Here we demonstrate that STAT5 is critical to maintain effector CD8+T cells. In this model, Stat5 is deleted in multiple tissues as the Mx1Cre transgene is expressed ubiquitously. It is possible that some of the effects we observe could be due to deletion of Stat5 in non-T cells. For instance, STAT5 signaling in dendritic cells (DCs) may influence survival of effector T cells as it has recently been shown that IL-7 can act on DCs to regulate the size of the CD4 T cell pool under lymphopenic conditions (33
). However, whether this axis operates under normal physiologic conditions or during viral infection remains unclear. If there were a non-T cell effect of Stat5 that was dominant, we would have expected to see similar effects on CD4+ and CD8+ T cell responses as expansion of both cells require DCs, and this was clearly not the case. Although we cannot completely rule out non-T cell effects of Stat5, our data clearly show that despite similar deletion of Stat5, CD8+ effector T cells rely on Stat5 considerably more than do effector CD4+ T cells. We were somewhat surprised to find that IL-15 was not required for maintaining effector CD4+ T cell responses, as a recent report showed that IL-15 contributes to survival of memory CD4+ T cells (34
). It is possible that IL-15 becomes a more prominent survival factor for memory CD4+ T cells than for effector CD4+ T cells. Indeed, we found that expression of CD122 was quite transient on effector CD4+ T cells (data not shown), and previous work showed that increased expression of CD122 on memory CD4+ T cells (34
), may facilitate their dependence on IL-15. We note that our data do not imply that IL-15 is not involved in effector CD4+ T cell survival, just that it is not strictly required. Thus, mechanisms maintaining effector CD4+ T cells remain unclear. Previous work has shown that Bcl-3, a NF-κB p50 family member, can promote survival of activated CD4+ T cells (35
). Further, activated CD4+ T cells have dramatically increased expression of A1 (1
), and A1 expression can be regulated by NF-κB signaling (36
). A1 can also antagonize Bim, making A1 a logical candidate for promoting effector CD4+ T cell survival. Future experiments will evaluate the requirement for A1 in the survival of effector CD4+ T cells.
Our data in effector CD8+ T cells are consistent with previous reports showing that in cell lines, IL-7 and IL-15 control expression of Bcl-2 via STAT5 (29
). However, other reports have suggested that STAT5 is not critical for Bcl-2 induction (40
). In one of these reports, mice were developed in which tyrosine 449 in the IL-7Rα gene was mutated to a phenylalanine (41
). The Y449F mutation incapacitates both STAT5 and PI-3K activation in response to IL-7 in lymphocytes and thymocytes (39
). Using T cells from these Y449F mice, the authors showed that although IL-7 was unable to induce detectable pSTAT5, it was able to increase expression of Bcl-2 (41
). From these data it was concluded that Bcl-2 upregulation was independent of STAT5 activation. However, at baseline, Bcl-2 levels were significantly lower in peripheral CD4+ and CD8+ T cells directly isolated from Y449F mice (41
), suggesting that, physiologically, IL-7RαY449 signaling controls expression of Bcl-2 in vivo
. Consistent with this, another group showed, in a thymocyte cell line, that Bcl-2 upregulation and STAT5 activation in response to IL-7 signaling required Y449F of IL-7Rα (39
). Furthermore, mice deficient in JAK3, which is required for IL-7 and IL-15 activation of STAT5, exhibited a profound loss of peripheral CD8+ T cells but had nearly normal levels of CD4+ T cells (42
). Moreover, JAK3-deficiency dramatically impaired Bcl-2 expression in CD8+CD4-, but not CD4+CD8- thymocytes (45
). Thus, the simplest explanation is that, under physiologic conditions, IL-7 and IL-15 require STAT5 to maintain Bcl-2 expression within CD8+ T cells.
Our data also suggest that STAT5 maintains Bcl-2 directly as cycloheximide failed to block Bcl-2 induction by IL-7 and IL-15. A previous paper showed in a pro-B cell line that cycloheximide blocked induction of Bcl-2 by IL-2, leading the authors to conclude that the effects of STAT5 on Bcl-2 induction by IL-2 were largely indirect (29
). However, in that study the effect of cycloheximide was only partial and these results were not reported to be significant. Our results in primary T cells suggest that the effects of IL-7 on Bcl-2 induction are direct and do not require new protein synthesis. These data are supported by a recent report showing that, in mast cells, STAT5 can bind to a site in intron 2 of the Bcl-2 gene (30
). Further work will be required to determine if cytokines promote STAT5 binding to this site in intron-2 in activated CD8+ T cells.
These data have implications for the development of T cell memory that emerges from the effector pool. We and others have previously shown that the pro-apoptotic molecule, Bim, is critical for limiting the numbers of effector T cells that can become memory T cells (20
). Here our data suggest that IL-7 and/or IL-15 signaling through Stat5 is likely critical to maintain levels of Bcl-2 sufficient to combat Bim and develop into memory T cells. Thus, cytokines may have use as potential vaccine adjuvants. Optimal vaccines would promote robust CD4+ and CD8+ T cell responses that would be long-lived. As we and others have shown, common gamma chain cytokines can significantly enhance CD4 and CD8 T cell responses (6
). Although these cytokines enhanced short-term T cell responses, their effects were transient once the cytokines were withdrawn. This makes sense given that survival of nearly all populations of T cells is achieved, at least in part, via competition for limiting amounts of cytokines. However, we note that, in general these studies were done in viral infections in which the T cell responses are extraordinarily robust. It remains unclear if cytokine adjuvants, given short-term, may enhance long-term memory responses under conditions in which less robust T cell responses are generated. This approach could be beneficial for enhancing sub-optimal vaccine responses which currently require several rounds of boosting.