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The Journal of Immunology, 2007, 179, 6734 -6740
Copyright © 2007 by The American Association of Immunologists, Inc.
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Dual Roles of IL-15 in Maintaining IL-7R{alpha}lowCCR7– Memory CD8+ T Cells in Humans via Recovering the Phosphatidylinositol 3-Kinase/AKT Pathway1

Hang-Rae Kim2, Kyung-A Hwang2 and Insoo Kang3

Department of Internal Medicine, Section of Rheumatology, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recently, we identified two subsets of CCR7– memory CD8+ T cells expressing high and low levels of the IL-7R {alpha}-chain (IL-7R{alpha}) that is essential for memory T cell survival in human peripheral blood. IL-7R{alpha}lowCCR7– memory CD8+ T cells that produce effector cytokines and perforin have impaired proliferation and survival in response to TCR triggering and IL-7, respectively. These findings raise a question of how such cells are sustained at significant numbers, >20% of peripheral CD8+ T cells, despite impaired IL-7- and TCR-mediated cell maintenance. In this study, we demonstrate that IL-7R{alpha}lowCCR7– memory CD8+ T cells have increased expression of IL-2/15R β-chain (IL-2/15Rβ), which is critical for IL-15 signaling, with enhanced gene expression of T box expressed in T cells (T-bet) and eomesodermin (eomes), transcriptional factors involved in IL-2/15Rβ expression compared with IL-7R{alpha}highCCR7– memory CD8+ T cells. Such a cytokine chain is functional as IL-7R{alpha}lowCCR7– memory CD8+ T cells proliferate considerably in response to IL-15. Furthermore, adding IL-15 to TCR triggering recovers impaired TCR-mediated proliferation of IL-7R{alpha}low memory CD8+ T cells via restoring the activation of the PI3K/AKT pathway. These findings indicate that IL-15 has dual roles in maintaining IL-7R{alpha}lowCCR7– memory CD8+ T cells via TCR-dependent and -independent mechanisms. Moreover, IL-15 can be useful in reviving impaired proliferative function of such memory CD8+ T cells with effector functions against infections and tumors via rescuing the PI3K/AKT pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunologic memory is a hallmark of the adaptive immune system. Naive T cells produced from the thymus proliferate and differentiate into effector T cells up on recognition of proper Ag. Although a large number of effector T cells eventually die, a small fraction becomes memory T cells providing a lifetime immune protection that is critical for host defense. The precise mechanisms for the generation and maintenance of memory T cells have been the subject of intense investigation and are not entirely understood. Ag persistency may help in maintaining memory T cell populations (1). However, studies show that Ag is not required for the maintenance of memory T cells (2). Memory T cells can survive and even slowly divide in the absence of Ag, suggesting that soluble factors such as cytokines can support this T cell compartment (3, 4, 5, 6). Indeed, several studies have demonstrated that IL-7 and IL-15, both members of the common cytokine receptor {gamma}-chain ({gamma}C)4 family cytokines, are essential in maintaining memory T cells using murine models (reviewed in Refs. 7, 8, 9).

IL-7 is produced by multiple stromal tissues, including epithelial cells in the thymus and bone marrow (8, 9). The biologic effect of IL-7 on target cells is controlled by the IL-7R complex that consists of two chains, the high-affinity IL-7R{alpha}-chain and {gamma}C (8, 9). IL-7 binding to the receptor promotes cell survival by up-regulating Bcl-2, an antiapoptotic molecule (8, 10). In mice, T cells expressing high levels of the IL-7R{alpha} chain survived better following infection compared with T cells expressing low levels of the same cytokine receptor chain (11), indicating that the essential role for this molecule in regulating memory T cell survival.

We recently identified two subsets of CD8+ T cells expressing IL-7R{alpha}high and IL-7R{alpha}low in effector memory (CD45RA–CCR7–) and CD45RA+ effector memory (EMCD45RA+; CD45RA+CCR7–) CD8+ T cells in human peripheral blood (12). IL-7R{alpha}lowCCR7– memory CD8+ T cells have limited proliferation and survival in response to TCR triggering and IL-7, respectively, whereas IL-7R{alpha}highCCR7– memory CD8+ T cells survive and proliferate vigorously under the same conditions (12). Although IL-7R{alpha}lowCCR7– memory CD8+ T cells are functionally exhausted cells with regard to TCR-mediated replication and IL-7-mediated survival, they represent >20% of the total peripheral CD8+ T cells in human adults (12). Taken together, these observations raise an intriguing question of how IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans are maintained in vivo. In the present study, we investigated a role for IL-15 in maintaining such cells since this cytokine is known to induce memory CD8+ T cell proliferation in mice (6, 13, 14) and animals genetically lacking IL-15 and the receptor for IL-15 do not have memory phenotype CD8+ T cells (15, 16).

IL-15 is produced largely by dendritic cells, monocytes, and stromal cells (17). The IL-15R complex consists of three chains, IL-15R{alpha}, IL-2/15Rβ, and {gamma}C chains. IL-15 binds the endogenous IL-15R{alpha} chain expressed by IL-15-producing cells and is trans-presented to the complex of IL-2/15Rβ and {gamma}C chains with the signaling capacity expressed on T cells (reviewed in Ref. 17). The recognition of IL-15 by the latter receptor chains leads to the activation of various signaling pathways including JAK1 and 3/STAT5 as well as PI3K/AKT (protein kinase B) (18). IL-15 has diverse functions that include promoting cytotoxicity and maintenance of memory CD8+ T cells as well as proliferation and differentiation of NK and NK T cells (17). In the current study, we have found that IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans express higher levels of IL-2/15Rβ and two transcriptional factors T-bet and eomes, which are involved in IL-2/15Rβ expression (19), compared with IL-7R{alpha}highCCR7– memory CD8+ T cells. IL-7R{alpha}lowCCR7– memory CD8+ T cells proliferate considerably in response to IL-15, indicating intact function of this cytokine receptor chain. Furthermore, we demonstrate that IL-7R{alpha}lowCCR7– memory CD8+ T cells have reduced signaling through the PI3K/AKT pathway in response to TCR triggering. IL-15 can restore this signaling pathway, leading to synergistic cell proliferation by IL-15 and anti-CD3 Ab stimulations even in the absence of CD28 costimulation. These findings indicate that IL-15 is involved in maintaining IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans via TCR-dependent and -independent mechanisms. Furthermore, our work suggests that IL-15 can be therapeutically useful in reviving the exhausted proliferative function of IL-7R{alpha}lowCCR7– memory CD8+ T cells specific for microorganisms and tumors via activating the PI3K/AKT pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and flow cytometry

This work was approved by the institutional review committee of Yale University. Human peripheral blood was drawn from healthy adult subjects after obtaining informed consent. As previously described (12), PBMCs were purified and stained with goat anti-human IL-7R{alpha} Abs (R&D Systems) followed by staining with donkey anti-goat IgG Abs (R&D Systems) and Abs to CD8 and CCR7 (BD Pharmingen). Cells were sorted into IL-7R{alpha}high and IL-7R{alpha}lowCCR7–CD8+ T cells using a FACSAria (BD Immunocytometry Systems). Some PBMCs were stained with Abs to IL-7R{alpha}, CD8, and CCR7 as well as Abs to IL-15R{alpha} (catalog no. MAB1471; R&D Systems), IL-2/15Rβ (catalog no. 554522; BD Biosciences), or isotype Abs (R&D Systems) followed by analyses on an LSRII (BD Immunocytometry Systems). Collected data were analyzed using FlowJo software (Tree Star).

Cell proliferation assay

Sorted cells were labeled with 0.5 µM CFSE (Molecular Probes) (12) and incubated for 7 days in a 96-well flat-bottom tissue culture plate coated with 10 µg/ml anti-CD3 Abs (BD Pharmingen) in the presence or absence of 10 µg/ml anti-CD28 Abs (BD Pharmingen), IL-15 (10 µg/ml; R&D Systems), and/or signaling molecule inhibitors (LY294002 (20 µM) and PD98059 (10 µM) from Cell Signaling Technology and AKT inhibitors II (25 µM) and IV (1 µM) from Calbiochem). Some cells were stimulated with IL-15 in the presence of Abs to IL-2/15Rβ (R&D Systems) or isotype controls (20). Cell proliferation was analyzed on a FACSCalibur (BD Immunocytometry Systems).

Western blot assay

As previously described (21), polystyrene latex microspheres (beads, diameter 6 µm; Polysciences) were coated with anti-CD3 Abs and used for cell stimulation. Sorted IL-7R{alpha}high and IL-7R{alpha}low memory CD8+ T cells (1 x 106 cells/200 µl) were rested overnight in RPMI 1640 medium with 10% FBS and stimulated with anti-CD3 Ab-coated beads (beads:cells, 2:1 ratio) in the presence or absence of IL-15 (50 ng/ml) for 1 h at 37°C. Some cells were pretreated with 20 µM LY294002 for 2 h before stimulation. Stimulated cells were lysed at 4°C for 30 min in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology) and analyzed by immunoblotting with a 1/500–1/2500 dilution of primary Abs (phospho-AKT (Thr308) and AKT from Cell Signaling Technology) followed by appropriate secondary Abs conjugated with HRP. Blots were visualized using an ECL system (Amersham Biosciences) or Supersignal West Femto Maximum Sensitivity Substrate (Pierce) followed by analysis of immunoblot density by TINA software, version 2.10e (Raytes). For reprobing, Abs were stripped off the membranes using Restore Western Blot Stripping buffer (Pierce).

Real-time PCR

cDNA was synthesized from total RNA isolated from cells. The PCR were performed by 1x SYBR Green mix (Qiagen). All results were normalized to β-actin expression. The following primer sequences were used for real-time PCR: IL-2/15Rβ forward, 5'-CCTGAATGTTTCAGACCACA-3' and reverse, 5'-ATAAGGAGACCGACTTGCAG-3'; t-bet forward, 5'-AGAAAGTCTTGGGCATGAAG-3' and reverse, 5'-TCAGGGATTAACACACATGC-3'; eomesodermin (eomes) forward, 5'-ACTTCTAGCACATTGCTCCC-3' and reverse, 5'-TTCCTCTGGTAAGAACCTCG-3'; β-actin forward, 5'-GAATCTCCGACCACCACTAC-3' and reverse, 5'-AAG GTGCTCAGGTCATTCTC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human IL-7R{alpha}lowCCR7– memory CD8+ T cells with limited TCR-mediated proliferative capacity express high levels of IL-15Rβ and proliferate in response to IL-15

We have investigated a potential role for IL-15 in maintaining human IL-7R{alpha}lowCCR7– memory CD8+ T cells which have decreased proliferation and survival in response to TCR triggering and IL-7, respectively. Although we had previously reported the expression of IL-15R{alpha} and IL-2/15Rβ (CD122) on IL-7R{alpha}high and IL-7R{alpha}low memory CD8+ T cells in peripheral blood from healthy human subjects, the expression levels of such molecules had not been directly compared between the two cell subsets (12). In the current study, we found that IL-7R{alpha}high and IL-7R{alpha}low memory CD8+ T cells express IL-15R{alpha}, which presents IL-15 to responding cells (17), at similar levels (Fig. 1, B and D). In contrast, the expression of IL-2/15Rβ, which is essential for transmitting IL-15 signaling in the cells (17), was significantly higher on IL-7R{alpha}lowCCR7– memory CD8+ T cells than on IL-7R{alpha}highCCR7– memory CD8+ T cells (Fig. 1, C and E).


Figure 1
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  		FIGURE 1. IL-15R{alpha} and IL-2/15Rβ expression by IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8 + T cells. PBMCs were stained with anti-CD8, CCR7, CD45RA, IL-7R{alpha}, IL-15R{alpha}, IL-2/15Rβ, or isotype Abs and analyzed on a flow cytometer (A–E). A, CCR7– memory CD8+ T cells have two different subsets of CD8+ T cells expressing high (IL-7R{alpha}high) and low (IL-7R{alpha}low) levels of IL-7R{alpha} (shaded histogram). B and C, Representative histograms of IL-15R{alpha} and IL-2/15Rβ (shaded histograms) and isotype control (open histograms) staining. D and E, Mean fluorescence intensity (MFI) of IL-15R{alpha} and IL-2/15Rβ staining is compared between IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells (n = 12). Values of p were obtained using Student’s two-tailed t test.

 
We next measured IL-2/15Rb gene expression as well as T-bet and eomes, transcriptional factors involved in expressing IL-2/15Rb (19), in IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells using real-time PCR. Indeed, the expression levels of these genes were higher in IL-7R{alpha}lowCCR7– memory CD8+ T cells than in IL-7R{alpha}highCCR7– memory CD8+ T cells (Fig. 2). This result suggests that the increased IL-2/15Rβ expression on the former cells is governed at the gene level in association with transcriptional factor T-bet and eomes as previously described in a mouse study (19).


Figure 2
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  		FIGURE 2. The gene expression of IL-2/15Rb, T-bet, and eomesodermin in IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells. PBMCs were stained with anti-CD8, CCR7, CD45RA, IL-7R{alpha}, IL-15R{alpha}, IL-2/15Rβ, or isotype Abs and sorted into IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells using a FACSAria. Total DNA was extracted from the sorted CD8+ T cell subsets and used for cDNA synthesis. The gene expression of IL-2/15Rb, T-bet, and eomesodermin (eomes) was measured by real-time PCR and all results were normalized to β-actin expression. Graphs show the mean ± SEM (n = 4) of the indicated genes.

 
To determine the biological significance of IL-2/15Rβ expression on IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans, we measured proliferation of these cells in the presence of IL-15 (500 pg/ml and 50 ng/ml) or PBS. The two subsets of memory CD8+ T cells proliferated considerably in response to high-dose IL-15 (50 ng/ml) but no detectable proliferation was noticed in response to low-dose IL-15 (500 pg/ml) or PBS (Fig. 3A). Also, IL-7R{alpha}high and IL-7R{alpha}lowCCR7–memory CD8+ T cells proliferated when they were stimulated with intermediate-dose IL-15 (5 ng/ml; Fig. 3B). Adding Abs to IL-2/15Rβ blocked the IL-15-mediated proliferation of IL-7R{alpha}high and IL-7R{alpha}lowCCD7– memory CD8+ T cells (data not shown), which further supports the biologic significance of their IL-2/15Rβ expression. Despite the difference in the expression of IL-15Rβ between IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells, their proliferative response to IL-15 was comparable (Fig. 3, A and B). This finding is different from the results of a mouse study where the proliferation of IL-15Rβhigh (CD122) and IL-15RβlowCD8+ T cells was measured in response to IL-15 and IL-15-inducing agents (LPS and (poly I:C)) (13). This discrepancy could be secondary to the difference in the species of study subjects (human vs mouse), proliferation system (in vivo vs in vitro), or time kinetics (day 3 vs day 7). In fact, measuring cell proliferation at day 4 of stimulation with IL-15 (50 ng/ml) showed comparable levels of proliferation in IL-7R{alpha}high and IL-7R{alpha}lowCCD7– memory CD8+ T cells (data not shown). Overall, our findings indicate that IL-7R{alpha}low CCR7– memory CD8+ T cells retain the capability to properly use IL-15 via expressing the IL-2/15Rβ chain and that IL-15 can be an essential factor for maintaining this cell subset independently of TCR triggering and IL-7.


Figure 3
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  		FIGURE 3. IL-15 restores limited TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells. PBMCs were stained with Abs to CD8, CCR7, and IL-7R{alpha} and sorted into IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells. A, IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells were labeled with CFSE and stimulated for 7 days with IL-15 or PBS. B, CFSE-labeled cells were incubated for 7 days in a 96-well flat-bottom tissue culture plate coated with anti-CD3 Abs (10 µg/ml) in the presence or absence of anti-CD28 Abs (10 µg/ml) and/or IL-15 (5 ng/ml). Results are representative data from five independent experiments. Numbers on histograms indicate the frequency of proliferating cells.

 
IL-15 restores impaired TCR-mediated proliferation of human IL-7R{alpha}lowCCR7– memory CD8+ T cells

We next determined whether IL-15 could restore impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans. This notion was based on the fact that the major source of IL-15 is APCs, which can also provide TCR triggering by presenting appropriate antigenic peptides to T cells (8, 17). IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells were sorted from PBMCs and stimulated with anti-CD3 Abs, anti-CD3/-CD28 Abs, IL-15 (5 ng/ml), or combinations thereof. As previously reported (12), IL-7R{alpha}lowCCR7– memory CD8+ T cells had decreased proliferation in response to anti-CD3 and anti-CD28 Ab stimulation compared with IL-7R{alpha}highCCR7– memory CD8+ T cells (Fig. 3B). Although such a phenomenon could stem from decreased CD28 expression on the former cells (12), our experiment showed that the proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells was still lower than in IL-7R{alpha}highCCR7– memory CD8+ T when cells were stimulated with anti-CD3 Abs alone (Fig. 3B). This observation suggests that IL-7R{alpha}lowCCR7– memory CD8+ T cells have an intrinsic alteration in TCR-mediated proliferation.

Of interest, adding IL-15 (5 ng/ml) to TCR triggering enhanced proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells (Fig. 3B), indicating the restoration of impaired TCR-mediated proliferation in this cell subset. A similar level of proliferation was noticed when high-dose IL-15 (50 ng/ml) was added, although the restoration of the proliferation was less in the presence of low-dose IL-15 (500 pg/ml; data not shown). Such an effect was independent of CD28 costimulation because IL-7R{alpha}lowCCR7– memory CD8+ T cells that were stimulated with anti-CD3 Abs/IL-15 or anti-CD3/anti-CD28 Abs/IL-15 had similar proliferation (Fig. 3B). The combination of IL-15 and anti-CD3/anti-CD28 Abs did not enhance the proliferation of IL-7R{alpha}high memory CD8+ T cells either, although adding this cytokine to anti-CD3 Ab alone moderately improved the proliferation of the same cells (Fig. 3B). In fact, the numbers of proliferating IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells were similar when they were stimulated with the combination of anti-CD3 Abs and IL-15. These observations indicate that IL-15 can restore impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells independently of CD28 costimulation and that such a mechanism can be essential for expanding and maintaining IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans.

The restoration of impaired TCR-mediated proliferation of human IL-7R{alpha}lowCCR7– memory CD8+ T cells by IL-15 is through activating the PI3K/AKT pathway

We investigated a potential underlying mechanism for restoring impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells by IL-15. The pathways involved in IL-15 signaling include JAK/STAT and PI3K/AKT (17, 18). The PI3K/AKT pathway also participates in TCR signaling (22). In fact, studies indicate that the PI3K/AKT pathway has a role in regulating the proliferation of T cells (23, 24). Thus, we measured activation (phosphorylation) of AKT in IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells in response to TCR triggering. IL-7R{alpha}lowCCR7– memory CD8+ T cells had decreased phosphorylation of AKT compared with IL-7R{alpha}highCCR7– memory CD8+ T cells in response to anti-CD3 Ab stimulation (Fig. 4), although both cells had similar levels of total AKT (Fig. 4). When IL-15 was added to anti-CD3 Ab stimulation in IL-7R{alpha}lowCCR7– memory CD8+ T cells, phosphorylation of AKT was enhanced (Fig. 4), indicating a role for the PI3K/AKT pathway in IL-15-mediated restoration of impaired proliferative function of IL-7R{alpha}lowCCR7– memory CD8+ T cells (Fig. 3B).


Figure 4
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  		FIGURE 4. IL-15 restores impaired TCR-mediated activation of the PI3K/AKT pathway. Sorted IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells were rested overnight and then stimulated with anti-CD3 Ab (10 µg/ml)-coated polystyrene beads (beads:cells ratio, 2:1) in the presence or absence of IL-15 (50 ng/ml) for 1 h at 37°C. Some cells were pretreated for 2 h with PI3K inhibitor LY294002 (20 µM) before stimulation. Cells were lysed following stimulation and cell lysates were analyzed by Western blotting using Abs to phospho-AKT (p-AKT, Thr308), AKT, and GAPDH. The relative immunoblot density of phospho-AKT was measured in different experiments (n = 3). A, Representative data. B, The results are shown as the mean ± SE (vertical bars). Values of p were obtained using Student’s two-tailed t test.

 
To further confirm the role of the PI3K/AKT pathway in restoring impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells by IL-15, these cells were stimulated with IL-15 and anti-CD3 Abs in the presence of PI3K and AKT inhibitors. Cells treated with PI3K inhibitor LY294002 had reduced cell proliferation, whereas the proliferation of cells treated with ERK inhibitor PD98059 remained unchanged (Fig. 5A). In addition, we treated IL-7R{alpha}lowCCR7– memory CD8+ T cells with two different AKT inhibitors. AKT inhibitor II inhibits activation of AKT without affecting other upstream or downstream kinase (25) and AKT inhibitor IV inhibits phosphorylation of AKT by targeting upstream kinase, 3-phosphoinositide-dependent protein kinase 1, but not PI3K (26). The doses we used in this study were based on our pilot study evaluating cytotoxic effects of the inhibitors (data not shown). Indeed, IL-7R{alpha}lowCCR7– memory CD8+ T cells treated with either AKT inhibitor had reduced proliferation in response to the combination of IL-15 and anti-CD3 Ab stimulations (Fig. 5B). These findings further support that the restoration of impaired TCR-mediated proliferation in IL-7R{alpha}lowCCR7– memory CD8+ T cells is dependent on the PI3K/AKT pathway.


Figure 5
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  		FIGURE 5. PI3K/AKT inhibitors block the restoration of impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells by IL-15. Sorted IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells were labeled with CFSE and stimulated for 7 days with anti-CD3 Abs (10 µg/ml) and IL-15 (5 ng/ml) in the presence or absence of (A) PI3K inhibitor LY294002 (20 µM) and MEK1 inhibitor PD980599 (10 µM) as a control inhibitor (B) as well as in the presence of AKT II (25 µM) or AKT IV (1 µM) inhibitors. Cell proliferation was analyzed on a FACSCalibur. Results are representative data from two to three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Two subsets of CCR7– memory CD8+ T cells expressing high and low levels of IL-7R{alpha} are present in human peripheral blood. The expression of effector cytokines and perforin by IL-7R{alpha}lowCCR7– memory CD8+ T cells indicates an important role of this cell subset in host defense against infections and tumors. Of interest, IL-7R{alpha}lowCCR7– memory CD8+ T cells have impaired proliferation and survival in response to TCR triggering and IL-7, respectively, which raises a question of how such cells are sustained at significant numbers, >20% of peripheral CD8+ T cells, and possibly expanded in the presence of antigenic stimulation. Our study demonstrates a role for IL-15 in expanding and maintaining IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans as evidenced by the findings that IL-15 induces proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells and restores impaired TCR-mediated proliferation of the same cell subset independently of CD28 costimulation via the PI3K/AKT pathway.

Based on our observations, we propose a model that supports the dual roles of IL-15 in maintaining IL-7R{alpha}lowCCR7– memory CD8+ T cells in humans (Fig. 6). In this model, IL-15 alone from IL-15-producing cells provides a proliferative signal to IL-7R{alpha}lowCCR7– memory CD8+ T cells via the IL-2/15Rβ and {gamma}C complex which leads to proliferation of polyclonal IL-7R{alpha}lowCCR7– memory CD8+ T cells regardless of their TCR specificity. This proliferation occurs in the absence (Fig. 6A, basal proliferation) or presence of infection or inflammation (Fig. 6B, bystander proliferation), although the latter condition with increased production of IL-15 can provide a stronger proliferative signal to IL-7R{alpha}lowCCR7– memory CD8+ T cells. For example, infection with a virus A can induce expansion of IL-7R{alpha}lowCCR7– memory CD8+ T cells which are not specific for this virus via IL-15-mediated bystander proliferation of the cells (Fig. 6B). We suspect that this is a mechanism explaining how IL-7R{alpha}lowCCR7– memory CD8+ T cells that have critical effector functions such as effector cytokine production and cytotoxicity are maintained at significant numbers in vivo in the absence of Ags and IL-7-mediated cell survival signals. Alternatively, a combination of IL-15 and antigenic stimulations induces massive proliferation of Ag-specific IL-7R{alpha}lowCCR7– memory CD8+ T cells (Fig. 6C). For instance, during infection with a virus B, IL-7R{alpha}lowCCR7– memory CD8+ T cells specific for virus B will rapidly proliferate as they receive both TCR triggering and IL-15 stimulation that synergistically increase cell proliferation (Fig. 6C). Through this mechanism, the immune system achieves rapid expansion of pathogen-specific effector CD8+ T cells, leading to the control of infection.


Figure 6
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  		FIGURE 6. Model for the dual roles of IL-15 in maintaining IL-7R{alpha}low CCR7– memory CD8+ T cells in vivo. IL-15 induces basal (A) and bystander (B) proliferation of polyclonal IL-7R{alpha}low CCR7– memory CD8+ T cells in the absence and presence of infection or autoimmunity, respectively. (C) A combination of IL-15 and Ag stimulation from infection or autoimmunity induces synergistic proliferation of Ag-specific IL-7R{alpha}low CCR7– memory CD8+ T cells.

 
In our study, we observed restoration of impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells by IL-15. Furthermore, such a restoration was dependent on activation of the PI3K/AKT pathway. The use of this pathway in promoting cell survival and proliferation in various cells by growth factors such as cytokines has been demonstrated (22, 27). However, our finding of activation of this pathway by IL-15 in particular in collaboration with anti-CD3 Abs in human primary CD8+ T cells was not previously reported. It is possible that decreased TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells is secondary to reduced expression of CD28, a costimulatory molecule providing signal two to T cells. However, the proliferation of this cell subset was considerably lower compared with that of IL-7R{alpha}highCCR7+CD8+ T cells when they were stimulated with anti-CD3 Abs alone. This finding suggests that impaired TCR-mediated proliferative capacity of such cell subset stems in part from an intrinsic alteration in the TCR signaling pathway. Thus, IL-15 likely restores this impairment by affecting the TCR signaling pathway. In fact, this notion is supported by our biochemical data showing enhanced activation of the PI3K/AKT pathway by a combination anti-CD3 Abs and IL-15 as well as blocking of such a combinational effect on cell proliferation by PI3K/AKT pathway inhibitors.

Our observations raise intriguing therapeutic possibilities. First, pathogen- and tumor-specific IL-7R{alpha}lowCCR7– memory CD8+ T cells that highly express perforin and produce effector cytokines can be expanded in vivo and in vitro by providing appropriate Ag and IL-15. This strategy would be particularly useful for rapidly promoting effector memory CD8+ T cell responses against microorganisms and tumors as well as for enhancing vaccine responses. In fact, the potential role for IL-15 for cancer therapy and vaccine development has been supported in animal studies (17, 28, 29, 30). For instance, mice that received tumor Ag-specific CD8+ T cells along with exogenous IL-15 treatment had a significantly delayed tumor relapse or complete tumor regression compared with mice that received tumor Ag-specific CD8+ T cells without IL-15. In fact, our study provides additional rationale for such a usage in humans (30). Also, IL-15 and the PI3K/AKT pathway could be a potential target for T cell-mediated autoimmune diseases where autoantigen-specific memory CD8+ T cells could be expanded by the combination of autoantigens and IL-15 (31). In fact, patients with systemic lupus erythematosus were found to have increased IL-15 levels (32, 33) and an expansion of memory CD8+ T cells expressing perforin correlating with disease activity (34). These findings suggest that the expansion of memory CD8+ T cells in systemic lupus erythematosus could be driven by IL-15 in combination with autoantigenic stimulation.

Although our current study shows a role for IL-15 in maintaining human IL-7R{alpha}lowCCR7– memory CD8+ T cells, the mechanism underlying for generating such cells needs to be determined. Previous studies suggest that TCR triggering and cytokines like IL-7 and IL-15 are involved in down-regulating IL-7R{alpha} expression on T cells in vitro (12, 35, 36). However, our recent study demonstrates the necessity of additional signal(s) for generating IL-7R{alpha}lowCCR7– memory CD8+ T cells in vivo as measured by the status of DNA methylation, a mechanism involved in regulating gene expression (37). In this study, resting human IL-7R{alpha}high and IL-7R{alpha}lowCCR7– memory CD8+ T cells had low and high levels of DNA methylation in the IL-7Ra gene promoter, respectively (37). However, stimulating IL-7R{alpha}highCD8+ T cells with TCR triggering and cytokines including IL-7 and IL-15 did not induce a change in DNA methylation in the IL-7Ra gene promoter despite decreased IL-7R{alpha} expression on stimulated cells. These findings suggest that inducing and maintaining low levels of IL-7R{alpha} expression on memory CD8+ T cells is a complicated process requiring stimulation more than TCR triggering, IL-7, and IL-15. We speculate that such stimulation could be provided by APCs or directly to the T cells in the setting of immune stimulations like infection.

Overall, the results of our study are novel by demonstrating that impaired TCR-mediated proliferation of IL-7R{alpha}lowCCR7– memory CD8+ T cells stems from a defect in activating the PI3K/AKT pathway and that such a defect can be restored by IL-15. This cytokine likely has dual roles in maintaining IL-7R{alpha}lowCCR7– memory CD8+ T cells that include inducing basal and bystander proliferation of the cells independently of TCR as well as synergistic expansion of Ag-specific IL-7R{alpha}lowCCR7– memory CD8+ T cells via the collaboration of IL-15 and TCR triggering. Lastly, our study also supports a potential clinical application of IL-15 in enhancing immune protection against infections and tumors in humans by promoting effector memory CD8+ T cell responses.


   Acknowledgment
 
We thank Dr. Mark Mamula for his critical review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the National Institute of Health (K08 AR49444-03), the American College of Rheumatology, the Arthritis Foundation, the American Foundation for Aging Research and Claude D. Pepper Older Americans Independence Center at Yale University School of Medicine (P30AG21342 National Institutes of Health/National Institute on Aging), and the General Clinical Research Center at Yale University (National Institutes of Health/National Center for Research Resources). Back

2 H.R.K. and K.A.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Insoo Kang, Section of Rheumatology TAC S541C, Yale School of Medicine, P.O. Box 208031, New Haven, CT 06520. E-mail address: Insoo.kang{at}yale.edu Back

4 Abbreviation used in this paper: {gamma}C, {gamma}-chain. Back

Received for publication May 14, 2007. Accepted for publication September 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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