The fact that T cells die in vitro at a much faster rate than in vivo suggests that there are survival factors in vivo that are absent in standard tissue culture medium. Several extracellular and intracellular factors have been identified that are capable of blocking or delaying apoptosis of T cells in vitro 1617192325515253
. Engagement of CD28 or any one of multiple cytokines can prevent or delay the death of T cells after activation. Several cytokines have also been reported to prolong survival of resting T cells in vitro. Although sufficient for survival in vitro, it is still unclear whether any of these factors are necessary for the survival of activated or resting T cells in vivo. It is likely that in vivo a T cell integrates multiple survival and death signals in its decision to proliferate, differentiate, survive, or die.
We previously showed that T cells activated in the presence of a proinflammatory environment induced by LPS were protected from deletion 18
. Although the LPS effect on the activated T cells was indirectly mediated by the proinflammatory cytokines IL-1α and TNF-α it was unclear whether the direct mediators of T cell survival were soluble or cell bound in this system. We previously reported that IL-6 produced by endothelial cells was a survival factor for resting T cells 16
. Since IL-6 is a proinflammatory cytokine, we were interested in its effects on activated T cells. We show in this paper that IL-6 could rescue only naive resting T cells and was ineffective at promoting survival of activated T cells or T cells bearing memory markers. Thus, the IL-6 effect is unlike the effect of the IL-2 family of cytokines (IL-2, IL-4, IL-7, and IL-15), which does rescue activated T cells 1951
and memory T cells ().
It is possible that these effects of IL-6 were not due to direct action of IL-6 on resting T cells, but rather due to induction by IL-6 of another molecule that itself was responsible for the differential survival of resting versus activated T cells. However, we do not think this is the case for several reasons. First, the IL-6 helped purified resting T cells survive. Therefore, if the effects of IL-6 were mediated by a second molecule, this would have had to be induced by IL-6 on resting T cells. Second, as shown earlier and discussed below, survival of resting versus activated T cells was accompanied by differential phosphorylation of Stat1, a signal transduction protein known to be affected by the IL-6 receptor. Third, one of the best-characterized factors induced by IL-6 is IL-4 39
. Although IL-4 is a survival factor for resting T cells, it also, unlike IL-6, acts as such for activated T cells. Moreover, we have shown that IL-6 is a survival factor for resting T cells from IL-4 knockout mice. Therefore, we do not believe the effects of IL-6 reported here are secondary to induction by IL-6 of some other factor.
We addressed whether the loss of sensitivity to IL-6 was restricted to the T cells that engaged antigen. We found that bystander resting T cells, isolated from the same mouse as the activated T cells, were rescued by IL-6. These data strongly suggested that engagement of the TCR was required for the mechanism driving the IL-6 insensitivity, and that cytokines or upregulated adhesion molecules after T cell activation in the mouse were insufficient to promote the IL-6 insensitivity.
A lot is known about signaling via the IL-6 receptor. The binding of the IL-6 receptor α chain to IL-6 initiates association with gp130 (the IL-6 receptor signaling subunit) to form an IL-6–specific signaling complex on the cell surface. After homodimerization and phosphorylation of gp130, there is recruitment and activation of several kinases that in turn phosphorylate and activate DNA binding proteins such as the Stats 4754
. IL-6 signaling is known to be mediated by the tyrosine kinases Jak1, Jak2, and Tyk2, as well as serine kinases 454755
. The activation of these kinases leads to phosphorylation of Stat1 on Tyr701 4748495056
and phosphorylation of Stat3 on both Tyr705
and Ser727 4655565758
. Immediately after phosphorylation, the Stat proteins form either homodimers or heterodimers and translocate to the nucleus where they bind DNA and drive transcription 47
. Inhibition of IL-6 receptor signaling at any step before phosphorylation of either Stat1 or Stat3 could result in the inhibition of all or some of the IL-6 effects on a cell.
With this information in mind, we tested whether IL-6 fails to prevent the death of activated T cells because of a defect in its signaling pathway in these cells. Activated T cells bore about half the number of IL-6 receptors per cell than resting T cells. However, we do not think this was responsible for the failure of IL-6 to rescue activated cells because IL-6 binding induced the phosphorylation of Stat3 on both Tyr 705 and Ser727 in both activated and resting T cells. Thus, the receptor on activated cells could deliver signals effectively.
Notably, engagement of IL-6 failed to induce phosphorylation of Stat1 tyrosines in activated T cells, whereas this phosphorylation occurred efficiently in resting T cells. This failure was not due to loss of Stat1 protein, and was thus caused by a real difference in signaling via the IL-6 receptor in activated T cells. The loss of Stat1 phosphorylation would prevent the transcription of genes that require Stat1 homodimer or Stat1/Stat3 heterodimer binding, but leave genes induced by Stat3 alone or in combination with other binding proteins intact. Thus, Stat1-induced genes are probably required for IL-6–mediated T cell survival. It is interesting to consider this result in light of our recent finding that type 1 IFNs slow the rates of death of activated but not resting T cells 59
. As type 1 IFNs also induce Stat1 phosphorylation, perhaps T cell death can be slowed by some combination of signaling by Stat1 and another, unknown factor(s) which is induced in resting T cells by IL-6 and in activated T cells by type 1 IFNs.
Mechanisms of IL-6 receptor signaling downregulation such as Stat masking 4142
, induction of the PIAS1 60
and PIAS3 proteins 46
, and the SOCS family of Jak binders 333435363738
have been recently described. The Stat masking and PIAS mechanisms of inhibition do not prevent the phosphorylation of Stats, whereas the SOCS inhibitors do. SOCS-1 has been reported to be an effective inhibitor of IL-6–induced Stat phosphorylation in a myeloid leukemia cell line and was shown to be upregulated by several cytokines 353738
. We are interested in determining whether SOCS-1 induction after T cell activation was responsible for the lack of IL-6–mediated survival or Stat1 phosphorylation in the activated T cells.
We did not find an induction of SOCS-1 mRNA after T cell activation. SOCS-1 was expressed in the T cells even before activation and could not be ruled out as a potential mediator of the insensitivity to IL-6 signaling found in many of the resting T cells. It is also possible that the functional regulation of these SOCS proteins is posttranscriptional or posttranslational. Currently, we are investigating the possibility that a SOCS-like inhibitor is recruited to one of the IL-6 receptor–associated Jaks after T cell activation. The selective inhibition of Stat1 signaling in the activated cells may be due to a lack of activation or recruitment of some but not all of the Jaks normally associated with gp130. This type of inhibition would allow recruitment and activation of some Stat proteins but not others.
In sum, these results suggest that some aspects of the IL-6 receptor are unaffected by T cell activation, whereas at least one signaling pathway is lost. This suggests that T cell activation leaves the T cells able to go through some, but not all, of the differentiative processes associated with IL-6. This is consistent with previous reports showing that IL-6 can induce differentiation of activated CD8+
T cells into cytotoxic T cells 61626364
, can promote activated CD4+
cell differentiation to the Th2 phenotype 39
, and with the data shown here, cannot promote the survival of activated T cells.
A probable teleological reason for the lack of IL-6 rescue of activated cells is to provide selective protection of nonspecific T cells during antigen-driven cell death in vivo. Many activated T cells die during immune responses to antigen, perhaps to avoid accumulation of once useful but now functionless cells, or to prevent shock during a second exposure to the same antigen. Insensitivity to IL-6 may contribute to the loss of activated cells, as their death would not be prevented by this cytokine. Naive T cells may depend on IL-6 for maintenance of survival in specific environments in vivo such as spleen, LNs, or during travel through the circulatory system. Many cell types, such as endothelial cells and fibroblasts, constitutively secrete low levels of IL-6, thus providing resting T cells with plenty of opportunities to engage IL-6 1665666768