The Pim kinase pathway contributes to survival signaling in primed CD8+ T cells upon CD27 costimulation

Stimulation of the costimulatory receptor CD27 by its ligand CD70 has proved important for the generation of primary and memory CD8(+) T cell responses in various models of antigenic challenge. CD27/CD70-mediated costimulation promotes the survival of primed T cells and thereby increases the size of effector and memory populations. In this paper, we reveal molecular mechanisms underlying the prosurvival effect of CD27. CD27 signaling upregulated expression of the antiapoptotic Bcl-2 family member Bcl-x(L). However, genetic reconstitution of Cd27(-/-) CD8(+) T cells with Bcl-x(L) alone or in combination with the related protein Mcl-1 did not compensate for CD27 deficiency in the response to influenza virus infection. This suggested that CD27 supports generation of the CD8(+) effector T cell pool not only by counteracting apoptosis via Bcl-2 family members. Genome-wide mRNA expression profiling indicated that CD27 directs expression of the Pim1 gene. Pim-1 is a serine/threonine kinase that sustains survival of rapidly proliferating cells by antiapoptotic and prometabolic effects that are independent of the mammalian target of rapamycin (mTOR) pathway. In TCR-primed CD8(+) T cells, CD27 could increment Pim-1 protein expression and promote cell survival throughout clonal expansion independent of the mTOR and IL-2R pathways. In addition, introduction of the Pim1 gene in Cd27(-/-) CD8(+) T cells partially corrected their defect in clonal expansion and formation of an effector pool. We conclude that CD27 may contribute to the survival of primed CD8(+) T cells by the upregulation of antiapoptotic Bcl-2 family members but also calls the Pim-1 kinase survival pathway into action.     

HIV-specific CD8+ T cells exhibit markedly reduced levels of Bcl-2 and Bcl-xL

Human immunodeficiency virus-specific CD8(+) T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-x(L) in HIV-specific CD8(+) T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8(+) T cells compared with CMV-specific and total CD8(+) T cells from HIV-infected individuals as well as total CD8(+) T cells from healthy donors. CD8(+) T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8(+) T cells from HIV-infected individuals. HIV-specific CD8(+) T cells also had significantly lower levels of Bcl-x(L) compared with CMV-specific CD8(+) T cells. Finally, IL-15 induces both Bcl-2 and Bcl-x(L) expression in HIV-specific and total CD8(+) T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-x(L) may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8(+) T cells and suggest a possible mechanism by which IL-15 increases their survival.     

ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo

During an acute immune response, CD8 T cells undergo rapid expansion followed by a contraction phase during which the majority of activated T cells die, leaving a few survivors to persist as memory cells. The regulation of T cell survival is critical at each stage of this response. 4-1BB, a TNFR family member, has been implicated in prolonging the survival of activated and memory CD8 T cells; however, the precise mechanisms by which 4-1BB sustains T cell survival are incompletely understood. Upon aggregation on T cells, 4-1BB associates with two TNFR-associated factors (TRAF), TRAF1 and TRAF2. TRAF2 is essential for downstream signaling from 4-1BB; however, the role of TRAF1 in 4-1BB signaling has not been elucidated and there have been conflicting data as to whether TRAF1 provides a positive or a negative signal in T cells. In this study, we report that TRAF1 plays a critical role in survival signaling downstream of 4-1BB during CD8 T cell expansion in response to viral infection in vivo. Further analysis reveals that TRAF1-deficient cells are impaired in their ability to up-regulate the prosurvival Bcl-2 family member Bcl-x(L) and show increased levels of the proapoptotic Bcl-2 family member Bim following 4-1BB signaling. TRAF1-deficient CD8 T cells fail to activate ERK in response to 4-1BB ligation and inhibition of ERK signaling downstream of 4-1BB in wild-type cells leads to increased Bim levels. Thus, TRAF1 has a prosurvival effect in CD8 T cells via the 4-1BB-mediated up-regulation of Bcl-x(L) and ERK-dependent Bim down-modulation.     

Suppressor of cytokine signaling 1 regulates IL-15 receptor signaling in CD8+CD44high memory T lymphocytes

T lymphocyte survival, proliferation, and death in the periphery are dependent on several cytokines. Many of these cytokines induce the expression of suppressor of cytokine signaling-1 (SOCS1), a feedback inhibitor of JAK kinases. However, it is unclear whether the cytokines that regulate T lymphocyte homeostasis are critically regulated by SOCS1 in vivo. Using SOCS1(-/-)IFN-gamma(-/-) mice we show that SOCS1 deficiency causes a lymphoproliferative disorder characterized by decreased CD4/CD8 ratio due to chronic accumulation of CD8+CD44(high) memory phenotype T cells. SOCS1-deficient CD8+ T cells express elevated levels of IL-2Rbeta, show increased proliferative response to IL-15 and IL-2 in vitro, and undergo increased bystander proliferation and vigorous homeostatic expansion in vivo. Sorted CD8+CD44(high) T cells from SOCS1(-/-)IFN-gamma(-/-) mice respond 5 times more strongly than control cells, indicating that SOCS1 is a critical regulator of IL-15R signaling. Consistent with this idea, IL-15 stimulates sustained STAT5 phosphorylation in SOCS1-deficient CD8+ T cells. IL-15 strongly induces TNF-alpha production in SOCS1-deficient CD8+ T cells, indicating that SOCS1 is also a critical regulator of CD8+ T cell activation by IL-15. However, IL-15 and IL-2 induce comparable levels of Bcl-2 and Bcl-x(L) in SOCS1-deficient and SOCS1-sufficient CD8+ T cells, suggesting that cytokine receptor signals required for inducing proliferation and cell survival signals are not identical. These results show that SOCS1 differentially regulates common gamma-chain cytokine signaling in CD8+ T cells and suggest that CD8+ T cell homeostasis is maintained by distinct mechanisms that control cytokine-mediated survival and proliferation signals.     

Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

CD4 T cell help plays an important role in promoting CD8 T cell immunity to pathogens. In models of infection with vaccinia virus (VV) and Listeria monocytogenes, CD4 T cell help is critical for the survival of activated CD8 T cells during both the primary and memory recall responses. Still unclear, however, is how CD4 T cell help promotes CD8 T cell survival. In this study, we first showed that CD4 T cell help for the CD8 T cell response to VV infection was mediated by IL-21, a cytokine produced predominantly by activated CD4 T cells, and that direct action of IL-21 on CD8 T cells was critical for the VV-specific CD8 T cell response in vivo. We next demonstrated that this intrinsic IL-21 signaling was essential for the survival of activated CD8 T cells and the generation of long-lived memory cells. We further revealed that IL-21 promoted CD8 T cell survival in a mechanism dependent on activation of the STAT1 and STAT3 pathways and subsequent upregulation of the prosurvival molecules Bcl-2 and Bcl-x(L). These results identify a critical role for intrinsic IL-21 signaling in CD8 T cell responses to an acute viral infection in vivo and may help design effective vaccine strategies.     

The roles of IL-12 in providing a third signal for clonal expansion of naive CD8 T cells

Stimulation of an effective in vitro or in vivo response by naive CD8 T cells requires three signals: TCR engagement, costimulation/IL-2, and a third signal that can be provided by IL-12. In addition to being required for acquisition of cytolytic function, IL-12 is required for optimal IL-2-dependent proliferation and clonal expansion. In experiments examining in vitro stimulation of naive CD8 T cells, IL-12 is shown to stimulate expression of the IL-2R alpha-chain (CD25) to much higher levels than are reached in response to just TCR and costimulation and/or IL-2. In addition, high CD25 expression is substantially prolonged in the presence of IL-12. As a consequence, the cells proliferate more effectively in response to low levels of IL-2. Examination of adoptively transferred TCR transgenic CD8 T cells responding to peptide Ag confirmed that IL-12 up-regulates CD25 in vivo, even when B7-mediated costimulation is largely blocked. TCR- and IL-2-dependent proliferation of CD8 T cells from mice deficient in CD25 was also found to increase in the presence of IL-12, indicating that CD25 up-regulation is not the only mechanism by which IL-12 increases clonal expansion of the cells. IL-2 and IL-12 both act to increase expression of both CD25 and the IL-12R, thus providing positive cross-regulation of receptor expression. These results suggest that when cross-priming dendritic cells present class I/Ag and costimulatory ligands, and produce IL-12, naive CD8 T cells will begin to produce IL-2 and both receptors will be optimally up-regulated to insure that an effective response is generated.     

Priming in the presence of IL-10 results in direct enhancement of CD8+ T cell primary responses and inhibition of secondary responses

Although IL-10 acts as an inhibitory cytokine for APC and CD4(+) T cell function, its effects on CD8(+) T cells are unclear. Additionally, little is known about whether initial priming in the presence of IL-10 can have long-lasting effects and influence subsequent CD8(+) T cell responses that occur in the absence of the cytokine. In the present study, we clarified the role of IL-10 during primary responses and examined whether exposure to IL-10 during initial priming of CD8(+) T cells impacted secondary responses. To determine the effect of IL-10 on Ag-specific T cell responses, peptide-pulsed IL-10R2(-/-) splenic dendritic cells were used to prime T cells from OT-I CD8(+) TCR transgenic mice. During the primary response, the presence of IL-10 resulted in enhancement of CD8(+) T cell numbers without detectable alterations in the kinetics or percentage of cells that underwent proliferation. A modest increase in survival, not attributable to Bcl-2 or Bcl-x(L), was also observed with IL-10 treatment. Other parameters of CD8(+) T cell function, including IL-2, IFN-gamma, TNF-alpha, and granzyme production, were unaltered. In contrast, initial exposure to IL-10 during the primary response resulted in decreased OT-I expansion during secondary stimulation. This was accompanied by lowered IL-2 levels and reduced percentages of proliferating BrdU(+) cells and OT-I cells that were CD25(high). IFN-gamma, TNF-alpha, and granzyme production were unaltered. These data suggest that initial exposure of CD8(+) T cells to IL-10 may be temporarily stimulatory; however, programming of the cells may be altered, resulting in diminished overall responses.     

On CD28/CD40 ligand costimulation, common gamma-chain signals, and the alloimmune response

Activation and robust expansion of naive T cells often require T cell costimulatory signals and T cell growth factors. However, the precise growth and costimulation requirements for activation and expansion of CD4(+) and CD8(+) T cells in vivo in allograft response are still not clearly defined. In the present study, we critically examined the role of CD28/CD40 ligand (CD40L) costimulation and the common gamma-chain (gamma(c)) signals, a shared signaling component by receptors for all known T cell growth factors (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21), in activation and expansion of CD4(+) and CD8(+) T cells in the allogeneic hosts. We found that CD28/CD40L costimulation and the gamma(c) signals are differentially involved in proliferation and clonal expansion of CD4(+) and CD8(+) T cells in response to alloantigen stimulation. CD8(+) T cells are highly dependent on the gamma(c) signals for survival, expansion, and functional maturation, whereas in vivo expansion of alloreactive CD4(+) T cells is largely gamma(c) independent. T cell costimulation via CD28 and CD40L, however, is necessary and sufficient for activation and expansion of CD4(+) T cells in vivo. In a skin transplant model, blocking both CD28/CD40L and the gamma(c) pathways induced prolonged skin allograft survival. Our study provides critical insights that the CD4 and CD8 compartments are most likely governed by distinct mechanisms in vivo, and targeting both costimulatory and gamma(c) signals may be highly effective in certain cytopathic conditions involving activation of both CD4(+) and CD8(+) T cells.     

Signal 3 availability limits the CD8 T cell response to a solid tumor

CD8 T cells need a third signal, along with Ag and costimulation, for effective survival and development of effector functions, and this can be provided by IL-12 or type I IFN. Adoptively transferred OT-I T cells, specific for H-2K(b) and OVA, encounter Ag in the draining lymph nodes of mice with the OVA-expressing E.G7 tumor growing at a s.c. site. The OT-I cells respond by undergoing limited clonal expansion and development of effector functions (granzyme B expression and IFN-gamma production), and they migrate to the tumor where they persist but fail to control tumor growth. In contrast, OT-I T cells deficient for both the IL-12 and type I IFN receptors expand only transiently and rapidly disappear. These results suggested that some signal 3 cytokine is available, but that it is insufficient to support a CTL response that can control tumor growth. Consistent with this, administration of IL-12 at day 10 of tumor growth resulted in a large and sustained expansion of wild-type OT-I cells with enhanced effector functions, and tumor growth was controlled. This did not occur when the OT-I cells lacked the IL-12 and type I IFN receptors, demonstrating that the therapeutic effect of IL-12 results from direct delivery of signal 3 to the CD8 T cells responding to tumor Ag in the signal 3-deficient environment of the tumor.     

Protein kinase C-theta is an early survival factor required for differentiation of effector CD8+ T cells

CD8(+) T cells are crucial for host defense against invading pathogens and malignancies. However, relatively little is known about intracellular signaling events that control the genetic program of their activation and differentiation. Using CD8(+) T cells from TCR-transgenic mice crossed to protein kinase C-theta (PKCtheta)-deficient mice, we report that PKCtheta is not required for Ag-induced CD8(+) T cell proliferation, but is important for T cell survival and differentiation into functional, cytokine-producing CTLs. Ag-stimulated PKCtheta(-/-) T cells underwent accelerated apoptosis associated with deregulated expression of Bcl-2 family proteins and displayed reduced activation of ERKs and JNKs. Some defects in the function of PKCtheta(-/-) T cells (poor survival and reduced Bcl-2 and Bcl-x(L) expression, CTL activity, and IFN-gamma expression) were partially or fully restored by coculture with wild-type T cells or by addition of exogenous IL-2, whereas others (increased Bim(EL) expression and TNF-alpha production) were not. These findings indicate that PKCtheta, although not essential for initial Ag-induced proliferation, nevertheless plays an important role in promoting and extending T cell survival, thereby enabling the complete genetic program of effector CD8(+) differentiation. The requirement for PKCtheta in different types of T cell-dependent responses may, therefore, depend on the overall strength of signaling by the TCR and costimulatory receptors and may reflect, in addition to its previously established role in activation, an important, hitherto unappreciated, role in T cell survival.     

IL-15 promotes the survival of naive and memory phenotype CD8+ T cells

IL-15 stimulates the proliferation of memory phenotype CD44(high)CD8(+) T cells and is thought to play a key role in regulating the turnover of these cells in vivo. We have investigated whether IL-15 also has the capacity to affect the life span of naive phenotype (CD44(low)) CD8(+) T cells. We report that IL-15 promotes the survival of both CD44(low) and CD44(high) CD8(+) T cells, doing so at much lower concentrations than required to induce proliferation of CD44(high) cells. Rescue from apoptosis was associated with the up-regulation of Bcl-2 in both cell types, whereas elevated expression of Bcl-x(L) was observed among CD44(high) but not CD44(low) CD8(+) cells. An investigation into the role of IL-15R subunits in mediating the effects of IL-15 revealed distinct contributions of the alpha- and beta- and gamma-chains. Most strikingly, IL-15R alpha was not essential for either induction of proliferation or promotion of survival by IL-15, but did greatly enhance the sensitivity of cells to low concentrations of IL-15. By contrast, the beta- and gamma-chains of the IL-15R were absolutely required for the proliferative and pro-survival effects of IL-15, although it was not necessary for CD44(high)CD8(+) cells to express higher levels of IL-15R beta than CD44(low) cells to proliferate in response to IL-15. These results show that IL-15 has multiple effects on CD8 T cells and possesses the potential to regulate the life span of naive as well as memory CD8(+) T cells.     

Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.     

Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells

Bcl-2 plays a critical role in regulating cell survival and apoptosis. We examined Bcl-2 expression in virus-specific CD8 T cells during the expansion, death, and memory phases of the T cell response following infection of mice with lymphocytic choriomeningitis virus (LCMV). Naive CD8 T cells expressed a basal level of Bcl-2 that was down-regulated in effector CD8 T cells just before the death phase. Bcl-2 levels remained low during the death phase but surviving memory CD8 T cells expressed higher levels of Bcl-2 than naive cells. These changes were shown to occur in LCMV TCR transgenic cells as well as virus-specific CD8 T cells in C57BL/6 and BALB/c mice identified by MHC class I tetramers. In all instances, memory CD8 T cells expressed higher levels of Bcl-2, suggesting that increased Bcl-2 expression plays a role in the long-term maintenance of memory CD8 T cells in vivo.     

TNF receptor type 2 (p75) functions as a costimulator for antigen-driven T cell responses in vivo

Naive T cells require costimulation for robust Ag-driven differentiation and survival. Members of the TNFR family have been shown to provide costimulatory signals conferring survival at distinct phases of the T cell response. In this study, we show that CD4 and CD8 T cells depend on TNFR type 2 (p75) for survival during clonal expansion, allowing larger accumulation of effector cells and conferring protection from apoptosis for a robust memory pool in vivo. We demonstrate using the MHC class I-restricted 2C TCR and MHC class II-restricted AND TCR transgenic systems that TNFR2 regulates the threshold for clonal expansion of CD4 and CD8 T cell subsets in response to cognate Ag. Using a novel recombinant Listeria monocytogenes (rLM) expressing a secreted form of the 2C agonist peptide (SIY) to investigate the role of TNFR2 for T cell immunity in vivo, we found that TNFR2 controls the survival and accumulation of effector cells during the primary response. TNFR2-/- CD8 T cells exhibit loss of protection from apoptosis that is correlated with diminished survivin and Bcl-2 expression. Null mutant mice were more susceptible to rLM-SIY challenge at high doses of primary infection, correlating with impaired LM-specific T cell response in the absence of TNFR2-mediated costimulation. Moreover, the resulting memory pools specific for SIY and listeriolysin O epitopes derived from rLM-SIY were diminished in TNFR2-/- mice. Thus, examination of Ag-driven T cell responses revealed a hitherto unknown costimulatory function for TNFR2 in regulating T cell survival during the differentiation program elicited by intracellular pathogen in vivo.     

Bcl-2 allows effector and memory CD8+ T cells to tolerate higher expression of Bim

As acute infections resolve, most effector CD8(+) T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8(+) T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8(+) T cells reported to have a longer lifespan (i.e., KLRG1(low)CD127(high)) have increased levels of Bcl-2 compared with their shorter-lived KLRG1(high)CD127(low) counterparts. Surprisingly, we found that these effector KLRG1(low)CD127(high) CD8(+) T cells also had increased levels of Bim compared with KLRG1(high)CD127(low) cells. Similar effects were observed in memory cells, in which CD8(+) central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8(+) effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8(+) T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8(+) T cells. Finally, we found that Bim levels were significantly increased in effector CD8(+) T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.     

p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-x(L). Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x(L) and its association with 14-3-3tau. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-x(L) and the increased association of Bad with 14-3-3tau. Our findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways.