The regulatory effects of IL-15, IL-7, and IL-2 on the development of memory CD8+ T cell responses has been well-documented in studies using different viral systems. In contrast, the role of IL-4—which is critical to the development of CD8+ T cells responses against certain tumors and parasites—remains poorly characterized. In the present study we used TCR transgenic CD8+ T cells specific for the epitope SIVPSAEQI of P. yoelii to compare normal and IL-4R KO CD8+ T cells with regard to the induction and maintenance of memory responses. During the first 4 d after immunization, IL-4R KO CD8+ T cells developed an apparently normal proliferative activity and differentiation profile. However, as the response develops there is a drastic reduction of memory IL-4R KO CD8+ T cells in nonlymphoid organs, and the establishment of memory populations in these tissues is affected severely. This loss is particularly striking because memory cells seem to develop normally in lymphoid tissues.
Experiments in which IL-4 was neutralized in vivo after immunization clearly demonstrated that the effect of this cytokine is exerted early during the first week after immunization, at the time when the activated cells migrate to nonlymphoid organs. These findings indicate that IL-4/IL-4R interactions that occur early after T cell activation are critical to establish long living memory CD8+ T cell populations in nonlymphoid organs at a critical point when homeostatic mechanisms are inducing a massive apoptosis on activated cells that results in a severe contraction of the CD8+ T cell response. Once CD8+ T cells enter a resting phase and begin to differentiate into memory cells, IL-4 seems to be dispensable. Together, these results demonstrate, for the first time, that IL-4 can modulate the differentiation of memory CD8+ T cells directly through the IL-4R that is expressed on these cells.
The results of the protection studies indicating that IL-4R KO CD8+
T cells lose their antiparasitic activity after failing to maintain memory cells in nonlymphoid organs suggest that this memory population—characterized by their capacity to reside in nonlymphoid tissue—represents the first line of defense against parasite infection in immune hosts. Adoptive transfer experiments using purified memory spleen cells revealed additional features of this memory population. Transfer of memory IL-4R KO cells obtained from spleen 25 d after immunization, a time point at which they are IL-4 independent and express CD62Lhi
, indicated that these cells can home to the liver and become capable of inhibiting parasite development as efficiently as normal memory cells. The results on the homing behavior of these cells are not unexpected because it is known that memory CD8+
T cells, regardless of their tissue origin or CD62 expression, home with similar efficiency to lymphoid and nonlymphoid organs after adoptive transfer (22
). It is unclear whether this migration behavior represents a physiologic trafficking pattern accelerated by the adoptive transfer, or if this is an abnormal effect that is induced by the ex vivo purification procedures and intravenous injection. Nevertheless, the fact that memory IL-4R KO cells can protect after adoptive transfer indicates that they are fully capable of inhibiting parasite development if they reside in the proper tissue compartments.
These findings appear to differ, at least in part, from those obtained in studies using vaccinia and lymphocytic choriomeningitis virus. In these viral systems, adoptively transferred “central memory” CD8+
T cells—defined by the expression of CD62Lhi
—represent the main protective cell subset (7
). In the malaria system, we found that adoptively transferred normal CD62Llo
memory cells display a comparable antiparasitic activity. Most importantly, the lack of protective activity by lymphoid resident CD62Lhi
IL-4R KO memory cells of unmanipulated mice suggests that this subset may not have a protective role that is as prominent as that observed in viral infections. This apparent discrepancy between the viral and parasite systems most likely reflects the biologic differences that exist between these microbial infections. Particularly important is the fact that the parasite stage that is susceptible to CD8+
T cells develops only in the liver and last for only 40–42 h, whereas viral infections affect several organs, including lymphoid tissues, and remain detectable for several days. In this situation, during viral infections, memory CD8+
T cells that reside in lymphoid organs may have more opportunities to be reactivated and become full effector cells, and mask the early protective activity of memory cells from nonlymphoid organs.
The selective effect of IL-4 in the development of memory CD8+
T cells in nonlymphoid organs is intriguing because it seems to differ from that of IL-7 and IL-15, both of which seem to affect the entire memory population at different time points (i.e., IL-7 helps to maintains memory populations after they have developed, whereas IL-15 seems to be important during T cell activation and after memory populations have been developed; references 8
). The molecular mechanism that underlies the effect of these cytokines of the γ-chain receptor family on CD8+
T cells remains poorly understood. Although the importance of IL-15/IL-15R interactions at different stages of the CD8+
T cell responses is well documented, little is known about the molecular basis of this cytokine effect. As for IL-7, recent studies indicate that this cytokine promotes the survival of memory CD8+
T cells by increasing the expression of antiapoptotic molecules, such as Bcl-XL
The mechanism by which the IL-4R modulates the development of memory CD8+
T cells in nonlymphoid organs remains to be elucidated. It is conceivable that the decrease of IL-4R KO memory cells in nonlymphoid organs may result from a reduced supply of memory cells from spleen, a selective migration of activated cells to lymphoid organs, or a decreased survival of memory cells in nonlymphoid organs. Our results show that the magnitude of normal and IL-4R KO CD8+
T cell responses at day 4 are identical in every organ. Moreover, at this time point the number of activated cells in each organ reaches their highest levels; this indicates that there is no lack of T cell migration from spleen or lymph nodes toward nonlymphoid tissues. After day 4 there is a pronounced reduction in the number of IL-4R KO CD8+
T cells in nonlymphoid organs. This may be explained by an accelerated emigration of activated cells toward lymphoid tissues facilitated by a high expression of CD62L, or it could be due to an increased death rate of activated cells in nonlymphoid organs. The idea that IL-4 enhances the survival of activated CD8+
T cells is supported by previous studies which described an antiapoptotic effect of IL-4 on CD4+
T cells (25
), and by our current findings that activated IL-4R KO CD8+
T cells express lower levels of the antiapoptotic molecule, Bcl-XL
when compared with activated normal cells. Further research is necessary to fully resolve this issue.
The existence of various cytokine-mediated mechanisms that affect the development of memory CD8+ T cell responses raises questions regarding the possible interactions that these cytokines may establish between themselves during this process. Perhaps these cytokines act in concert—with a certain degree of specialization—to perform distinct tasks at different developmental phases of the T cell response. Alternatively, it is conceivable that these cytokine/cytokine receptor interactions might represent redundant functions that ensure a proper development and establishment of memory cells. Perhaps more important, it has yet to determined whether the same set of cytokines participates in the development of all antimicrobial CD8+ T cell responses. A “one size fits all” model of CD8+ T cell development may be too simplistic; the degree of participation of each cytokine may vary depending on the virulence of the infectious pathogen, the tissue compartments that are affected, or the pathology caused by the infection. A better characterization of the mechanisms by which these cytokines sustain and regulate the development of memory CD8+ T cells should open new avenues for research that are aimed at manipulating this differentiation process with a view to enhance the efficacy of T cell responses induced by vaccination.