Evidence of a novel IL-2/15R beta-targeted cytokine involved in homeostatic proliferation of memory CD8+ T cells

The homeostasis of memory CD8+ T cells is regulated by cytokines. IL-15 is shown to promote the proliferation of memory CD8+ T cells, while IL-2 suppresses their division in vivo. This inhibitory effect of IL-2 appears to occur indirectly, through other cell populations including CD25+CD4+ T cells; however, the details of this mechanism remain unclear. In this study, we show that 1) both Ag-experienced and memory phenotype CD8+ T cells divided after the depletion of IL-2 in vivo; 2) this division occurred normally and CD44(high)IL-2/15Rbeta(high) CD8+ T cells generated after IL-2 depletion in IL-15 knockout (KO) and in IL-7-depleted IL-15 KO mice; 3) surprisingly, the blockade of IL-2/15Rbeta signaling in IL-2-depleted IL-15 KO mice completely abolished the division of memory CD8+ T cells, although the only cytokines known to act through IL-2/15Rbeta are IL-2 and IL-15; and 4) the expression of IL-2/15Rbeta molecules on memory CD8+ T cells was required for their division induced by IL-2 depletion. These results demonstrate that the depletion of IL-2 in vivo induced memory CD8+ T cell division by an IL-15-independent but by an IL-2/15Rbeta-dependent mechanism, suggesting the existence of a novel IL-2/15Rbeta-utilizing cytokine that acts directly on memory CD8+ T cells to promote cell division.     

Generation and growth of CD28nullCD8+ memory T cells mediated by IL-15 and its induced cytokines

Accumulation of CD28(null)CD8+ T cells and the defects of these cells in response to antigenic stimulation are the hallmarks of age-associated decline of T cell function. However, the mechanism of these age-associated changes is not fully understood. In this study, we report an analysis of the growth of human CD28(null) and CD28+CD8+ memory T cells in response to homeostatic cytokine IL-15 in vitro. We showed that 1) there was no proliferative defect of CD28(null)CD8+ memory T cells in response to IL-15 compared with their CD28+ counterparts; 2) stable loss of CD28 expression occurred in those actively dividing CD28+CD8+ memory T cells responding to IL-15; 3) the loss of CD28 was in part mediated by TNF-alpha that was induced by IL-15; and 4) CCL4 (MIP-1beta), also induced by IL-15, had a significant inhibitory effect on the growth of CD28(null) cells, which in turn down-regulated their expression of CCL4 receptor CCR5. Together, these findings demonstrate that CD28(null)CD8+ memory T cells proliferate normally in response to IL-15 and that IL-15 and its induced cytokines regulate the generation and growth of CD28(null)CD8+ T cells, suggesting a possible role of IL-15 in the increase in CD28(null)CD8+ T cells that occurs with aging.     

Profound enhancement of the IL-12/IL-18 pathway of IFN-gamma secretion in human CD8+ memory T cell subsets via IL-15

Human memory CD8(+) T cell subsets, termed central memory and effector memory T cells, can be identified by expression of CD45RA, CD62 ligand (CD62L), and CCR7. Accordingly, functional differences have been described for each subset, reflecting unique roles in immunological memory. The common gamma-chain cytokines IL-15 and IL-7 have been shown to induce proliferation and differentiation of human CD8(+) T cell subsets, as well as increased effector functions (i.e., cytokines, cytotoxicity). In this study, we observed that addition of IL-15 or IL-7 to cultures of human CD8(+) T cells profoundly enhanced the IL-12-IL-18 pathway of IFN-gamma production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction of IFN-gamma by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior in its ability to enhance IL-12-IL-18-induced IFN-gamma, without evidence of a synergistic effect between IL-15 and IL-7. We also observed that IL-15- and IL-7-mediated enhancement of IL-12-IL-18-induced IFN-gamma production was a functional property of effector memory CD8(+) T cells. Despite a lack of association between cell division and acquisition of IL-12-IL-18-induced IFN-gamma, down-regulation of CD62L expression correlated well with increased IL-12-IL-18-induced IFN-gamma. Purified central memory T cells stimulated with IL-15 and IL-7 down-regulated CD62L and acquired potent IL-12-IL-18-induced IFN-gamma similar to effector memory T cells. Thus, in addition to its known role in development of T cell memory, IL-15 may amplify memory CD8(+) T cell effector functions by increasing sensitivity to proinflammatory cytokine stimulation.     

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.     

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.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands

The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

An IFN-gamma-dependent pathway controls stimulation of memory phenotype CD8+ T cell turnover in vivo by IL-12, IL-18, and IFN-gamma

Unlike naive T cells, memory phenotype (CD44(high)) T cells exhibit a high background rate of turnover in vivo. Previous studies showed that the turnover of memory phenotype CD8(+) (but not CD4(+)) cells in vivo can be considerably enhanced by products of infectious agents such as LPS. Such stimulation is TCR independent and hinges on the release of type I IFNs (IFN-I) which leads to the production of an effector cytokine, probably IL-15. In this study, we describe a second pathway of CD44(high) CD8(+) stimulation in vivo. This pathway is IFN-gamma rather than IFN-I dependent and is mediated by at least three cytokines, IL-12, IL-18, and IFN-gamma. As for IFN-I, these three cytokines are nonstimulatory for purified T cells and under in vivo conditions probably act via production of IL-15.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells

Naive T cells undergo slow homeostatic proliferation in response to T cell lymphopenia, which is also called lymphopenia-induced proliferation (LIP). IL-7 is critically required for this process, but previous studies suggested IL-15 was expendable for LIP of naive CD8 T cells. In contrast, we show that IL-15 is important for sustained CD8 T cell proliferation and accumulation in a lymphopenic setting, as revealed by truncated LIP in IL-15(-/-) hosts. At the same time, we find that IL-12 enhances LIP by acting directly on the CD8 T cells and independently of IL-15, suggesting distinct pathways by which cytokines can regulate homeostatic proliferation. Interestingly, the memory-phenotype CD8 T cell generated by LIP in IL-15(-/-) hosts are phenotypically distinct from the rare endogenous memory-phenotype cells found in IL-15(-/-) animals, suggesting these cells are generated by different means. These findings demonstrate that cytokine requirements for LIP change during the process itself, illustrating the need to identify factors that regulate successive stages of lymphopenia-driven proliferation.     

Exposure to IL-15 and IL-21 enables autoreactive CD8 T cells to respond to weak antigens and cause disease in a mouse model of autoimmune diabetes

Autoreactive CD8(+) T lymphocytes play a key role in the pathogenesis of several autoimmune diseases. It is not yet well understood how autoreactive CD8(+) T cells, which express TCRs with low reactivity toward self-Ags, gain the ability to respond to autoantigens to cause disease. Previously, we have shown that prior stimulation of CD8(+) T cells with synergistic combinations of cytokines produced by the innate immune response, such as IL-21 and IL-15, induces Ag-independent proliferation. Such "cytokine-primed" CD8 T cells displayed increased responsiveness to limiting quantities of the cognate Ag. In this paper, we report that prior stimulation with IL-15 and IL-21 also enables CD8(+) T cells to respond to weakly agonistic TCR ligands, resulting in proliferation, cytokine secretion, and cytolytic activity. Using a transgenic mouse model of autoimmune diabetes, we show that cytokine-primed autoreactive CD8(+) T cells induce disease following stimulation by weak TCR ligands, but their diabetogenic potential is dependent on continuous availability of IL-15 in vivo. These findings suggest that inflammatory cytokines could facilitate the triggering of autoreactive CD8(+) T cells by weak autoantigens, and this mechanism may have important implications for autoimmune diseases associated with microbial infections and chronic inflammation.     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

Differential production of IL-23 and IL-12 by myeloid-derived dendritic cells in response to TLR agonists

The recently delineated role for IL-23 in enhancing Th-17 activity suggests that regulation of its expression is distinct from that of IL-12. We hypothesized that independent TLR-mediated pathways are involved in the regulation of IL-12 and IL-23 production by myeloid-derived dendritic cells (DCs). The TLR 2 ligand, lipoteichoic acid (LTA), the TLR 4 ligand, LPS, and the TLR 7/8 ligand, resimiquod (R848), induced production of IL-23 by DCs. None of these TLR ligands alone induced significant IL-12 production, except when combined with IFN-gamma or other TLR ligands. Notably, IL-23 production in response to single TLR ligands was inhibited by IL-4. DCs treated with single TLR agonists induced IL-17A production by allogeneic and Ag-specific memory CD4(+) T cells, an effect that was abrogated by IL-23 neutralization. Moreover, these DCs stimulated IL-17A production by tumor peptide-specific CD8(+) T cells. In contrast, DCs treated with dual signals induced naive and memory Th1 responses and enhanced the functional avidity of tumor-specific CD8(+) T cells. These results indicate that distinct microbial-derived stimuli are required to drive myeloid DC commitment to IL-12 or IL-23 production, thereby differentially polarizing T cell responses.     

Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells

IL-15 and IL-15Ralpha are required for generation of memory-phenotype CD8 T cells in unimmunized mice. However, the role of IL-15 in primary expansion and generation of Ag-specific memory CD8 T cells in vivo has not been investigated. We characterized the CD8 T cell response against vesicular stomatitis virus (VSV) in IL-15(-/-) and IL-15Ralpha(-/-) mice. Surprisingly, IL-15 was required for primary expansion of VSV-specific CD8 T cells. The generation of VSV-specific memory CD8 T cells was also impaired without IL-15 signaling, and this defect correlated with a decrease in memory CD8 T cell turnover. Despite minimal proliferation without IL-15, a subset of memory cells survived long-term. IL-15Ralpha expression was low on naive CD8 T cells, up-regulated on Ag-specific effector cells, and sustained on memory cells. Thus, IL-15 was important for the generation and the subsequent maintenance of antiviral memory CD8 T cells.     

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.     

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.