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Blood, 1 July 2006, Vol. 108, No. 1, pp. 11-18. Prepublished online as a Blood First Edition Paper on March 16, 2006; DOI 10.1182/blood-2006-01-0144.
PLENARY PAPERS EBV-associated mononucleosis leads to long-term global deficit in T-cell responsiveness to IL-15From the Cancer Research United Kingdom (CRUK) Institute for Cancer Studies and the Medical Research Council (MRC) Centre for Immune Regulation, University of Birmingham, United Kingdom.
In mice, interleukin-7 (IL-7) and IL-15 are involved in T-cell homeostasis and the maintenance of immunologic memory. Here, we follow virus-induced responses in infectious mononucleosis (IM) patients from primary Epstein-Barr virus (EBV) infection into long-term virus carriage, monitoring IL-7 and IL-15 receptor (IL-R) expression by antibody staining and cytokine responsiveness by STAT5 phosphorylation and in vitro proliferation. Expression of IL-7R  was lost from all CD8+ T cells, including EBV epitope-specific populations, during acute IM. Thereafter, expression recovered quickly on total CD8+ cells but slowly and incompletely on EBV-specific memory cells. Expression of IL-15R was also lost in acute IM and remained undetectable thereafter not just on EBV-specific CD8+ populations but on the whole peripheral T- and natural killer (NK)-cell pool. This deficit, correlating with defective IL-15 responsiveness in vitro, was consistently observed in patients up to 14 years after IM but not in patients after cytomegalovirus (CMV)-associated mononucleosis, or in healthy EBV carriers with no history of IM, or in EBV-naive individuals. By permanently scarring the immune system, symptomatic primary EBV infection provides a unique cohort of patients through which to study the effects of impaired IL-15 signaling on human lymphocyte functions in vitro and in vivo. (Blood. 2006;108:11-18)
The immune system relies on tightly controlled mechanisms to regulate the size of lymphocyte pools. From work in mouse systems, cytokines of the common  chain (c) family appear to play an important role in T-cell homeostasis.1 In particular, interleukin-7 (IL-7) is required for the maintenance and homeostatic proliferation of both naive and memory T cells2,3 and is thought to act principally through up-regulation of antiapoptotic proteins. In parallel, IL-15 is involved in the generation and homeostatic (antigen-independent) maintenance of memory CD8+ T cells through acting as a growth, and possibly also as an antiapoptotic, factor.4-6 T-cell responsiveness to these cytokines is in each case determined by expression of the relevant receptor on the cell surface. The IL-7 receptor is a heterodimer consisting of an chain (CD127) that binds IL-7 with high-affinity (Ka = 10-10 M) and the common c (CD132). By contrast, the IL-15 receptor involves a private chain, a  subunit (CD122) shared with the IL-2 receptor, and the common c. The chain alone binds to IL-15 with high affinity (Ka = 10-11 M), and in this case the / complex alone can also bind the cytokine, albeit with much lower affinity.7 Both the IL-7 and IL-15 receptors signal via Janus kinase (JAK) family members; in both cases, the chain signals via JAK1 and the c via JAK3, leading to the nuclear translocation of STAT3 and STAT5, respectively.8
Of particular interest is the role that these cytokines may also play in the regulation of antigen-dependent responses. In mouse models, this is best studied in the context of CD8+ T-cell responses induced by viral infection. Thus, IL-15-deficient mice show reductions in the initial expansion of effector CD8+ T cells that typify acute viral infection,9,10 implying a role for this cytokine in the generation and proliferation of reactive cells. IL-15R Here, we have used the model of Epstein-Barr virus (EBV) infection to ask to what extent such findings mirror events occurring during the human T-cell response to viral challenge. Primary EBV infection, as seen in infectious mononucleosis (IM) patients, is associated with a marked CD8+ T-cell expansion that is largely virus-specific and has been well characterized in terms of immunodominant EBV lytic and latent cycle epitopes.15,16 We therefore followed IM patients prospectively, from the time of acute IM through convalescence into the asymptomatic virus carrier state, and characterized both EBV-specific and total T-cell population for cytokine receptor expression and for cytokine responsiveness.
Donors Cases of IM were identified on clinical grounds and confirmed by high leukocyte counts. EBV-associated cases were identified by heterophile antibody positivity and high EBV DNA loads in peripheral blood mononuclear cells (PBMCs). Cytomegalovirus (CMV)-associated cases were identified among heterophile antibody-negative adult mononucleosis patients by the presence of CMV-specific IgM antibodies, with subsequent isotype switching to IgG, and high CMV DNA loads in plasma.17 Individual IM patients were bled in the acute phase and/or at different time points after infection (between 1 month and 14 years); all patients included in the study had shown resolution of acute disease symptoms within 4 weeks of the initial acute phase. Blood samples were also taken from healthy donors, most of whom were in the same age range (18-30 years) as the EBV-IM patients. These included 30 individuals known to have been EBV and/or CMV carriers for at least 5 years and to have no prior history of IM, 30 individuals with no serologic evidence of prior EBV infection, and 5 individuals with no serologic evidence of prior CMV infection. In each case, PBMCs were isolated from heparinized blood and aliquots of cells were cryopreserved. Ethics approval for this study was granted from the South Birmingham Health Authority Local Research Ethics Committee. Tetramers HLA class I tetramers were prepared for the following epitopes/HLA combinations identified in earlier work15,18,19: EBV lytic cycle epitopes: GLCTLVAML/HLA-A*0201, YVLDHLIVV/HLA-A*0201, RAKFKQLL/HLA-B*0801, and EPLPQGQLTAY/HLA-B*3501; EBV latent cycle epitopes: CLGGLLTMV/HLA-A*0201, FLRGRAYGL/HLA-B*0801, QAKWRLQTL/HLA-B*0801, YPLHEQHGM/HLA-B*3501, and HPVGE-ADYFEY/HLA-B*3501; and CMV epitopes: NLVPMVATV/HLA-A*0201, VLEETSVML/HLA-A*0201, ELRRKMMYM/HLA-B*0801, ELKRK-MIYM/HLA-B*0801, QIKVRVDMV/HLA-B*0801, IPSINVHHY/HLA-B*3501, and TPRVTGGGAM/HLA-B*0701. Throughout this paper, the nomenclature for the epitopes has been abbreviated to the first 3 amino acids (as underlined in the preceding sentence). Peptides were purchased from Alta Biosciences (University of Birmingham, Birmingham, United Kingdom) and phycoerythrin (PE)-conjugated tetramers were produced as described.15 Cell staining
In stainings that included tetramers, cells were first incubated with a pretitrated concentration of PE-conjugated tetramer (
In examining mononuclear cell subsets for receptor expression, PBMCs were first exposed to human IL-R IL-7- and IL-15-induced STAT5 phosphorylation PBMCs were exposed to PE-labeled tetramer for 15 minutes at 37°C in the presence of recombinant IL-7 or IL-15 (R&D Systems) at doses up to 100 ng/mL, then washed and fixed in 2% formaldehyde in phosphate-buffered saline (PBS; Gibco, Paisley, United Kingdom) for 10 minutes at room temperature. Cells were subsequently stained with Tricolour-labeled anti-CD8, then permeabilized with ice-cold 90% methanol, stained intracellularly for 30 minutes at room temperature using rabbit anti-human phospho STAT5 mAb (Tyr694; Cell Signaling Technology, Beverly, MA) followed by FITC-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates), and analyzed by flow cytometry. In some cases, IL-15-induced STAT5 phosphorylation of total CD8+ T cells was assayed as described on PBMCs and in parallel on CD8+ purified T cells and CD14-depleted PBMCs from the same donors (Miltenyi Biotech, Bergisch Gladbach, Germany). CD8+ T cells were purified by positive selection (CD8 microbeads; Miltenyi Biotech), and CD14+ cell depletion was performed using mAb-coated magnetic beads according to the manufacturer's instructions (CD14 microbeads; Miltenyi Biotech). IL-7- and IL-15-supplemented PBMC cultures
PBMCs were resuspended in culture medium, consisting of RPMI (Gibco), 10% vol/vol fetal calf serum (FCS; Gibco), and recombinant human IL-7 or IL-15 (R&D Systems) at a final concentration of 1 ng/mL, with or without the addition of anti-CD3/CD28 coated-beads (1 bead/cell; Dynal, Compiegne, France) as a costimulus. Cells were counted at days 3, 5, and 7 and each time recultured at 1 x 106 cells/mL; a final count was made at day 10. Levels of IL-7R Statistical analysis Statistical analysis was performed using GraphPad prism software (San Diego, CA). Data were compared using a Mann-Whitney test and significant differences verified with 95% confidence intervals.
IL-7R and IL-15R expression on EBV-specific CD8+ T cells
Figure 1 shows data from a typical experiment comparing an IM patient, IM141, studied in the acute phase of disease and again 3 months and 2 years later, with a healthy HLA-B*0801-matched EBV carrier who had no history of IM. Tetramer staining identifies CD8+ T cells reactive to 2 B*0801-restricted viral epitopes, the lytic epitope RAK (Figure 1A) and the latent epitope FLR (Figure 1B), and reveals degrees of expansion and subsequent contraction of these virus-specific responses that are typical of primary EBV infection.15 For both epitope-specific populations, IL-7R
Figure 2 summarizes the cumulative results on EBV-specific CD8+ T cells from 8 IM patients followed prospectively from the time of acute infection, from another 15 IM patients studied only in the acute phase of IM, and from 22 IM patients from whom only later bleeds (1 to 14 years after IM) were available. They are compared with data from 22 healthy EBV carriers, none of whom had any history of symptomatic primary EBV infection. Overall, the phenotypes of 5 EBV lytic and 5 EBV latent epitopes, restricted through either HLA-A*0201, B*0801, or B*3501, were examined. All were consistently low for IL-7R
IL-7R and IL-15R expression on PBMC cell subsets
We noted from Figure 1 that the absence of IL-15R
To ask whether other pathogens inducing clinically apparent mononucleosis might likewise have long-lasting effects on cytokine receptor expression, we studied 4 individuals who presented with severe IM-like symptoms and large CD8+ T-cell expansions caused by primary CMV infection. As shown in Figure 3C, recovery from acute primary CMV infection was accompanied by rising levels of both IL-7R and IL-15R on total CD8+ T cells, approaching the levels seen in healthy CMV carriers and in CMV-naive individuals. In one of these CMV-IM patients, we were able to study the CD8 response to a CMV-coded epitope (the B7-restricted lytic epitope TPR from the pp65 protein) in acute phase and at 3 and 12 months later. As shown in Figure S1 (available on the Blood website; see the Supplemental Figures link at the top of the online article), TPR-specific T cells gradually recovered expression of both cytokine receptors, following the trend seen for receptor expression on CD8+ T cells as a whole. These findings strongly suggest that the long-term deficit in IL-15R expression seen following classic IM is not a consequence of CD8+ T-cell hyperexpansion, per se, but is a specific marker of individuals with a history of the EBV-associated disease.
From IL-15R staining profiles such as Figure 3A, the lymphocyte pool of post-IM patients contained a small subset of CD8- cells that were IL-15R+. To identify these cells, we costained unfractionated PBMCs for the receptor and for subset markers. Data from 2 post-IM patients (studied 5 and 14 years after their acute infection) and from 2 healthy EBV carriers are shown in Figure 4. The post-IM patients expressed IL-15R on both CD19+ B cells and CD14+ monocytes at levels equivalent to that seen in healthy carriers, but failed to express IL-15R on CD8+ T cells, CD4+ T cells, or CD56+ natural killer (NK) cells. We consistently observed these differences in assays on PBMCs from 11 post-IM patients versus 9 healthy carriers, using 2 different anti-IL-15R-specific mAbs.
Functional CD8+ T-cell responses to IL-7 and IL-15 in relation to receptor status
To determine whether cytokine receptor expression levels correlated with responsiveness, we exposed PBMCs from post-IM patients and healthy virus carriers to 1 ng/mL IL-7 or IL-15 and then assayed for cytokine-triggered phosphorylation of STAT5 as a marker of response. Figure 5 shows data from a representative IM patient, the HLA-A*0201-positive IM81, studied in acute phase and 14 years after infection, and from an HLA-B*0801-positive EBV carrier control. Responses to IL-7 among CD8+ T cells (Figure 5A) were barely detectable in the acute IM81.1 sample, whether focusing on cells specific for EBV lytic (YVL) or latent (CLG) epitopes or on the total CD8 population. However, in the IM81.5 sample taken 14 years later, the majority of cells in both epitope-specific memory populations and in the CD8 population as a whole showed STAT5 phosphorylation at levels indistinguishable from the healthy carrier. Of importance, parallel assays using IL-15 as the stimulus (Figure 5B) clearly showed that the absence of IL-15R
Figure 6 summarizes data from further experiments comparing responses (in 11 long-term post-IM patients versus 18 healthy carrier controls) to increasing cytokine concentrations, mean levels of response being expressed in each case as the percentage of cells showing STAT5 phosphorylation in EBV epitope-specific and in total CD8+ T-cell populations. Responses to IL-7 (Figure 6A) titrated similarly in the 2 types of donor, with responses just detectable at 0.01 ng/mL IL-7 and peaking at 10 to 100 ng/mL; the only discernable difference was that the maximal response in the total CD8 T-cell population was consistently lower in post-IM patients than in healthy carriers. However, responses to IL-15 were quite different (Figure 6B): while healthy carriers gave titration curves similar to those seen for IL-7, with significant levels of STAT5 phosphorylation detectable at 0.1 ng/mL IL-15 and almost maximal levels at 10 ng/mL, the post-IM donors began to show some responses only at 10 ng/mL or higher. From these titration curves, estimates of cytokine doses required for half-maximal responses showed that post-IM donor CD8+ T cells (whether the EBV-specific or the total population) were more than 20-fold less sensitive to IL-15 than healthy carrier cells. This is consistent with IL-15R -negative post-IM CD8+ T cells binding cytokine via the low-affinity / complex of the IL-15R.7 Indeed, we confirmed by specific mAb staining that CD8+ T cells from post-IM donors did express both and chains at levels in the same range as CD8+ T cells from healthy carriers (data not shown).
Note that these previous assays were conducted on whole PBMC populations that contain monocytes. This was a potential concern since, in mouse systems, monocytes have been shown to bind IL-15 through their expression of the IL-15R
Finally, we studied the in vitro proliferative response to 1 ng/mL IL-7 and IL-15, comparing PBMCs from long-term post-IM patients with those from healthy carriers, in each case with or without anti-CD3/CD28 mAb-coated beads as a stimulus. As shown in Figure 7A, there was a detectable proliferative response to IL-7 in post-IM PBMC cultures, with or without CD3/CD28 costimulation, although this was reproducibly less than that observed in healthy carriers (Figure 7A). However, while PBMCs from healthy carriers maintained cell numbers when cultured in IL-15 alone and proliferated well in costimulated cultures, in both situations post-IM PBMCs showed significant levels of cell death within the first 3 days; thereafter, there was some proliferation in costimulated cultures but progressive death in IL-15 alone (Figure 7B). In the same experiments, we monitored cytokine receptor expression at the beginning of cell culture and in viable cells harvested 3 and 10 days after CD3/CD28 costimulation in the presence of cytokine. As shown in Figure S2A, in both post-IM and healthy carrier cultures, expression of IL-7R was rapidly down-regulated on CD8+ and on CD8- lymphocyte populations by day 3, but then recovered to reach high levels by day 10; at that time, the majority of cells in the cultures were in fact CD8+ T cells. In the IL-15 and CD3/CD28-costimulated cultures, the healthy carrier showed a similar response, with down-regulation of IL-15R expression by day 3 followed by recovery at day 10. However, even after such combined cytokine and CD3/CD28 costimulation, there was never any induction of IL-15R on cells from post-IM donors (Figure S2B).
Prompted by studies showing a role for IL-7 and IL-15 in the development and maintenance of CD8+ memory T cells to viral infection in mouse systems,4,24 the present work explored the analogous situation in humans by following young adults in whom primary EBV infection is manifest as IM. With regard to IL-7, as for the primary responses in mice,11 most of the highly expanded CD8+ T-cell population seen in the blood of acute IM patients, including defined EBV epitope-specific populations, has down-regulated IL-7R expression. Thereafter, IL-7R expression on the total CD8 population recovered to healthy control donor levels within a few months, by which time activated cells have disappeared from the blood and the circulating CD8+ T-cell pool has returned within the normal range.15,25 Of interest, EBV epitope-specific CD8+ T cells in the blood of post-IM patients take 2 or more years to acquire the levels of IL-7R expression shown by equivalent memory populations in healthy EBV carriers. This contrasts with the situation in mice that have controlled and cleared lymphocytic choriomeningitis virus (LCMV) infection, where IL-7R up-regulation appears to be an immediate identifier of cells entering memory.4,11,13 The data from post-IM patients are in fact closer to those reported for mice carrying persistent low-grade LCMV infection, where virus-specific CD8+ T cells express only low levels of IL-7R and appear to be dependent upon continual antigenic stimulation rather than upon IL-7-mediated homeostatic signals for their maintenance.26
Parallels with mouse models broke down when the study turned to IL-15R
These findings raised the possibility that transient downregulation of IL-15R
Acute primary EBV infection, when manifest as IM, appears to be unique in producing a global down-regulation of IL-15R
These in vitro studies leave open the question as to what the biologic consequences of IL-15R
We are very grateful to Dr Naeem Khan (Birmingham) for providing CMV-restricted tetramers; to Dr Mark Wills (Cambridge) for access to PBMCs from post-CMV-IM patients; and to Drs Dorothy Crawford (Edinburgh), Jan Gratama (Rotterdam), Eric Robinet (Besançon), Cliona Rooney (Houston), and Tim Wellinger (Oxford) for providing PBMCs from EBV-naive donors. The authors have no conflicting financial interests.
Submitted January 12, 2006; accepted February 11, 2006.
Prepublished online as Blood First Edition Paper, March 16, 2006; DOI 10.1182/blood-2006-01-0144.
Supported by the Medical Research Council, United Kingdom. D.S. has benefited from a Lavoisier grant from the Ministère des affaires étrangères, France.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: A. B. Rickinson, CRUK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston Birmingham B15 2TT, United Kingdom; e-mail: a.b.rickinson{at}bham.ac.uk.
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Abstract
Introduction
0.5 µg/mL) at 37°C for 15 minutes and then washed, and all further steps were conducted on ice. Cells were exposed to human IL-R
