The CD8+ T cell compartment of human CMV-seropositive individuals characteristically contains a high proportion of cells that express NK cell receptors (NKRs) which may contribute to the surveillance of virus-infected cells. To test whether this enhanced expression is a direct and immediate result of CMV infection, we used DNA microarrays to analyze putative changes in the RNA expression level of 39 NKRs in CMV-specific CD8+ T cells of renal transplant recipients experiencing primary CMV infection. Already in the acute phase of infection 29 NKRs were induced, of which 19 remained high 1 year after cessation of viral replication. Activating and inhibitory NKRs were induced to a similar extent. Detailed longitudinal flow cytometric analyses confirmed NKR changes at the protein level. Strikingly, a strong induction of CD94 on CD3+ T cells was observed with surface expression of activating CD94dim NKG2C dimers appearing before inhibitory CD94bright NKG2A ones. After the acute phase of infection, the balance between inhibitory and activating receptors did not change. Thus, CMV infection induces a rapid and lasting change in the expression of NKRs on human CD8+ T cells.

Cytomegalovirus is a persistent β herpes virus that in the Western world infects 60–90% of the population. In immunocompetent individuals, primary CMV infection is infrequently diagnosed because of the lack of prototypical symptoms. Immunocompromised patients, however, may experience severe complaints due to pulmonary and gastrointestinal pathology (1). In the suppression of initial CMV replication and the maintenance of viral latency, both NK cells and T cells have been implicated (2). CMV appears to be unique among the persisting viruses in that it stably augments the number of circulating resting cytolytic T cells (3). Within the CD4+ pool, these cells are characterized by the absence of CD28 and the presence of cytolytic granules containing perforin and granzyme B (4). For a fraction of these cells, specificity for CMV peptides presented by HLA class II molecules can indeed be demonstrated (5). Also, the CD8+ T cell compartment of CMV carriers contains an expanded fraction of perforin- and granzyme-expressing cells (6). These cells have been characterized by various phenotypic markers (for a review, see Ref. 7) and CMV peptide-specific CD8+ T cells are often contained within this distinctive subset (3). Frequently, CMV-specific CD8+ T cells in the latency stage lack the lymph node-homing receptor CCR7 and the costimulatory receptors CD27 and CD28, but express CD57 and CD45RA. Longitudinal studies in kidney transplant recipients have indicated that the acquisition of this phenotype by Ag-specific CD8+ T cells is a stepwise process that continues for an extended period after primary CMV infection (8). Because these cells typically lack expression of the classical costimulatory receptors, it has been suggested that other surface molecules might contribute to their activation upon encountering virus-infected cells (9, 10, 11).

NK cell receptors (NKRs)4 have first been described as surface receptors on NK cells that bind to specific HLA class I molecules (12, 13, 14, 15). These receptors may belong to either the Ig- or C-type lectin superfamilies and transmit, upon binding their ligands, inhibitory or activating signals to the cell’s interior. The quality of the signal depends on the composition of the intracellular parts of the individual receptors or their associated molecules. Additionally, NK cells express natural cytotoxic receptors (NCR), like NKp44 and NKp46, for which no ligands have been conclusively defined but that appear to function as strong activators of NK cell function. Apart from NK cells, expression of NKRs is also found on the resting cytotoxic effector cells of CMV carriers (16). In agreement with this, transsectional cohort studies have shown that CMV infection leaves an imprint in the NKR repertoire on T cells (17). Whether NKRs on T cells regulate recognition and subsequent elimination of CMV-infected cells is not clear. Still, certain NKRs are able to down-modulate cytotoxicity induced via TCR/CD3 or to inhibit cytolysis after cross-linking other NKRs (18, 19, 20, 21). Triggering of the C-type lectin receptor NKG2D on Ag-specific CD8+ cells does not trigger cytokine production, calcium mobilization, nor cytotoxicity as it does in NK cells, but, instead, it augments cytotoxic and proliferative responses of T cells (9). Other NKRs may lower activation thresholds (22) and thereby function as costimulatory receptors for T cells. Ibegbu et al. (23) described the presence of the inhibitory C-type lectin KLRG1 in combination with CD57 on terminally differentiated memory CD8+ cells. These cells secrete cytokines but have a poor replicative proliferation, suggesting a modifying role of KLRG1 in these CD8+ cells.

Elegant studies in mice have revealed that NKRs are able to modify the functional competence of virus-specific T cells in vivo. CD94-NKG2A, an inhibitory receptor of the C-type lectin class, was shown to induce anergy in a polyomavirus-specific CD8+ T cell population, contributing to the development of tumors (24). Thus, NKRs on T cells may be indispensable for an adequate balance of activation and control of T cells during viral infection.

The development of CMV-specific T cell responses can longitudinally be followed in recipients of kidney allografts that encounter CMV for the first time as a result of virus reactivation from the transplanted organ (25). We used DNA microarrays combined with flow cytometry to obtain a comprehensive view on the regulation of NKR expression on CMV-induced CD8+ T cells. Specifically, we asked whether 1) NKR expression is a direct and immediate consequence of CMV infection and 2) whether the balance between activating NKRs (aNKRs) and inhibitory NKRs (iNKRs) changes between the acute and latent stage.

Renal transplant recipients were treated with basiliximab prophylactically and with basic immunosuppressive drug therapy consisting of prednisolone, mycophenolate mofetil, and cyclosporin. Thirteen of 15 patients experienced a primary CMV infection. Antiviral therapy consisted of ganciclovir. Cells from three patients were used in microarray analyses. Longitudinal samples from 10others were used for detailed flow cytometry. Two CMV-seronegative renal transplant patients who did not develop primary CMV infection were used to verify whether the phenotypical changes were indeed inflicted by CMV infection rather than by the transplantation itself. Patient characteristics are listed in Table I. All patients gave written informed consent and the study was approved by the local medical ethical committee.

Table I.

Patient characteristics

PatientAge (Years)Days to First Positive PCRNo. of Days with CMV+ PCRHLA Typing of Recipient (R) and Donor (D)Assays
69 31 219 R: A2/3 B7/39(16) DR15(2)/12(5) Microarray 
    D: A2/23(9) B62(15)/49(21) DR11(5)/13(6)  
35 45 99 R: A2/3 B7/35 DR1/15(2) Microarray 
    D: A2/11 B7/8 DR4/15(2)  
27 62 144 R: A1/3 B7/35 DR15(2)/4 Microarray 
    D: A1/3 B7/35 DR4  
35 47 68 R: A11/3 B51(5)/39(16) DR1/15(2) FACS 
    D: A11/28 B51(5)/27 DR1/9  
73 25 122 R: A1/2 B7/60(40) DR13(6) FACS 
    D: A1 B63(15)/8 DR17(3)/13(6)  
42 46 118 R: A26(10)/32(19) B44(12)/8 DR3/12(5) FACS 
    D: A1/31(19) B39(16)/8 DR17(3)/8  
35 25 79 R: A3/11 B7/51(5) DR15(2)/14(6) FACS 
    D: A11/2 B51(5) DR12(5)/14(6)  
47 28 83 R:A1/3 B7/8 DR15(2)/3 FACS 
    D: A3/11 B7/45(12) DR15(2)/4  
30 29 80 R: A1/2 B8/8 DR17(3) FACS 
    D: A2 B8/41 DR3/7  
10 33 NA R: A3/24(9) B51(5)/55(22) DR4/13(6) FACS 
    D: A2/24(9) B51(5)/55(22) DR13(6)/14(6)  
11 49 NA R: A2/3 B60(40)/13 DR13(6)/7 FACS 
    D: A1/3 B8/13 DR3/7  
12 32 96 14 R: A28 B60(40)/44(12) DR1/13(6) FACS 
    D: A2/28 B44(12)/57(22) DR1/7  
13 26 33 37 R: A3/31(19) B51(5) DR3/14(6) FACS 
    D: A2/3 B27/61(40) DR16(2)/17(3)  
14 32 61 45 R: A11/31(19) B51(5)/60(40) DR1/4 FACS 
    D: A2/3 B13/51(5) DR1/7  
15 32 49 74 R: A3/11 B7/48 DR15(2) 15(2) FACS 
    D: A3/11 B7/18 DR15(2)/3  
PatientAge (Years)Days to First Positive PCRNo. of Days with CMV+ PCRHLA Typing of Recipient (R) and Donor (D)Assays
69 31 219 R: A2/3 B7/39(16) DR15(2)/12(5) Microarray 
    D: A2/23(9) B62(15)/49(21) DR11(5)/13(6)  
35 45 99 R: A2/3 B7/35 DR1/15(2) Microarray 
    D: A2/11 B7/8 DR4/15(2)  
27 62 144 R: A1/3 B7/35 DR15(2)/4 Microarray 
    D: A1/3 B7/35 DR4  
35 47 68 R: A11/3 B51(5)/39(16) DR1/15(2) FACS 
    D: A11/28 B51(5)/27 DR1/9  
73 25 122 R: A1/2 B7/60(40) DR13(6) FACS 
    D: A1 B63(15)/8 DR17(3)/13(6)  
42 46 118 R: A26(10)/32(19) B44(12)/8 DR3/12(5) FACS 
    D: A1/31(19) B39(16)/8 DR17(3)/8  
35 25 79 R: A3/11 B7/51(5) DR15(2)/14(6) FACS 
    D: A11/2 B51(5) DR12(5)/14(6)  
47 28 83 R:A1/3 B7/8 DR15(2)/3 FACS 
    D: A3/11 B7/45(12) DR15(2)/4  
30 29 80 R: A1/2 B8/8 DR17(3) FACS 
    D: A2 B8/41 DR3/7  
10 33 NA R: A3/24(9) B51(5)/55(22) DR4/13(6) FACS 
    D: A2/24(9) B51(5)/55(22) DR13(6)/14(6)  
11 49 NA R: A2/3 B60(40)/13 DR13(6)/7 FACS 
    D: A1/3 B8/13 DR3/7  
12 32 96 14 R: A28 B60(40)/44(12) DR1/13(6) FACS 
    D: A2/28 B44(12)/57(22) DR1/7  
13 26 33 37 R: A3/31(19) B51(5) DR3/14(6) FACS 
    D: A2/3 B27/61(40) DR16(2)/17(3)  
14 32 61 45 R: A11/31(19) B51(5)/60(40) DR1/4 FACS 
    D: A2/3 B13/51(5) DR1/7  
15 32 49 74 R: A3/11 B7/48 DR15(2) 15(2) FACS 
    D: A3/11 B7/18 DR15(2)/3  

NA, Not applicable.

Allophycocyanin-conjugated HLA-A2 tetramer loaded with the CMV pp65-derived NLVPTMVATV peptide and allophycocyanin-conjugated HLA-B7 tetramer loaded with the CMV pp65-derived TPRVTGGGAM peptide were obtained from Sanquin.

PBMC were isolated from heparinized blood using standard density gradient centrifugation and subsequently cryopreserved in liquid nitrogen until the day of analysis.

To isolate naive CD8+ cells, cells were isolated from buffy coats from healthy donors by a two-step procedure. After Ficoll, CD8+ T cells were isolated by CD8+ microbeads (Miltenyi Biotec) and stored overnight at 4°C in 10% (v/v) serum-containing medium. CD8+ T cells were then labeled with CD27-FITC (7C9, homemade), CD45RA-RD1 (Coulter), and CD8-allophycocyanin (BD Pharmingen) and FACS sorted using a FACS Aria (BD Biosciences) in naive CD8+ T cells (CD8+CD45RAhighCD27high). To isolate CMV-specific CD8+ effector cells at the peak of the CMV response, PBMC were stained with HLA-DR-FITC (BD Biosciences, CD38-PE (BD Biosciences), and CD8-allophycocyanin (BD Pharmingen). HLA-DRhighCD38highCD8+ were sorted using a FACS Aria (BD Biosciences).

To obtain CMV-specific CD8+ cells in the latency phase, PBMC obtained from 40 to 60 wk after transplantation (long term, 1 year postinfection (p.i.)) were stained with allophycocyanin-conjugated tetramers and subsequently allophycocyanin microbeads (Miltenyi Biotec) were used to isolate the cells. Upon reanalysis, the purified populations contained between 95 and 97% tetramer-binding cells.

RNA was isolated using the nucleospin RNA isolation kit (Machery-Nagel) according to the manufacturer’s instructions. mRNA was amplified using the MessageAmp II Kit (Ambion). Labeling, hybridization, and data extraction were performed at ServiceXS (Leiden, The Netherlands). Briefly, 100 ng of total RNA was mixed with 1 μl of T7 oligo(dT) primer in a total volume of 12 μl. Primer and template were denatured by incubating at 70°C for 10 min and annealed by putting the reaction tubes on ice. The first-strand reaction was performed by adding 8 μl of Reverse Transcription Master Mix (containing 10× First Strand buffer, RNase inhibitor, dNTP mix, and reverse transcriptase) and incubating at 42°C for 2 h. Second- strand cDNA synthesis was done by adding 63 μl of Nuclease-Free Water, 10 μl of 10× second-strand buffer, 4 μl of dNTP mix, 2 μl of DNA polymerase, and 1 μl of RNase H and incubating at 16°C for 2 h. cDNA purification was done according to the manufacturer’s protocol (Ambion). In vitro transcription was initiated by addition of 2 μl of aaUTP Solution (50 mM), 12 μl of ATP, CTP, and GTP mix (25 mM), 3 μl of UTP Solution (50 mM), 4 μl of T7 10× reaction buffer, and 4 μl of T7 enzyme mix and incubated at 37°C for 9 h. Machery-Nagel RNA Clean up mini-spin columns were used for purification of the cRNA. Dye coupling reaction was performed using 5 μg of amino allyl aRNA in 3.33 μl, 5 μl of DMSO, and 1.66 μl of NaCO3 buffer added to the monoreactive dye, prepared according to the manufacturer’s protocol (Amersham Biosciences). After an incubation at room temperature for 60 min, 4.5 μl of 4 M hydroxylamine was added and incubated at room temperature for 15 min. Dye-labeled aRNA was purified with Machery-Nagel RNA Clean up mini-spin columns and the samples were checked on concentration and dye incorporation on the Nanodrop ND-1000. Hybridization was performed with 600 ng of each labeled target along with fragmentation and hybridization buffer at 60°C for 17 h onto Human Whole Genome (WHG) 44K Oligo Microarrays from Agilent Technologies per the manufacturer’s protocol.

The microarray slides were washed following the user manual instructions and scanned using the Agilent dual-laser DNA microarray scanner. Default settings of Agilent Feature Extraction preprocessing protocols were used to obtain normalized expression values from the raw scans. Exact protocol and parameter settings are described in the Agilent Feature Extraction Software User Manual 7.5 (http://chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=37629). The default Agilent normalization procedure, called Linear & Lowess, was applied. Rosetta Resolver (Rosetta Biosoftware, Seattle, WA) was used for analysis of the data.

PBMC were washed in PBS containing 0.01% (w/v) NaN3 and 0.5% (w/v) BSA (PBA). PBMC (500,000) were incubated with appropriate concentrations of tetrameric complexes in a small volume for 30 min at 4°C, protected from light. Then fluorescent-labeled conjugated mAbs (concentrations according to the manufacturer’s instructions) were added and incubated for 30 min at 4°C, protected from light. For analysis of expression of surface markers, the following mAbs were used in different combinations: CD56-allophycocyanin, NKG2D-allophycocyanin (BD Pharmingen), KIR2DS1/L1-PE (CD158a and h, clone EB6), KIR2DL2/L3/S2-PE (CD158b and j, clone GL183), NKp44-PE, NKp46-PE, NKG2A-PE (all from Beckman Coulter), NKG2C-PE (R&D Systems), CD27-FITC (homemade clone 3A12 for FACS analysis and homemade clone 7C9 for FACS sorting), CD27-PE (clone L128), CD3-PerCP-Cy5.1, CD94-PE, CD94-allophycocyanin, NKB1 (KIR3DL1)-FITC, CD38-PE, CD45RA-FITC, anti-HLA-DR-FITC (all BD Biosciences). Cells were washed in PBA and analyzed using a FACSCalibur flow cytometer (BD Biosciences) or FACS Canto flow cytometer (BD Biosciences) and CellQuest software (BD Biosciences) or FlowJo software (Tree Star).

From patients 4 and 8 (see Table I), CD8+CD158+ cells were sorted at two time points: at the peak of the viral load (T1) and 2 mo later (T2: patient 4) or 6 mo later (T2: patient 8) when the virus was not detectable. RNA was isolated and quantitative PCR was performed on cDNA with the LightCycler System (Roche Diagnostics) in microcapillary tubes with a QuantiTect SYBR green PCR kit solution (Qiagen). After a 15-min denaturation step at 95°C, 50 PCR cycles of 15 s at 94°C, 30 s at 58°C, 45 s at 72°C, and 5 s at 79°C were performed. To confirm the purity and specificity of the reaction, a melting curve analysis was performed at the end of the PCR by slowly increasing (0.1°C/s) the temperature of the reaction from 65 to 95°C. A standard curve was established with a dilution of purified PCR product from 10 to 107 molecules. GAPDH was used as an internal reference. The killer Ig-like receptor (KIR) primers used were either the same as those published by Uhrberg et al. (26) or one nucleotide modifications thereof: KIR2DS2, 5′-CTGCACAGAGAGGGGAAGTA-3′; reverse, 5′-ACGCTCTCTCCTGCCAA-3′; KIR2DL2, 5′-CATGATGGGGTCTCCAAA-3′; reverse, 5′-GCCCTGCAGAGAACCTACA-3′; KIR2DL3, 5′-CCACTGAACCAAGCTCCG-3′; reverse, 5′-GCAGGAGACAACTTTGGATCA-3′; and GAPDH, 5′-GGACCTGACCTGCCGTCTAG-3′; reverse, 5′-CCACCACCCTGTTGCTGTAG-3′.

Trend plotting was performed in a Rosetta Resolver with a fold change cutoff of ≥2 and a one-way ANOVA test with a p value cutoff of ≤0.05 with the Benjamini and Hochberg false-discovery rate correction to select sequences with differences across the different cell populations. The paired, nonparametric Wilcoxon-signed rank test was used for analysis of genes that differ between different cell populations. The nonparametric Mann-Whitney U test was used for analysis of differences between aNKRs and iNKRs. Linear regression analysis was performed to show the relationship between NKG2A and CD94bright expression. Significance was indicated by p < 0.05.

To analyze regulation of NKR gene expression by CMV in humans, we took advantage of the fact that dynamic change in virus-specific T cell populations can carefully be tracked in CMV-seronegative renal transplant recipients that experience a primary CMV infection due to the transplantation of a CMV-carrying donor kidney. As described earlier (8), circulating CMV-tetramer-binding CD8+ cells emerge ∼2–4 wk after the first positive CMV PCR, but their number is too low to obtain enough cells for microarray analyses. To be able to include enough cells eligible for analysis, we isolated all virus-activated CD8+ T cells by sorting CD8+HLA-DR+CD38+ T cells. We previously showed that these activated lymphocytes appear at a high frequency early in the anti-CMV response and that at this point all CMV-tetramer+ cells are contained within this population (8). To obtain virus-specific cells in the latency stage, CMV-specific pp65 tetramer+ cells were isolated from the same patients between 40 and 60 wk after the primary response. At this time point, no CMV could be detected in the blood by sensitive PCR. Because we used the two-color microarray Agilent WHG chips to perform RNA expression profiling, the expression level of each gene in a specific population is given as a ratio relative to the expression level of naive CD8+ cells obtained from healthy donors (subset CD8+ cells/naive CD8+ cells).

Microarray analysis revealed a number of changes in gene expression levels of several molecules (Fig. 1). The changes in mRNA expression levels of Ki-67, CD28, CCR7, CD27, CD127, perforin, granzyme B, and IFN-γ were confirmed at the protein level by FACS analyses; Ki-67, a proliferation marker, was only elevated during the peak of viral replication. In contrast, CD28, CD27, CD127, and CCR7 expression was low on virus-specific T cells. Finally, the cytolytic proteins granzyme B and perforin and the antiviral cytokine IFN-γ were high in virus-specific cells.

FIGURE 1.

Confirmation of the microarray results by FACS analyses of selected CD8+ T cell proteins that are known to be regulated during a primary CMV infection. Graphs show the RNA fold changes found by microarray analysis (as compared with naive CD8+ cells) for the genes indicated. The protein confirmation of the selected genes is performed by flow cytometry. Density plots are gated on CD8+ T cells. Depicted on the x-axis is the pp65 CMV-specific tetramer staining, on the y-axis is the protein as indicated. peak, Peak of CMV infection, 1 year; ∼1 year after CMV infection. Quadrants depicted as percentages of total CD8+ T cells. Shown is one representative patient (patient 15).

FIGURE 1.

Confirmation of the microarray results by FACS analyses of selected CD8+ T cell proteins that are known to be regulated during a primary CMV infection. Graphs show the RNA fold changes found by microarray analysis (as compared with naive CD8+ cells) for the genes indicated. The protein confirmation of the selected genes is performed by flow cytometry. Density plots are gated on CD8+ T cells. Depicted on the x-axis is the pp65 CMV-specific tetramer staining, on the y-axis is the protein as indicated. peak, Peak of CMV infection, 1 year; ∼1 year after CMV infection. Quadrants depicted as percentages of total CD8+ T cells. Shown is one representative patient (patient 15).

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On the Agilent’s WHG array, 39 NKR-related sequences were present. Changes in their relative expression levels are represented in Table IIand Fig. 2. Three distinct expression profiles could be distinguished. First, the expression of only eight NKRs such as LILRA4 or KIR3DL3 did not show significant changes (Table II). Second, a limited number of receptors, including KIR2DS2, KIR2DS4, and KIR3DL2, were up-regulated during the peak of the CMV infection but returned to baseline levels in the latency stage (Fig. 2,A). However, the majority of NKR genes were induced early in the response and remained high in the follow-up. Two NKRs (LAIR1 and LILRP2) were only induced in the latent state (Table II).

Table II.

Changes in NKR during primary CMVa

Alternative NameNaivebPeakc1 YeardmAbse
aNKR      
 MHC class I specific      
  KIRs      
   KIR2DS2 CD158j 3,6 1,4 GL183f 
   KIR2DS4 CD158l 1,2 6,4 1,9  
   KIR3DL5A CD158f 1,1 2,3 1,1  
  Leukocyte Ig-like      
   LILRA1 LIR6/CD85l 0,9 10,6 13,4  
   LILRA3 ILT6/LIR4/CD85e 0,9 2 2,9  
   LILRA4 ILT7 0,9 1,3 1,3  
   LILRA5 CD85f 0,9 3,7 2,3  
 Non-MHC class I specific      
  NCRs      
   NCR1 NKp46 0,9 47,4 39,1 NKp46 
   NCR2 NKp44 1,1 0,9 0,9 NKp44 
   NCR3 NKp30 1,1 1,4 0,9  
  C-type lectin receptors      
   KLRC2 NKG2C 9,8 7,8 NKG2C 
   KLRK1 NKG2D 3,6 2,3 NKG2D 
   KLRC3 NKG2E 2,8 1,2  
   KLRC4 NKG2F 2,2 1,9  
   KLRF1  0,9 5 12,8  
   CLEC2B AICL 3,8 2,2  
  Other      
   CD16 FcyIIIa 0,9 8,5 14  
   CD244 2b4 1,1 13,5 10,6  
   NKG7  8,5 14,9  
iNKR      
 MHC class I specific      
  KIRs      
   KIR2DL1 CD158a 0,8 6 1,6 EB6g 
   KIR2DL2 CD158b1 5 1,5 GL183f 
   KIR2DL4 CD158d 2,9  
   KIR3DL1 CD158e1/NKB1 2,9 1,1 NKB1 
   KIR3DL2 CD158k 1,1 7,1 1,4  
   KIR3DL3 CD158z 1,1 1,3  
  Leukocyte Ig-like receptors      
   LILR1  0,9 2,3 2,4  
   LILRB1 ILT2/LIR1/CD85j 28,3 61,4  
   LILRB2 ILT4/LIR2/CD85d 1,6 13,9 17,9  
   LILRB3 ILT5/LIR3/CD85a 2,1 2,2  
   LILRB4 ILT3/LIR5/CD85k 0,9 1,1  
   LILRB5 LIR8/CB85c 0,9 1,4  
 Non-MHC class I specific      
  NCRs      
   None      
  C-type lectin receptors      
   KLRA1  2,2 2,4  
   KLRB1 CD161 9,3 17,1  
   KLRC1 NKG2A/CD159A 1,1 9 8 NKG2A 
   KLRG1 MAFAL 0,9 11,5 14,5  
  Other      
   LAIR1  1,1 2,6  
   LAIR2  1,2 11,8 34,4  
Either aNKR or iNKR      
  Leukocyte Ig-like receptors      
   LILRP2 ILT10/CD85m 1,5 2,1  
  C-type lectin receptors      
   KLRD1 CD94 36,5 38,9 CD94 
Alternative NameNaivebPeakc1 YeardmAbse
aNKR      
 MHC class I specific      
  KIRs      
   KIR2DS2 CD158j 3,6 1,4 GL183f 
   KIR2DS4 CD158l 1,2 6,4 1,9  
   KIR3DL5A CD158f 1,1 2,3 1,1  
  Leukocyte Ig-like      
   LILRA1 LIR6/CD85l 0,9 10,6 13,4  
   LILRA3 ILT6/LIR4/CD85e 0,9 2 2,9  
   LILRA4 ILT7 0,9 1,3 1,3  
   LILRA5 CD85f 0,9 3,7 2,3  
 Non-MHC class I specific      
  NCRs      
   NCR1 NKp46 0,9 47,4 39,1 NKp46 
   NCR2 NKp44 1,1 0,9 0,9 NKp44 
   NCR3 NKp30 1,1 1,4 0,9  
  C-type lectin receptors      
   KLRC2 NKG2C 9,8 7,8 NKG2C 
   KLRK1 NKG2D 3,6 2,3 NKG2D 
   KLRC3 NKG2E 2,8 1,2  
   KLRC4 NKG2F 2,2 1,9  
   KLRF1  0,9 5 12,8  
   CLEC2B AICL 3,8 2,2  
  Other      
   CD16 FcyIIIa 0,9 8,5 14  
   CD244 2b4 1,1 13,5 10,6  
   NKG7  8,5 14,9  
iNKR      
 MHC class I specific      
  KIRs      
   KIR2DL1 CD158a 0,8 6 1,6 EB6g 
   KIR2DL2 CD158b1 5 1,5 GL183f 
   KIR2DL4 CD158d 2,9  
   KIR3DL1 CD158e1/NKB1 2,9 1,1 NKB1 
   KIR3DL2 CD158k 1,1 7,1 1,4  
   KIR3DL3 CD158z 1,1 1,3  
  Leukocyte Ig-like receptors      
   LILR1  0,9 2,3 2,4  
   LILRB1 ILT2/LIR1/CD85j 28,3 61,4  
   LILRB2 ILT4/LIR2/CD85d 1,6 13,9 17,9  
   LILRB3 ILT5/LIR3/CD85a 2,1 2,2  
   LILRB4 ILT3/LIR5/CD85k 0,9 1,1  
   LILRB5 LIR8/CB85c 0,9 1,4  
 Non-MHC class I specific      
  NCRs      
   None      
  C-type lectin receptors      
   KLRA1  2,2 2,4  
   KLRB1 CD161 9,3 17,1  
   KLRC1 NKG2A/CD159A 1,1 9 8 NKG2A 
   KLRG1 MAFAL 0,9 11,5 14,5  
  Other      
   LAIR1  1,1 2,6  
   LAIR2  1,2 11,8 34,4  
Either aNKR or iNKR      
  Leukocyte Ig-like receptors      
   LILRP2 ILT10/CD85m 1,5 2,1  
  C-type lectin receptors      
   KLRD1 CD94 36,5 38,9 CD94 
a

Bold numbers represent fold changes of significantly changed genes according to the criteria described in Materials and Methods.

b

Naïve: CD8+CD45+CD27+.

c

Peak: CD8+CD38+HLA-DR+.

d

One year: CMV-specific tetramer pp65+ cells.

e

mAbs used in FACS analyses.

f

Clone GL183 detects KIR2DS2/2DL2/2DL3.

g

Clone EB6 detects KIR2DS1/L1.

FIGURE 2.

Two expression profiles of significantly regulated NKRs upon primary CMV infection. Trend plots are made with the Rosetta Resolver software; bold lines connect significantly changed NKRs. Background lines represent all gene expression data of the microarray. A, Transient up-regulation during acute phase of infection. B, Up-regulation during the acute phase and ∼40 wk p.i.

FIGURE 2.

Two expression profiles of significantly regulated NKRs upon primary CMV infection. Trend plots are made with the Rosetta Resolver software; bold lines connect significantly changed NKRs. Background lines represent all gene expression data of the microarray. A, Transient up-regulation during acute phase of infection. B, Up-regulation during the acute phase and ∼40 wk p.i.

Close modal

Remarkably, KIRs, that bind epitopes on HLA-C and HLA-B class I alleles were almost exclusively present in the second profile, e.g., induced during the acute phase of the viral infection, but absent in CMV pp65-specific tetramer-positive CD8+ cells in the renal transplant patients 1 year p.i. The other classes of receptors did not show predominance for a particular expression profile.

To visualize the CMV-induced changes on NKR gene expression in CD8+ T cells, we calculated a cumulative fold change and the mean fold change of all receptors. When all NKR genes were grouped together, a strong increase of NKR expression was observed both in the acute and latency stages (Fig. 3,A). Of the 39 NKR genes on the chip, 19 are activating, 18 are inhibiting, while 2 have yet an unknown function. CD94 forms heterodimers with either inhibiting NKG2A or activating NKG2C. Separating the NKRs in aNKRs and iNKRs revealed a similar expression pattern for both types of receptors (Fig. 3 B). Since a small change level in RNA can have a major impact at the protein level, we also counted the number of aNKRs and iNKRs that changed during infection. This showed that there was no selective induction of either aNKRs or iNKRs at a particular stage; at the peak of CMV, 16 aNKRs, 12 iNKRs and CD94 were elevated, whereas 11 aNKRs, 8 iNKRs, and 2 “unknown function” NKRs (LILRP2 and CD94) were up-regulated after ∼1 year of infection. Thus, the array data did not provide any indication that the balance between activating and inhibitory receptor genes differs between virus-specific cells in the acute vs the latent stage. It should be mentioned that in the acute phase CMV-specific CD8+ T cells containing activated T cells were studied, while in the latent phase pp65-tetramer+ CMV-specific cells were studied. This could potentially influence the results, although it is not expected to radically change the results since tetramer+ T cells also have a CD38+HLA-DR+ phenotype in the acute phase.

FIGURE 3.

Acquisition and maintenance of iNKRs and aNKRs during primary CMV infection detected by microarray. A, Cumulative fold change was calculated by summing all of the fold changes of genes that change ≥2 at the peak and/or 1 year p.i. observed from aNKRs (n = 15) or iNKRs (n = 13); all NKRs (n = 31, including unknown NKRs). *, p < 0.001; Wilcoxon-signed rank test. Multiple observations and genes that did not change ≥ (n = 8) were ignored. B, Mean fold changes and SD were calculated. #, Mann-Whitney U test.

FIGURE 3.

Acquisition and maintenance of iNKRs and aNKRs during primary CMV infection detected by microarray. A, Cumulative fold change was calculated by summing all of the fold changes of genes that change ≥2 at the peak and/or 1 year p.i. observed from aNKRs (n = 15) or iNKRs (n = 13); all NKRs (n = 31, including unknown NKRs). *, p < 0.001; Wilcoxon-signed rank test. Multiple observations and genes that did not change ≥ (n = 8) were ignored. B, Mean fold changes and SD were calculated. #, Mann-Whitney U test.

Close modal

To obtain a more thorough insight into the detailed kinetics of NKR expression, four-color flow cytometric analyses were performed. Due to the limited number of fluorescence channels, we chose to analyze the NKR phenotype in combination with CD3, CD56, and CD27 markers. CD27 was included to monitor the maturation of virus-specific cells by the gradual loss of CD27. The combination of CD3 and CD56 proved to be a good combination to distinguish between virus-specific T cells, including CD4+ and CD8+ T cells, and NK cells.

Representative FACS plots of one patient are shown in Fig. 4 and the data of six patients analyzed are represented in Fig. 5. A clear induction of NKR expression was observed when the viral load reached its maximum. Although expression on CD3+ cells was low (0.4 ± 0.07%) and remained low after transplantation (up to 1.9 ± 0.4%), a consistent and significant increase of KIR2DS1/KIR2DL1 (detected by the EB6 mAb, further referred to as EB6+; Fig. 5,A) was observed when CMV DNA became detectable. Although with clearly different kinetics, consistent increases in cell surface expression were also found for KIR2DS2/2DL2/2DL3 (detected by the GL183 mAb, further referred to GL183+; Fig. 5,B), KIR3DL1 (NKB1; Fig. 5,C), NKG2D (Fig. 5,D), the NCRs NKp44 and NKp46 (Fig. 5, E and F), and CD94 (Fig. 6,A). In two CMV-seronegative individuals that received a CMV-negative donor kidney, no changes in NKR expression were observed (data not shown), showing that the alterations in NKR expression were indeed a consequence of the CMV infection and not related to the transplantation procedure. In the follow-up periods of ∼1 year, we did not observe a specific association of NKR with CD27 expression, indicating that induction of these molecules is independent from other differentiation steps of human CMV-specific CD8+ T cells (Fig. 4).

FIGURE 4.

Induction of NKRs on CD3+ cells during primary CMV infection detected by FACS analysis. Dot plots are gated on CD3+ T cells in which the CD27 marker is plotted against the staining for EB6 (KIR2DS1/KIR2DL1), GL183 (KIR2DL2, KIR2DL3, KIR2DS2), NKB1 (KIR3DL1), NKG2D, NKp44, NKp46, or CD94. Quadrant percentages depicted as percentages from CD3+ cells. Shown is one representative patient (patient 4). PCR viral load is given as copies per ml. Time is defined as weeks from the peak of the PCR viral load. All stainings are CD27-FITC, NKR-PE, CD3-PerCP, and CD56-allophycocyanin, except in the NKB1 staining, which is FITC labeled and CD27-PE is used.

FIGURE 4.

Induction of NKRs on CD3+ cells during primary CMV infection detected by FACS analysis. Dot plots are gated on CD3+ T cells in which the CD27 marker is plotted against the staining for EB6 (KIR2DS1/KIR2DL1), GL183 (KIR2DL2, KIR2DL3, KIR2DS2), NKB1 (KIR3DL1), NKG2D, NKp44, NKp46, or CD94. Quadrant percentages depicted as percentages from CD3+ cells. Shown is one representative patient (patient 4). PCR viral load is given as copies per ml. Time is defined as weeks from the peak of the PCR viral load. All stainings are CD27-FITC, NKR-PE, CD3-PerCP, and CD56-allophycocyanin, except in the NKB1 staining, which is FITC labeled and CD27-PE is used.

Close modal
FIGURE 5.

Induction of NKRs on CD3+ T cells during primary CMV infection detected by FACS analysis. A–F, Percentages are calculated as means and SEM from T cells in six patients with a primary CMV infection. Time is defined as weeks from the peak of the PCR viral load determined for each patient separately. G and H, Quantitative PCR for KIR2DL2/DL3/DS2, which can be detected by flow cytometric staining with GL138 mAb. G, Patient 4, T1; peak CMV, T2; week 4 mo after peak. H, Patient 8, T1; peak CMV, T2; 6 mo after peak.

FIGURE 5.

Induction of NKRs on CD3+ T cells during primary CMV infection detected by FACS analysis. A–F, Percentages are calculated as means and SEM from T cells in six patients with a primary CMV infection. Time is defined as weeks from the peak of the PCR viral load determined for each patient separately. G and H, Quantitative PCR for KIR2DL2/DL3/DS2, which can be detected by flow cytometric staining with GL138 mAb. G, Patient 4, T1; peak CMV, T2; week 4 mo after peak. H, Patient 8, T1; peak CMV, T2; 6 mo after peak.

Close modal
FIGURE 6.

CD94dim, CD94bright, NKG2A, and NKG2C on T cells during primary CMV. Total CD94 (A), CD94dim (B), and CD94bright (C) expression on CD3+ T cells. D, Linear regression analysis was performed between CD94bright and NKG2A expression on CD3+ cells (R2 = 0.98). NKG2A (E) and NKG2C (F) expression on CD8+ cells. Shown is one representative patient of three patients. NKG2A (G) and NKG2C (H) on CMV-specific CD8+ pp65-tetramer+ cells. Shown is one representative patient (patient 4). Dot plots are gated on vital cells. Numbers indicate the percentages within the corresponding quadrants from the gated population. Time is defined as weeks from the peak of the PCR viral load determined for each patient separately. PreTx, Pretransplantation; n.a., not applicable.

FIGURE 6.

CD94dim, CD94bright, NKG2A, and NKG2C on T cells during primary CMV. Total CD94 (A), CD94dim (B), and CD94bright (C) expression on CD3+ T cells. D, Linear regression analysis was performed between CD94bright and NKG2A expression on CD3+ cells (R2 = 0.98). NKG2A (E) and NKG2C (F) expression on CD8+ cells. Shown is one representative patient of three patients. NKG2A (G) and NKG2C (H) on CMV-specific CD8+ pp65-tetramer+ cells. Shown is one representative patient (patient 4). Dot plots are gated on vital cells. Numbers indicate the percentages within the corresponding quadrants from the gated population. Time is defined as weeks from the peak of the PCR viral load determined for each patient separately. PreTx, Pretransplantation; n.a., not applicable.

Close modal

We investigated whether the presence of KIR ligands corresponded to the elevation of specific KIRs during the primary CMV infection. No evidence for this assumption was found, because patients 5, 8, and 9 nor their transplanted kidneys expressed HLA-Bw4, the ligand for NKB1, but still NKB1 was strongly induced in these individuals (data not shown).

The increase in GL183+ was very prominent, as at the end of the follow-up up to 13% of the circulating CD3+ pool expressed this marker. Because the Ab GL183 recognizes both activating (KIR2DS2) and inhibitory members (KIR2DL2 and DL3) of the KIR family, we analyzed, by quantitative PCR, whether CMV induced the selective outgrowth of a monoclonal population expressing only one KIR species. CD8+GL183+ cells from two patients (patients 4 and 8) at two time points were obtained and in these cells all three transcripts could be detected with values that are in the normal range of KIR-purified cells (Fig. 5 G). This shows that the strongly expanded GL183+ T cell population does not represent the outgrowth of a monoclonal KIR+ population.

The increase in CD94 expression was quite prominent on CD3+ T cells (Figs. 4 and Fig. 6,A), and we therefore analyzed in more detail whether the molecule would operate in transmitting activating and/or inhibiting signals. CD94 can only exert its signaling function when heterodimerized with either NKG2A (inhibiting dimer) or NKG2C (activating dimer). The intensity of CD94 expression is, as reported by Arlettaz et al. (27), high in conjunction with NKG2A but low when bound to NKG2C. During the first weeks of the CMV response, predominantly CD94dim cells appeared in the circulation (Fig. 6,B) and somewhat later CD94bright cells became detectable (Fig. 6,C). As expected (27), we found a correlation between the CD94bright and NKG2A expression in our samples (Fig. 6,D). Because the majority of CD94-positive T cells are CTLs, we continued the analysis on CD8+ T cells. Already before CMV infection, NKG2C is present on a small subset of CD8+ cells, whereas NKG2A is almost absent on CD8+ cells. Confirming the differential rise in the dimerization pattern of CD94 with NKG2C and NKG2A, we found that a NKG2C increase on CD8+ cells could be detected shortly after the start of the infection (2 wk before CMV peak; Fig. 6,F), whereas the increase of NKG2A started later in the response (Fig. 6,E). To analyze the expression of these dimers on established virus-specific cells, pp65 tetramers were used to analyze cells of a HLA-A2+ individual. The inhibitory CD94:NKG2A dimer was found on the majority of the CMV-specific tetramer+ cells irrespective of the stage of infection (Fig. 6,G). In marked contrast, the activating NKG2C dimer was induced on the CD3+ cells but conspicuously absent from the tetramer-binding cells (Fig. 6 H). The absence of CD94/NKG2C on cells with proven virus-specificity is reminiscent of earlier findings showing that tetramer-binding cells also lack EB6 and GL183 (CD158a,h and CD158b, j expression) (16).

In this study, we show the impact of a primary CMV response on NKR virus-induced CD8+ T lymphocytes. Using a microarray approach, we found that mRNA expression levels of different members of all NKR families, including KIRs, leukocyte Ig-like receptors, NCRs, and C-type lectin receptors in virus-induced CD8+ T cells, increased during the infection. Many of these genes remained increased up to 50 wk after the peak of the initial viral burst when the virus load had long dropped below detection by sensitive PCR.

Changes in KIR2DL1/S1 (EB6), KIR2DL2/S2 (GL183), and KIR3DL1 (NKB1) mRNA were observed in the acutely activated CD8+HLA-DR+CD38+ cells, but expression levels in the pp65 tetramer CD8+ cells obtained during latency were comparable to those of naive CD8+ T cells. This observation is in agreement with previous observations (16, 28) where expression of these KIRs was found on the CD8+CD45RA+CD27 effector-type cells typical for CMV- infected individuals present during CMV infection but absent on the pp65 tetramer-binding cells. The reason for this difference is unclear. CD8+CD45RA+CD27 only expand to high numbers after CMV infection (6) and it therefore seems likely that many CMV-reactive cells are contained within this subset. Elegant studies by Picker et al. (29) have shown that the T cell response to CMV is very robust and is directed against many peptides of the virus. Studies until now have mainly focused on pp65-specific T cells and therefore it is possible that CMV-specific CD8+ T cells, reactive toward epitopes generated from other viral proteins, will have different phenotypes with regard to, for example, KIR.

NKR expression on CD8+ T cells is not associated with other latent viruses such as EBV and HIV (28), suggesting that expression of these molecules has a particular function in the anti-CMV response. Since CMV-specific T cells generally reach later differentiation stages than T cells specific for other persisting viruses (3), one might assume that the expression of NKRs is an integral part of the late differentiation phenotype. However, our array data appear to argue against this notion. Up-regulation of a vast number of NKRs already takes place during the early phase of the response when cells have an activated early differentiation phenotype (16). Rather, our data suggest that the expression of NKRs as a consequence of CMV infection is an early and adaptive response of the CD8+ effector cell pool to the virus. Whether specific viral products and/or the virus-induced modulation of HLA expression have created an evolutionary necessity for this broad expression of the NKR repertoire will have to be resolved.

The early up-regulation of the NCR NKp44 was found by flow cytometry but was not revealed by the microarrays. This is perhaps not surprising since the up-regulation of NKp44 in CD3+ cells is transient and low, reaching a maximum at the peak of the viral load of only mean 1.7%. Two weeks after the peak of the viral load, the frequency of NKp44-expressing cells is already decreasing in most patients. It is possible that the patients selected for the RNA isolation had too low levels to be detectable by microarray analysis. In any case, it suggests that NKp44, as in NK cells (30, 31), is preferentially expressed on the mitogenically activated cells that can only be found early in infection (8).

CD94 was found to be expressed on both CD3+ cells (protein) and CD8+ CMV-specific cells (RNA). CD94 forms heterodimers with NKG2x proteins (where x can be A, B, C, E, or H). The NKG2A/B/CD94 is an inhibitory receptor, whereas the NKG2C/CD94 and NKG2E/H/CD94 are activating receptors (32). Arlettaz et al. (27) reported that CD94bright expression is associated with NKG2A and CD94dim is associated with NKG2C, although also other NKG2 proteins may dimerize with CD94. Our observation that CD94dim was primarily enhanced during the acute phase of the infection, whereas CD94bright accounted for the latent phase was confirmed by the quick rise in NKG2C followed by robust expansion of NKG2A which continued to expand for at least 40 wk after the peak of the viral load. A strict correlation between microarray data and surface expression of NKG2C was not apparent in our analyses.

Indeed, it has been reported (27, 33) that if NKG2A is expressed, no surface NKG2C expression could be observed even with abundant transcription of NKG2C. This discrepancy might be due to different affinities of CD94 for binding partners and a preference to dimerize with NKG2A. Also in our analyses, whereas NKG2A was expressed at the cell surface of tetramer+ cells, no NKG2C expression could be detected in apparent contrast with the high NKG2C mRNA content detected in the microarray analysis.

The coordinated changes of aNKRs and iNKRs on recently primed and late differentiated CD8+ T cells at different time points during the viral infection suggest that NKRs contribute to the fine tuning of antiviral effector functions, keeping the delicate balance between efficient destruction of virus-infected cells and loss of organ function due to immunopathology. This idea is in line with several observations on the function of KIRs and leukocyte Ig-like receptors showing that they can, for example, down-regulate killing efficiency (34), inhibit cytokine production (35), and weaken proliferative potential (28) of CD8+ T cell clones. Henel et al. (36) recently demonstrated that with low Ag concentrations, KIR expression on memory CD4+ T cells uncoupled granule release from gene activation, reasoning that a general inflammation is inhibited to circumvent pathological events. In view of this, it can be reasoned that in immune reactions against persistent viruses, like CMV, a negative regulation of general inflammation is essential to prevent damage by continued activation of T lymphocytes.

Cohort studies have indicated that the NKR profile on NK and T cells is imprinted by CMV (17). We here have demonstrated a strong induction of NKRs on T cells as a direct result of CMV infection. These NKRs might provide essential control mechanisms for the activation of T cells by regulating costimulatory signals, dampening T cell activation when viral loads diminishes, and controlling harmful activation to the host during latent viral infections.

We thank Dr. Perry Moerland for assistance with the bioinformatics and statistics of the microarray analysis.

The authors have no financial conflict of interest.

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 by Grant C03.2034 from the Dutch Kidney Foundation (to A.v.S.). R.A.W.v.L. was supported by VICI Grant 918.46.606 from The Netherlands Organization of Scientific Research.

4

Abbreviations used in this paper: NKR, NK cell receptor; NCR, natural cytotoxic receptor; p.i., postinfection; KIR, killer Ig-like receptor; aNKR, activating NKR; iNKR, inhibitory/inhibiting NKR.

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