Chain Cytokines IL-2, IL-7, IL-15, and IL-21 Induce the Expression of Programmed Death-1 and Its Ligands

The Common γ-Chain Cytokines IL-2, IL-7, IL-15, and IL-21 Induce the Expression of Programmed Death-1 and Its Ligands

This information is current as Audrey L. Kinter, Emily J. Godbout, Jonathan P. McNally, of October 2, 2021. Irini Sereti, Gregg A. Roby, Marie A. O'Shea and Anthony S. Fauci J Immunol 2008; 181:6738-6746; ; doi: 10.4049/jimmunol.181.10.6738 http://www.jimmunol.org/content/181/10/6738 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2008/10/31/181.10.6738.DC1 Material http://www.jimmunol.org/ References This article cites 54 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/181/10/6738.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Common ␥-Chain Cytokines IL-2, IL-7, IL-15, and IL-21 Induce the Expression of Programmed Death-1 and Its Ligands

Audrey L. Kinter,1 Emily J. Godbout, Jonathan P. McNally, Irini Sereti, Gregg A. Roby, Marie A. O’Shea, and Anthony S. Fauci

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 ␥-chain (␥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 mono- Downloaded from cytes/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 ␥c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by ␥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 ␥c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that ␥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 http://www.jimmunol.org/ cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host. The Journal of Immunology, 2008, 181: 6738–6746.

rogrammed death (PD)-12 and its ligands, PD-L1 (B7- prove T cell counts and function in certain individuals with lym- H1) and PD-L2 (B7-DC), have been shown to play an phopenia and/or immune dysfunction or in conjunction with vac- P important role in peripheral tolerance (1–3). Addition- cines (14–25). A potential complication of this therapeutic ally, the PD-1 axis has been implicated in the down-regulation modality is the fact that the administration of ␥c cytokines can be

of virus-specific T cell responses in the context of certain associated with prolonged or excessive peripheral T cell expan- by guest on October 2, 2021 chronic viral infections (4, 5). PD-1 ligation has been shown to sion/activation, increasing the possibility for immune-mediated inhibit TCR-mediated events (1, 6, 7) and has been associated damage to the host (26–31). In this regard, little is known con- with apoptosis in some (8–10), but not all, studies (6, 11). PD-1 cerning the mechanisms whereby the host avoids immune-medi- is up-regulated on lymphocytes following TCR-mediated acti- ated damage during prolonged periods of cytokine-driven T cell vation and remains elevated in the context of persistent Ag- activation and expansion. specific immune stimulation, whether the immune responses are In the present study, we investigated whether cytokine-mediated directed at self or foreign Ags (2, 3, 12, 13). However, little is immune activation can induce PD-1 and PD-1 ligand expression. known regarding PD-1 expression in the context of Ag nonspe- Of numerous cytokines tested, only IL-2, IL-7, IL-15, and IL-21 cific T cell stimulation by cytokines. were found to directly induce the expression of PD-1 and PD-L1 The common ␥-chain (␥c) cytokines IL-2, IL-7, IL-15, and, to a on purified T cells as well as PD-1 ligands on APCs. These in vitro lesser extent, IL-21 play a critical role in the support of T cell data were supported by data obtained on PBMC of HIV-infected proliferation, survival, and function during both immune responses individuals receiving in vivo IL-2 immunotherapy. Data regarding and homeostatic peripheral expansion (HPE). Additionally, several the effects of PD-1 ligation in functional assays suggest that PD-1 of these cytokines are currently being administered in vivo to im- induced by IL-2, IL-7, and IL-15 does not compromise cytokine- driven events (signaling, proliferation, or survival), but may serve to limit certain T cell effector functions upon TCR engagement and Laboratory of Immunoregulation, National Institute of Allergy and Infectious Dis- thus interfere with immune-mediated damage. eases, National Institutes of Health, Bethesda, MD 20892 Received for publication May 5, 2008. Accepted for publication September 5, 2008. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Isolation of PBMC and T cell subsets with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Audrey Kinter, Laboratory of Lymphophereses were performed on HIV-negative and HIV-positive sub- Immunoregulation, National Institute of Allergy and Infectious Diseases, National jects under National Institutes of Health-approved protocols (National In- Institutes of Health, Building 10, Room 6A33, MSC-1576, 9000 Rockville Pike, stitutes of Health 02-I-0202 and 81-I-0164), and PBMC were obtained by Bethesda, MD 20892. E-mail address: [email protected] Hypaque-Ficoll density centrifugation. Total T cells were isolated using 2 Abbreviations used in this paper: PD, programmed death; ␥c, ␥-chain; HPE, ho- negative immunomagnetic bead selection with an Ab cocktail (Stem Cell meostatic peripheral expansion; MFI, mean fluorescence intensity; M/M, monocytes/ Technologies), and in certain experiments CD45RA-negative (memory) macrophages; PD-L, ligand of PD; TCM, central memory T; TEff, effector T; TEM, subsets were isolated using immunomagnetic beads (Invitrogen/Dynal). effector memory T. PBMC from HIV-positive individuals receiving IL-2 (3–7.5 M IU s.c./bid; www.jimmunol.org The Journal of Immunology 6739 Downloaded from http://www.jimmunol.org/

FIGURE 1. Cytokine-mediated induction of PD-1 expression on T cells contained in PBMC and on purified T cells. A, Total PBMC were treated with a variety of cytokines or anti-CD3 plus anti-CD28 for 7 days and PD-1 expression was assessed on CD4ϩ and CD8ϩ T cells. Data represent the means Ϯ SD of 15 independent experiments. B–E, The ␥c cytokines IL-2, IL-7, IL-15, and IL-21 directly induced PD-1 expression on purified T cells. Purified T cells were stimulated with 100 ng or U/ml of all cytokines tested in A; only cytokines that significantly up-regulated PD-1 on purified T cells are shown. by guest on October 2, 2021 B, Representative flow plots of PD-1 expression on purified T cells induced by cytokines and anti-CD3/CD28 (day 6). PD-1ϩ cell frequency (C) and MFI (D)inCD4ϩ and CD8ϩ subsets in purified T cells stimulated with ␥c cytokines or anti-CD3/CD28 are shown; p values refer to comparison with untreated p Ͻ 0.05; ●, p Ͻ 0.005). Data represent the means Ϯ SD of eight independent experiments. E, Kinetics of cytokine ,ء) cells unless otherwise indicated and anti-CD3/CD28-induced PD-1 expression on purified T cells (CD8ϩ shown; CD4ϩ kinetics similar). Data represent the means Ϯ SD of six independent experiments.

5-day cycle) were collected/frozen on day 1 before the first IL-2 adminis- PD-L2. Frozen PBMC obtained from HIV-positive subjects receiving IL-2 tration and day 5 before the last two doses. All patients received IL-2 while in vivo were thawed (all time points per patient were assayed at the same also receiving effective antiretroviral therapy, leading to a suppression of time) and stained for PD-1, PD-L1, PD-L2, CD3, CD4, CD8, CD19, CD14, viral load to Ͻ50 copies/ml. Ki67, and HLA-DR. All mAbs were purchased from BD Pharmingen unless otherwise indicated. Cellular stimulation Freshly isolated PBMC were cultured at 106/ml in media (RPMI 1640 plus STAT5 and Lck activation 1 mM HEPES buffer and penicillin/streptomycin with 10% human AB serum) alone or with IL-1␤ (5–100 ng/ml), IL-2 (5–200 IU/ml), IL-4 (5– Purified human T cells were prestimulated with IL-2 or anti-CD3/CD28 100 ng/ml), IL-6 (5–100 ng/ml), IL-7 (5–100 ng/ml), IL-8 (5–100 ng/ml), for 6 days. Harvested cells were restimulated with IL-2 plus tosylacti- IL-10 (5–100 ng/ml), IL-12 (25–200 ng/ml), IL-15 (5–100 ng/ml), IL-18 vated beads (Invitrogen/Dynal) coated with hIgG or PD-L2 hIgG (2 ␮ 7 ␮ 7 (5–100 ng/ml), IL-21 (5–200 ng/ml), TGF-␤ (5–100 ng/ml), IFN-␥ (5–100 g/10 beads) or with beads coated with anti-CD3 (4 g/10 beads)/ ␮ 7 ng/ml), TNF-␣ (2–50 ng/ml), or anti-human CD3 (5 ␮g/ml) plus anti- anti-CD28 (2 g/10 beads) or isotype plus PD-L2 or hIgG (3:1 bead- human CD28 (2 ␮g/ml) (BD Pharmingen). All cytokines were purchased to-cell ratio). Cells were stimulated for 15 (Lck) or 45 (STAT5) min at from the National Cancer Institute Biological Resource Branch Preclinical 37°C and then stained with phospho-specific Y505 Lck (BD Pharmin- Repository (Frederick, MD) or R&D Systems. Cells were monitored for gen, clone 4/LCK-Y505), phospho-specific Y694 STAT-5 (BD Pharm- PD-1 expression during a 10–16-day period. For all subsequent analyses, ingen clone 47), PD-1 (BD Pharmingen, clone MIH4), and CD8 (BD unless otherwise indicated, PBMC, purified total T cells, or CD45RAϪ Pharmingen) using eBioscience’s Fix/Perm solution per the manufac- (memory) T cells were cultured in media plus IL-2 (200 IU/ml), IL-7 (25 turer’s instructions. ng/ml), IL-15 (100 ng/ml), IL-21 (100 ng/ml), or anti-CD3/CD28 (as above). Functional assays Phenotyping Proliferation. Freshly isolated, purified T cells, or PBMC (candida) were plated (105/well) in 96-well plates either 1) coated with isotype PD-1 and PD-L expression and naive/memory subset phenotypes were as- hIgG or hPD-L1-IgG or hPD-L2-IgG chimeric proteins (5 ␮g/ml, R&D sessed using anti-human PD-1 (clone MIH4), PD-L1 (clone MIH1; eBio- Systems) or 2) in the presence of neutralizing anti-PD-L1 Ab (5 ␮g/ml; science), or PD-L2 (clone MIH18; eBioscience), CD45RA, CD62L, CD3, eBioscience). Cells were then stimulated with plate-bound anti-CD3 CD4, and CD8. PBMC were stained for CD3, CD19, CD14, and PD-L1 or (0.2 ␮g/ml)/anti-CD28 (0.4 ␮g/ml), Candida albicans (5 ␮g/ml), or 6740 COMMON ␥-CHAIN CYTOKINES INDUCE PD-1 AND ITS LIGANDS Downloaded from

FIGURE 2. Characterization of IL-2-, IL-7-, IL-15-, and IL-21-mediated induction of PD-1 expression on purified T cells. Purified T cells were ␥ Ϯ stimulated with c cytokines or anti-CD3/anti-CD28 and stained for CD8, CD45RA, CD62L, and PD-1. Data represent the means SD of eight http://www.jimmunol.org/ ϩ ϩ Ϫ ϩ independent experiments. A, Representative flow plot of gating for naive (TN; CD45RA CD62L ), central memory (TCM; CD45RA CD62L ), effector Ϫ Ϫ ϩ Ϫ ϩ high ϩ memory (TEM; CD45RA CD62L ) and effector (TEff; CD45RA CD62L ) T cell subsets and PD-1 and PD-1 populations. B, Comparison of PD-1 .(joined), p Ͻ 0.05, CD8ϩ vs CD4ϩ T cells) ء ;p Ͻ 0.05, treated vs untreated ,ء) cell frequencies in CD8ϩ and CD4ϩ T cell subsets at day 6 poststimulation C, Purified CD45RA-negative (memory) T cells were stimulated with various concentrations of cytokines, and PD-1 expression was assessed in CD4ϩ and CD8ϩ subsets 6–7 days posttreatment. Values in bold represent the cytokine concentrations used in all other experiments. Data are representative of three independent experiments.

3 ϩ with ␥c cytokines. Proliferation was assessed by 16-h [ H]thymidine While IL-12 induced PD-1 on CD8 T cells in the context of by guest on October 2, 2021 uptake at day 6 poststimulation. PBMC (Fig. 1A and supplemental Fig. 1),3 only the ␥c cytokines Cytokine production and apoptosis. Purified T cells received an initial IL-2, IL-7, IL-15, and IL-21 were found to directly induce PD-1 ␥ ␮ stimulation with c cytokines or anti-CD3/CD28 (5/2 g/ml) for 5–8 days. expression on purified T cells (Fig. 1, B–E). ␥c cytokines increased Cells were washed, restimulated with plate-bound anti-CD3/CD28 (0.2/0.4 ϩ ␮ ␮ the frequency of PD-1 cells (Fig. 1C) and modestly increased the g/ml) plus hIgG, PD-L1-hIgG, or PD-L2-hIgG (5 g/ml) and tested in ϩ parallel for annexin V or cytokine production. For cytokine production, PD-1 mean fluorescence intensity (MFI) (Fig. 1D) on both CD4 brefeldin A (GolgiPlug; BD Pharmingen) was added 2 h after restimula- (IL-21 had no effect on MFI) and CD8ϩ T cells. A significant tion, and 4 h later cells were stained for PD-1, CD8, and intracellular IL-2 difference in cytokine-induced PD-1 expression between CD4ϩ and IFN-␥ as per the manufacturer’s recommendations. Apoptosis was ϩ assessed at 6 h after restimulation by staining for PD-1, annexin V (BD and CD8 T cells was seen only with IL-21 treatment (Fig. 1, C Pharmingen), CD4, and CD8. In certain apoptosis experiments, prestimu- and D). ␥c cytokines induced PD-1 expression on purified T cells lated T cells were exposed to PD-L1 or PD-L2 IgG in the absence of more slowly than did anti-CD3/CD28 stimulation, but effects were anti-CD3; however, results are not shown but were similar to those re- long lasting and increased over time, with the exception of IL-21 ported below. (cells did not survive beyond 6–8 days) (Fig. 1E, similar results ϩ Statistical analyses were obtained for CD4 T cells). ␥c cytokine treatment of purified T cells increased the frequency of PD-1ϩ CD8ϩ and CD8Ϫ Paired Student’s t test and Spearman’s nonparametric correlation were ϩ Ϫ ϩ (CD4 ) cells in the central (TCM, CD45RA CD62L ) and effec- used for statistical analyses. Ϫ Ϫ ϩ tor (TEM, C45RA CD62L ) memory subsets; PD-1 effector cell ϩ Ϫ Results (TEff, CD45RA CD62L ) frequencies were also elevated, but few In vitro cytokine-mediated induction of PD-1 expression events were present in this gate (Fig. 2, A and B). While there were significant differences in the percentages of PD-1ϩ CD8ϩ vs Induction of PD-1 expression has been previously described only CD4ϩ T cells, there were no significant differences in the response under conditions of TCR stimulation. In the present study, we (fold induction vs untreated) to any stimulation condition in any investigated whether cytokine-mediated immune activation can high subset. The frequency of PD-1 TEM cells was increased by contribute to PD-1 expression. PBMC were stimulated with a va- IL-2, IL-15, and IL-21, whereas anti-CD3/CD28 stimulation in- riety of cytokines and T cells were monitored for PD-1 expression. duced PD-1high expression in all T cell subsets (supplemental Fig. Of those cytokines tested, IL-2, IL-7, IL-12, IL-15, and IL-21 in- 2A). Of note, the TEff population had a higher frequency of PD- duced significant levels of PD-1 expression above background (un- 1high cells than did other subsets under both untreated and cyto- ␤ ␣ treated cells), whereas proinflammatory (IL-1 and TNF- ; IL-6, kine-treated conditions (supplemental Fig. 2A). Moderate cytokine IL-8 data not shown), immunosuppressive (TGF-␤; IL-10, data not shown), and certain immunoregulatory (IL-4 and IFN-␥; IL-18, data not shown) cytokines had no significant effect (Fig. 1A). 3 The online version of this article contains supplemental material. The Journal of Immunology 6741

FIGURE 3. ␥c cytokines induce the expression of PD-1 ligands. PBMC or purified T cells were treated with ␥c cytokines or anti-CD3/anti-CD28 and stained for CD3, CD19, CD14, and PD-L1 or PD-L2. Data are the means Ϯ SD obtained from five independent exper- p Ͻ 0.05 comparing treated to untreated ,ء) iments cells). A, Representative flow plots of PD-L1 or PD-L2 expression in CD3ϩ (left; gated on CD19Ϫ lymphocytes) and CD14ϩ (middle, right; gated on M/M) PBMC following stimulation with IL-2 or anti- CD3/CD28. B, The percentage of PD-L1ϩ and PD-L2ϩ M/M stimulated (day 3) in the context of PBMC. C, Comparison of the PD-L1 MFI on T cells vs CD14ϩ M/M stimulated in the context of PBMC (● (joined), p Ͻ 0.01 comparing T cells vs M/M). D, The percentage of PD-L1ϩ B cells (CD19ϩ in PBMC) and purified T cells day 6 poststimulation. Downloaded from

concentrations (6 ng/ml) were effective in up-regulating PD-1 on cytokines on PD-1 ligand expression have not been reported. In purified memory (CD45RAϪ) T cells; however, IL-2 and IL-15 the context of total PBMC, ␥c cytokines increased the fre- http://www.jimmunol.org/ were most effective at high concentrations (Fig. 2C). quency and, with the exception of IL-21, the MFI of PD-L1ϩ monocytes/macrophages (M/M) (Fig. 3, B and C). CD3ϩ In vitro cytokine-mediated induction of PD-1 ligand PBMC (T cell) expression of PD-L1 (MFI; Fig. 3C) and fre- expression quency (data not shown) were modestly, but significantly, ele- The functional consequences of PD-1 expression on T cells vated by all ␥c cytokines except IL-21. However, PD-L1 ex- clearly rely on the interaction of these cells with PD-1 ligand- pression (MFI) on T cells remained significantly lower than on expressing cells. PD-L2 (B7-DC) expression is largely re- M/M under all conditions (Fig. 3C). The frequency, but not the ϩ stricted to dendritic cells and activated macrophages, whereas MFI (data not shown), of PD-L2 M/M was increased by IL-2, by guest on October 2, 2021 PD-L1 (B7-H1) is expressed more promiscuously and can be IL-15, and IL-21 (Fig. 3B); no PD-L2 was observed on T or B found on a variety of hematopoietic and nonhematopoietic cells (data not shown). All ␥c cytokines, with the exception of cells, particularly in the tissue (32, 33). While PD-L1 and IL-21, induced PD-L1 expression on purified T cells, and IL-21 PD-L2 have been shown to be up-regulated on both professional was the only ␥c cytokine to up-regulate PD-L1 on B (CD19ϩ) and nonprofessional APC upon stimulation with TLR ligands cells in PBMC (Fig. 3D). Of interest, treatment of purified T and certain cytokines (32, 33), such as IFN-␥, the effects of ␥c cells with IL-2, IL-7, and IL-15 increased the frequency of

FIGURE 4. The expression of PD-1 and its ligands is increased following IL-2 administration (5 days) to HIV-infected individuals. PD-1ϩ (A) cell frequency and (B) MFI on CD4ϩ and CD8ϩ T cells. C, Percentage of PD-L1ϩ T cells and M/M (CD14ϩ) and percentage of PD-L2ϩ M/M. D, PD-1ϩ CD8ϩ and CD8Ϫ (CD4ϩ)T cells are proliferating (proliferation Ag, Ki67ϩ) in vivo following IL-2 administration. Data are of samples obtained before and on day 5 of IL-2 administration when the individuals (n ϭ 5) were receiving antiretroviral therapy and had viral loads Ͻ50 copies/ml before the start of and during the IL-2 cycle. 6742 COMMON ␥-CHAIN CYTOKINES INDUCE PD-1 AND ITS LIGANDS

FIGURE 5. Effect of PD-1 engagement on proliferation and signal transduction: role of exogenous and endogenous PD-1 ligand. A, Proliferation of puriﬁed T cells (Candida proliferation was assessed in PBMC) cultured with IL-2, IL-7, IL-15, IL-21, or plate-bound Downloaded from anti-CD3/anti-CD28 in the presence of plate-bound hIgG control, PD-L1 IgG, or PD-L2 IgG (data are the means and SD of eight independent experiments). B, Proliferation of puriﬁed T cells stimulated via TCR (anti- CD3/CD28; immobilized on plate) or ␥c cytokines in the presence of isotype control or neutralizing anti- ϭ

PD-L1 Ab (n 6 independent experiments). C, Puriﬁed http://www.jimmunol.org/ T cells prestimulated with IL-2 or anti-CD3/anti-CD28 for 6 days. Cells then received a secondary stimulation with IL-2 or anti-CD3/anti-CD28 in the presence of hIgG or PD-L2 IgG-coated beads. The frequency of active Lck-Pϩ (TCR complex signal; left) and STAT5-Pϩ (␥c cytokine signal; right) cells was assessed at 15 and 45 min, respectively. (Data are means Ϯ SD of ﬁve p Ͻ 0.05 comparing hIgG ,ء (.independent experiments vs hPD-L IgG-treated cells). by guest on October 2, 2021

PD-L1 on all memory/effector and naive T cell subsets (sup- also increased. Of note, IL-2 therapy was associated with elevated plemental Fig. 2B). frequencies of PD-1ϩ T cells expressing the proliferation Ag Ki67 (Fig. 4D), suggesting that IL-2-mediated induction of PD-1 or its In vivo IL-2-mediated induction of PD-1 and PD-1 ligand ligands did not interfere with the ability of PD-1ϩ cells to prolif- expression erate in vivo in response to this cytokine. To determine whether cytokines can modulate the PD-1 axis in ␥ vivo, PD-1 and PD-L expression was assessed on PBMC isolated Functional consequences of c cytokine-mediated modulation from HIV-positive subjects (n ϭ 5; HIV viral load Ͻ50) before of the PD-1 axis and during (day 5) administration of IL-2 immunotherapy. Fol- Several of these ␥c cytokines are currently being used, or are being lowing IL-2 administration, the frequency of activated (HLA- considered, as modalities for immunotherapy (16, 23, 25) or as DRϩ) PD-1ϩ CD4ϩ and CD8ϩ T cells, and the MFI of PD-1ϩ adjuvants in the context of vaccination (34–36). It is therefore CD8ϩ T cells, was signiﬁcantly elevated (Fig. 4, A and B). Fur- important to determine the potential role of the PD-1 axis in mod- thermore, the frequency of M/M and T cells expressing PD-L1 ulating the activity and/or survival of ␥c cytokine-stimulated (Fig. 4C, left) and of M/M expressing PD-L2 (Fig. 4C, right) was lymphocytes. Engagement of PD-1 has been shown to inhibit The Journal of Immunology 6743 Downloaded from FIGURE 6. The effect of ␥c cytokines and exogenous PD-1 ligands on anti-CD3/CD28-induced cytokine production. Puriﬁed T cells were pretreated with IL-2, IL-7, IL-15, or plate-bound anti-CD3/anti-CD28 for 6 days. Cells were then restimulated with plate-bound anti-CD3/anti-CD28 in the presence of plate-bound hIgG, PD-L1 IgG, or PD-L2 hIgG and stained for IFN-␥, IL-2, CD4, and CD8. A, Representative plot of IL-2 and IFN-␥ expression by total T cells receiving no prior stimulation or prestimulated with cytokine (IL-15) or anti-CD3/CD28 and then restimulated with anti-CD3/CD28 in the absence of PD-1 ligand (hIgG). B, The frequency of IFN-␥ϩ, IL-2ϩ, and IFN-␥ϩ/IL-2ϩ cells following restimulation with anti-CD3/CD28 in the absence or presence of PD-1 ligands. In the absence of PD-1 ligand (hIgG), ␥c cytokine pretreated T cells exhibit a higher frequency of IFN-␥ϩ, IL-2ϩ, and IFN-␥ϩ/IL-2ϩ cells ● Ͻ Ͻ ϩ compared with cells receiving no prior stimulation ( , p 0.005) or anti-CD3/CD28 pretreatment (p 0.05 for all intracellular cytokine (ICC) http://www.jimmunol.org/ populations; not shown), The presence of PD-L1 IgG or PD-L2 IgG at restimulation reduced the frequency of IFN-␥ϩ (left), IL-2ϩ (right), and IFN-␥ϩ/ p Ͻ 0.05 comparing PD-L hIgG ,ء .IL-2ϩ (middle) T cells under all prestimulation conditions. Data are means Ϯ SD of seven independent experiments to control hIgG-treated cells.

TCR-stimulated T cell proliferation and effector function (1, 6, 37, the time of secondary stimulation inhibited signaling mediated by 38) and has also been associated with increased apoptosis (8–10); the TCR complex (Fig. 5C, left; Lck) but not by IL-2 (Fig. 5C, any of these effects could have negative consequences on cytokine- right; STAT5). mediated T cell expansion and survival. Having determined that PD-1 engagement does not directly in- by guest on October 2, 2021 Proliferation was assessed in purified T cells stimulated with terfere with ␥c cytokine-mediated proliferation or signaling events, ␥c cytokines or TCR stimuli (anti-CD3/CD28 and recall Ag (C. the effects of PD-1 ligation on the survival and function ␥c cyto- albicans in PBMC)) in the presence of hIgG control, PD-L1- kine and anti-CD3/CD28 pretreated T cells following restimula- hIgG, or PD-L2 hIgG (Fig. 5A) or in the presence of neutral- tion via TCR was evaluated. Purified ␥c cytokine and anti-CD3/ izing anti-PD-L1 Ab (Fig. 5B). IL-2, IL-7, and IL-15 induced CD28 prestimulated T cells were harvested at time points of strong significant proliferation of purified T cells; IL-21-treated cells PD-1 expression (6–8 days poststimulation), washed, and then did not survive well at day 6 and thus did not incorporate thy- restimulated with anti-CD3/CD28 in the presence of hIgG (con- midine (Fig. 5A). The presence of either PD-L1 IgG or PD-L2 trol), PD-L1 IgG, or PD-L2 IgG. Cells were then tested in parallel IgG at the time of initial stimulation significantly suppressed for cytokine production (intracellular cytokine (ICC)) and apopto- anti-CD3/CD28, and the presence of PD-L1 IgG suppressed sis (annexin V) after 5–6 h. Candida-stimulated proliferation; however, neither PD-L1 nor ␥c cytokine pretreated T cells produced high levels of IL-2 and PD-L2 IgG significantly reduced ␥c cytokine-induced prolifer- IFN-␥ following restimulation with anti-CD3/CD28 in the absence ation (Fig. 5A). In addition, we investigated the role of endog- of PD-1 ligands (hIgG control) (Fig. 6). The frequency of IL-2ϩ, enous T cell-associated PD-L1 in proliferation by conducting IFN-␥ϩ, and IL-2ϩ/IFN-␥ϩ T cells following restimulation in the assays using purified T cells in the presence of neutralizing presence of hIgG was significantly higher in ␥c cytokine pretreated anti-PD-L1 Ab. Anti-PD-L1 Ab had no significant effect on the cells compared with cells not receiving prior stimulation and with ability of purified T cells to proliferate in response to either anti-CD3/CD28 prestimulated cells ( p ϭ 0.04–0.0004; not immobilized anti-CD3/CD28 or ␥c cytokines (Fig. 5B), sug- shown) (Fig. 6B). However, the presence of either PD-L1 or gesting that endogenous T cell-associated PD-L1 does not sig- PD-L2 IgG during restimulation dramatically reduced the fre- nificantly inhibit proliferation under these in vitro conditions. quency of IFN-␥ϩ (Fig. 6B, left), IL-2ϩ/IFN-␥ϩ (Fig. 6B, middle), To verify that PD-1 engagement does not inhibit ␥c cytokine- and IL-2ϩ (Fig. 6B, right) populations in T cells prestimulated induced intracellular signaling events, STAT5 activation (␥c cy- with either ␥c cytokines or anti-CD3/CD28. There were no sig- tokine signaling; IL-2) and Lck activation (TCR complex-signal- nificant differences between PD-L1 and PD-L2 IgG for this effect. ing; anti-CD3-CD28) was determined in the presence PD-L2 IgG. PD-1 engagement has been described to induce apoptosis in Purified T cells were pre-stimulated with IL-2 or anti-CD3/CD28 certain studies, while other studies find PD-1 to simply be a marker (6 days) to induce PD-1 expression. Cells were then exposed to for susceptibility to apoptosis (6, 8–11). To investigate the impact hIgG or PD-L2 IgG at the time of secondary stimulation with IL-2 of cytokine-induced PD-1 on apoptosis, pretreated (as above) T or anti-CD3/CD28 and stained for active (phosphorylated) Lck or cells were washed, restimulated with anti-CD3/CD28 in the pres- STAT5. Whether PD-1 was initially induced by IL-2 or by anti- ence of hIgG, PD-L1, or PD-L2 IgG, and apoptosis (surface an- CD3/CD28 pre-treatment, engagement of PD-1 by PD-L2 IgG at nexin Vϩ) was assessed in the PD-1high (MFI Ͼ 100), PD-1mid 6744 COMMON ␥-CHAIN CYTOKINES INDUCE PD-1 AND ITS LIGANDS

sion has not been investigated. The present study demonstrates that the ␥c cytokines IL-2, IL-7, IL-15, and IL-21 directly induce the PD-1 axis in purified T cells and PBMC of healthy subjects in vitro and in PBMC of HIV-infected subjects receiving IL-2 immunotherapy in vivo. The results of in vitro functional assays suggest that engagement of ␥c cytokine-induced PD-1 does not suppress ␥c cytokine-mediated signal transduction, T cell expansion, or enhancement of function and survival, but does significantly inhibit certain effector functions (cytokine production) of cytokine-stimulated T cells upon subsequent TCR triggering. This is the first report, to our knowledge, of ␥c cytokine-mediated up-regulation of the PD-1 axis. Although the list of cytokines screened in the present study for the ability to modulate PD-1 was not exhaustive, the ability to induce PD-1 expression on purified T cells was limited to certain ␥c cytokines (IL-2, IL-7, IL-15, and IL-21; Fig. 1). While all these cytokines play a role in promoting T cell expansion, survival, or function during normal immune responses, IL-7 and IL-15 play a critical role in HPE (20, 22, 27, 39–47). The induction of PD-1 expression in vitro by ␥c cytokines was restricted to memory (T and T ) and effector T cells (Fig. Downloaded from FIGURE 7. Cytokine prestimulated T cells exhibit reduced apoptosis, EM CM 2). In this regard, it has been reported that memory, but not naive, and this effect is not influenced by the presence of PD-L1 or PD-L2 hIgG. Purified T cells were treated as in Fig. 6 and apoptosis (surface annexin T cell populations can undergo expansion that is cytokine driven Vϩ) was assessed at 6 h poststimulation in the PD-1high, PD-1mid, and and TCR-MHC-independent, at least in mice (39, 40, 45). Of in- PD-1Ϫ T cell populations as indicated in A. B, The frequency of annexin terest, naive T cell subsets did respond to IL-2, IL-7, and IL-15, Vϩ cells in all PD-1 subsets in the presence of control hIgG; apoptosis in as did memory/effector subsets, by up-regulating PD-L1 (sup- the PD-1high, PD-1mid, and PD-1Ϫ subsets was significantly different under plemental Fig. 2B). IL-2, IL-7, and IL-15 increased both the http://www.jimmunol.org/ ;p Ͻ 0.001). C, The frequency of annexin Vϩ cells frequency and, to a lesser extent, the MFI of PD-L1ϩ T cells ,ء) all conditions tested in PD-1high (right) and PD-1mid (left) subsets in the presence of hIgG, however, PD-L1 remained at much lower levels (MFI) than PD-L1 IgG, or PD-L2 IgG. IL-2, IL-7, or IL-15 pretreatment results in those seen on M/M (Fig. 3). We found no significant inhibitory mid high reduced apoptosis in the PD-1 populations and IL-7 in the PD-1 role of T cell-associated PD-L1 in purified T cell cultures (Fig. populations, and these effects are not diminished in the presence of PD-L1 ϩ 5); however, in our in vitro conditions PD-L1 T cells are not comparing cytokine-treated vs untreated T ,ء ;or PD-L2 hIgG (p Ͻ 0.05 cells; ●, comparing cytokine vs anti-CD3/anti-CD28 prestimulated T cells; presenting Ag. In studies demonstrating suppression via PD- p ϭ NS comparing hIgG vs PD-L IgG for all treatments). Data are L1-B7.1 (48, 49) or PD-L1-PD-1 (1, 6, 7), TCR is co-engaged Ϯ either by beads or alloreactive T cells. Only IL-21 significantly means SD of eight independent experiments. by guest on October 2, 2021 induced PD-L1 on B cells (Fig. 3), consistent with its effect on B cell activation/differentiation (44, 50). PD-L1 and, to a lesser Ϫ (80 Ͼ MFI Ͼ 20) and PD-1 (MFI Յ 10) T cell populations (Fig. extent, PD-L2 expression on M/M in PBMC in vitro (Fig. 3) 7A). When restimulated with anti-CD3/CD28 in the absence of the and in vivo (HIV-positive subjects receiving IL-2 immunother- PD-1 ligand (control hIgG), the frequency of apoptosis differed apy; Fig. 4) was dramatically up-regulated by ␥c cytokines. ␥c significantly based on the level of PD-1 expression (PD-1high Ͼ cytokines did not induce PD-1 ligand expression on purified Ϫ PD-1mid Ͼ PD-1 subsets) under all conditions tested (Fig. 7B); M/M in vitro, and the effect on M/M in PBMC was found to be similar results were observed in cells not receiving a secondary largely IFN-␥-dependent (data not shown). TCR stimulation (data not shown). ␥c cytokine pretreatment re- The lack of an inhibitory effect of PD-1 engagement on ␥c cy- sulted in a small, but significant, reduction in apoptosis within the tokine-mediated signaling or downstream functional events in PD-1mid population (Fig. 7C, left) compared with anti-CD3/ vitro (Fig. 5) is not surprising based on our understanding of PD- CD28 pretreatment or no prior treatment; only IL-7 reduced 1-mediated immunosuppression. Our data are consistent with pre- apoptosis in the PD-1high (Fig. 7C, right) T cell population. Of vious studies demonstrating that the inhibitory effects of PD-1 en- ϩ note, the reduction of apoptosis in PD-1 populations by cyto- gagement may require TCR-triggered SHP-2 recruitment to the kine pretreatment was maintained even in the presence of cytoplasmic tail of PD-1 and that PD-1 ligation by itself does not PD-L1 or PD-L2 IgG (Fig. 7C). generate a suppressive signal (7). Furthermore, PD-1 inhibitory effects appear to require that the TCR and PD-1 signals be deliv- Discussion ered in both temporal and physical proximity (7). Our observation ␥c cytokines play an important role in peripheral T cell expansion, that PD-L1 Ig or anti-PD-L1 neutralizing Ab does not alter cyto- function, and survival, and therefore several of these cytokines are kine-driven proliferation data is supported by the observation of currently being administered in vivo as immunotherapy in certain increased proliferation (Ki67 expression) in PD-1ϩ T cells follow- diseases (16, 25), as well as adjuvants in vaccination. However, ␥c ing in vivo administration of IL-2 to HIV-infected subjects (Fig. cytokine administration and HPE under lymphopenic conditions 4D). Of interest, IL-7 and IL-15 have previously been found to can be associated with prolonged or excessive peripheral T cell rescue proliferation of T cells stimulated with anti-CD3 in the expansion/activation, increasing the possibility for immune-medi- presence of PD-L1 (51), suggesting that PD-1 engagement does ated damage to the host (26–31). The PD-1 axis has been shown not inhibit proliferation driven by these cytokines even if TCR to play an important role in controlling the potentially harmful triggering is contributing to the expansion of T cells in vivo. In activity of peripheral T cells that are reactive to self Ags and per- contrast, ␥c cytokine pretreatment did render TCR-triggered ef- sistent foreign Ags (2, 6, 13); however, the role of PD-1 in main- fector function (measured by cytokine (IL-2/IFN-␥) secretion; Fig. taining tolerance during cytokine-driven T cell activation/expan- 6) of T cells sensitive to PD-1-mediated suppression. The ability of The Journal of Immunology 6745

PD-1 engagement to induce apoptosis remains controversial (6, 8, 4. Freeman, G. J., E. J. Wherry, R. Ahmed, and A. H. Sharpe. 2006. Reinvigorating 9, 11). Our data suggest that PD-1 does not mediate apoptosis, exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade. J. Exp. Med. 203: 2223–2227. even in the context of TCR signaling, but rather that the level of 5. Grakoui, A., E. John Wherry, H. L. Hanson, C. Walker, and R. Ahmed. 2006. PD-1 expression (MFI) correlates with susceptibility to apoptosis Turning on the off switch: regulation of anti-viral T cell responses in the liver by (Fig. 7). Consistent with the pro-survival effects of these ␥c cyto- the PD-1/PD-L1 pathway. J. Hepatol. 45: 468–472. 6. Latchman, Y., C. R. Wood, T. Chernova, D. Chaudhary, M. Borde, I. Chernova, kines (21, 39–41, 52), pretreatment of T cells with ␥c cytokines, Y. Iwai, A. J. Long, J. A. Brown, R. Nunes, et al. 2001. PD-L2 is a second ligand particularly IL-7, moderately reduced apoptosis within the PD-1ϩ for PD-1 and inhibits T cell activation. Nat. Immunol. 2: 261–268. 7. Chemnitz, J. M., R. V. Parry, K. E. Nichols, C. H. June, and J. L. Riley. 2004. cell populations and this effect was maintained even in the pres- SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of ence of PD-1 ligands. Of note, the protective effects of cytokines programmed death 1 upon primary human T cell stimulation, but only receptor on cell death seen in this study were less than those reported by ligation prevents T cell activation. J. Immunol. 173: 945–954. 8. Ishida, Y., Y. Agata, K. Shibahara, and T. Honjo. 1992. Induced expression of other studies in which cytokines were present during stimulation/ PD-1, a novel member of the immunoglobulin gene superfamily, upon pro- apoptosis assessment. grammed cell death. EMBO J. 11: 3887–3895. Whether ␥c cytokine-induced PD-1 influences T cell function 9. Petrovas, C., J. P. Casazza, J. M. Brenchley, D. A. Price, E. Gostick, W. C. Adams, M. L. Precopio, T. Schacker, M. Roederer, et al. 2006. PD-1 is a upon subsequent TCR triggering is particularly relevant in the set- regulator of virus-specific CD8ϩ T cell survival in HIV infection. J. Exp. Med. ting of cytokines administered as immunotherapy or as vaccine 203: 2281–2292. adjuvants (14–25). While modest (6 ng/ml) concentrations of ␥c 10. Dong, H., S. E. Strome, D. R. Salomao, H. Tamura, F. Hirano, D. B. Flies, P. C. Roche, J. Lu, G. Zhu, K. Tamada, et al. 2002. Tumor-associated B7–H1 cytokines induced PD-1 expression on memory T cells in vitro promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. (Fig. 2), normal in vivo levels of these cytokines may be insuffi- 8: 793–800. cient, except perhaps in tissue microenvironments, to induce the 11. Vibhakar, R., G. Juan, F. Traganos, Z. Darzynkiewicz, and L. R. Finger. 1997.

Activation-induced expression of human programmed death-1 gene in T-lym- Downloaded from PD-1 axis at a functionally relevant level. Furthermore, note that T phocytes. Exp. Cell Res. 232: 25–28. cell restimulations were conducted using low concentrations of 12. Velu, V., S. Kannanganat, C. Ibegbu, L. Chennareddi, F. Villinger, anti-CD3 and anti-CD28 in the presence of considerable concen- G. J. Freeman, R. Ahmed, and R. R. Amara. 2007. Elevated expression levels of inhibitory receptor programmed death 1 on simian immunodeficiency virus-spe- trations of PD-1 ligands. These in vitro conditions may reflect cific CD8 T cells during chronic infection but not after vaccination. J. Virol. 81: conditions existing during the interaction between T cells and non- 5819–5828. professional APC expressing high levels of PD-1 ligands. Thus, 13. Keir, M. E., L. M. Francisco, and A. H. Sharpe. 2007. PD-1 and its ligands in T-cell immunity. Curr. Opin. Immunol. 19: 309–314. http://www.jimmunol.org/ our data need not conflict with the use of ␥c cytokines as adjuvants 14. Gu, X. X., F. Y. Yue, C. M. Kovacs, and M. A. Ostrowski. 2007. The role of cytokines which signal through the common ␥ chain cytokine receptor in the in the context of vaccination or as immunotherapy to enhance Ag- ϩ ϩ specific T cell responses (14–25). However, these data may pro- reversal of HIV specific CD4 and CD8 T cell anergy. PLoS ONE 2: e300. 15. Ferrari, G., K. King, K. Rathbun, C. A. Place, M. V. Packard, J. A. Bartlett, vide some insight into the optimal timing of cytokine administra- D. P. Bolognesi, and K. J. Weinhold. 1995. IL-7 enhancement of antigen-driven tion (and thus PD-1 axis induction) relative to TCR triggering activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp). Clin. Exp. Immunol. 101: 239–248. (vaccines or persistent pathogens whose replication can be mod- 16. Levy, Y. 2006. Cytokine therapies in HIV infection. Med. Sci. 22: 751–754. ulated by treatment) (53, 54). Furthermore, the inability to detect 17. Lori, F., D. B. Weiner, S. A. Calarota, L. M. Kelly, and J. Lisziewicz. 2006. an improvement of T cell function following ␥c cytokine admin- Cytokine-adjuvanted HIV-DNA vaccination strategies. Springer Semin. Immu- istration may be influenced by the timing, relative to cytokine ad- nopathol. 28: 231–238. 18. Pett, S. L., and A. D. Kelleher. 2003. Cytokine therapies in HIV-1 infection: by guest on October 2, 2021 ministration, of when cells are reexposed to Ag and assayed for present and future. Expert Rev. Anti Infect. Ther. 1: 83–96. function (53, 54). 19. Hryniewicz, A., D. A. Price, M. Moniuszko, A. Boasso, Y. Edghill-Spano, ␥ S. M. West, D. Venzon, M. Vaccari, W. P. Tsai, E. Tryniszewska, et al. 2007. In summary, the present study provides evidence that the c Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus cytokines IL-2, IL-7, IL-15, and IL-21, known to play an important level in simian immunodeficiency virus mac251-infected macaques. J. Immunol. role in peripheral T cell activation and/or expansion and survival, 178: 3492–3504. 20. Moroz, A., C. Eppolito, Q. Li, J. Tao, C. H. Clegg, and P. A. Shrikant. 2004. up-regulate both PD-1 and its ligands in vitro and in vivo. Of note, IL-21 enhances and sustains CD8ϩ T cell responses to achieve durable tumor the expression of PD-1/PD-1 ligand does not appear to negatively immunity: comparative evaluation of IL-2, IL-15, and IL-21. J. Immunol. 173: impact the ability of T cells to expand, function, or survive in 900–909. ␥ 21. Kovacs, J. A., R. A. Lempicki, I. A. Sidorov, J. W. Adelsberger, I. Sereti, response to further c cytokine exposure, but does render them W. Sachau, G. Kelly, J. A. Metcalf, R. T. Davey, Jr., J. Falloon, et al. 2005. susceptible to PD-1 ligand-mediated suppression of TCR-triggered Induction of prolonged survival of CD4ϩ T lymphocytes by intermittent IL-2 function. It remains to be established whether induction of the therapy in HIV-infected patients. J. Clin. Invest. 115: 2139–2148. ␥ 22. Zeng, R., R. Spolski, S. E. Finkelstein, S. Oh, P. E. Kovanen, C. S. Hinrichs, PD-1 axis by any of these c cytokines plays a role in maintaining C. A. Pise-Masison, M. F. Radonovich, J. N. Brady, N. P. Restifo, et al. 2005. tolerance in vivo under conditions of significant cytokine-driven T Synergy of IL-21 and IL-15 in regulating CD8ϩ T cell expansion and function. cell activation/expansion, such as during immunotherapy and, in J. Exp. Med. 201: 139–148. 23. Kovacs, J. A., M. Baseler, R. J. Dewar, S. Vogel, R. T. Davey, Jr., J. Falloon, some cases, HPE in lymphopenic individuals. M. A. Polis, R. E. Walker, R. Stevens, N. P. Salzman, et al. 1995. 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