γc family cytokines, it is also possible that the finding either applies to only a minor subset of memory CD4 cells or is a reflection of a capacity to engage aberrant compensatory survival mechanisms that arise when responses to γc cytokines are genetically impaired (18
). In either case, γc-deficient T cells might utilize other cytokines that become available, and these cytokines may promote homeostatic division under conditions of lymphopenia to sustain long-term memory.
Consistent with previous conclusions that CD4 cells do not depend solely on γc family cytokines for proliferation during an Ag-induced response (18
), we found that naive TCR transgenic CD4 cells could be comparably expanded by cognate Ag and that the resulting effector cells distributed normally in both IL-7–sufficient and IL-7–deficient recipients. Our results suggest that IL-7 is not critical for survival of CD4 cells during Ag-induced activation or division, or for their survival during dissemination to nonlymphoid sites. However, the primed population disappeared from both lymphoid and nonlymphoid tissues only under conditions of IL-7 deprivation. Thus, we envision that IL-7 is necessary for the survival of developing memory cells. Because our previous analyses demonstrate that cytokines are not required for activated CD4 cells to return to rest and acquire properties of memory cells (28
), it is less likely that IL-7 also regulates the differentiation of memory cells. IL-7 is not only produced by stromal cells in lymphoid tissues but also by epithelium, liver, and skin (14
). Thus, it is possible that local availability of IL-7 can support the survival of memory cells in many different tissues, and that the decay we observed in nonlymphoid sites is not due to T cell migration/recirculation that results in IL-7 withdrawal only in the lymphoid compartment.
Although it could be argued that memory CD4 cells primed in lymphopenic animals might be atypical because of their induction and maintenance in an environment where survival factors may be elevated due to the lack of normal consumption, our finding that transgenic CD4 cells induced in normal recipients showed a similar dependence on IL-7 for survival suggests that this is an unfounded concern. In addition, when polarized Th1 and Th2 cells were induced under conditions where fully differentiated effectors are generated, and rested to induce a memory-like population (28
), we observed similar susceptibility to IL-7 withdrawal in vivo (unpublished data). Thus, recently primed CD4 cells also appear to be highly susceptible to sudden withdrawal of IL-7. Consistent with the antiapoptotic effects of IL-7, our data suggest that IL-7 may play a key role in preventing the demise of effectors by activated T cell autonomous death (41
), thereby promoting CD4 cell survival during the effector to memory transition. Thus, IL-7 may be an important survival factor for early memory CD4 cells as well as for maintaining long term memory CD4 cells.
Because we studied TCR transgenic memory CD4 cells generated by specific immunization protocols, it is possible that the findings may not be generally typical for memory CD4 cells. However, our observations that transgenic memory CD4 cells with either central or effector memory phenotypes can utilize IL-7 for persistence and that transgenic and polyclonal naive and memory CD4 cells have comparable IL-7 dependence are striking ( and ). Together with previous papers on naive and memory CD8 cells (11
), our results support the hypothesis IL-7 is a key cytokine for the physiologic survival of resting peripheral T cells and in this capacity contributes to regulation of the pool sizes of naive and memory cells for both the CD4 and CD8 subsets. Nevertheless, alterations in cytokine receptor expression during an immune response and the relative abundance of various cytokines in the local milieu may determine changes in the preferential usage of growth and/or survival factors.
It remains unclear why IL-7 appears to function to mediate survival but not homeostatic proliferation of memory CD4 cells. However, this finding is reminiscent of the fact that IL-2 and IL-4, in addition to IL-7, transduce signals that promote survival of resting T cells without inducing division (45
). Furthermore, a recent analysis of polyclonal memory phenotype CD4 cells suggests that TCR-mediated signals can support homeostatic cycling of memory CD4 cells in the absence of IL-7 but that IL-7 is required in addition for optimal survival (19
). Our results also suggest that survival and homeostatic turnover of T cells need not be linked in vivo. Mediators that might exclusively promote turnover of memory CD4 cells have yet to be identified, and it is possible that multiple cytokines could exhibit redundancy in this regard. Importantly, our data suggest that an essential step in the maintenance of CD4 cell memory is the provision of survival signals from cytokines, and that IL-7 may play a prominent role in this process.