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Keywords:

  • Cytomegalovirus;
  • KIR;
  • CD85j;
  • NKG2D;
  • T cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements

The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3 K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCRγδ+ and TCRαβ+CD8+ T lymphocytes, but it has been also detected in CD4+ T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4+ T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4+NKG2D+ T lymphocytes that coexpressed perforin. NKG2D was detected in CD28 and CD28dull subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4+ T cells, triggering proliferation and cytokine production (i.e. IFN-γ and TNF-α). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4+ T lymphocytes and thus may have a role in the response against infected HLA class II+ cells displaying NKG2D ligands.

Abbreviations:
aNKR:

activating NKR

HCMV:

human cytomegalovirus

KIR:

killer immunoglobulin-like receptor

KLR:

killer lectin-like receptor

NKR:

natural killer receptor

RA:

rheumatoid arthritis

ULBP:

UL16-binding proteins

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements

Some T lymphocyte subsets share with NK cells the expression of inhibitory and activating receptors specific for HLA class I molecules, such as killer immunoglobulin-like receptors (KIR), CD94/NKG2A, CD94/NKG2C and ILT2 (LIR1, CD85j) 14. NK cell receptors (NKR) have been detected in TCRγδ+ cells as well as TCRαβ+ CD8+ and CD4+ lymphocytes with an effector-memory phenotype 58, including virus-specific cytotoxic T lymphocytes (CTL) 9, 10. The molecular basis regulating NKR expression along T cell differentiation is not completely understood, but there is evidence supporting the involvement of cytokines and TCR-dependent signals, and it has been hypothesized that NKR expression results from chronic antigenic stimulation 6, 11.

Inhibitory NKR (iNKR) are believed to prevent T cell-mediated autoreactivity and counterbalance the action of activating NKR (aNKR) 6, 12. Most aNKR (i.e KIR and CD94/NKG2C) are coupled by DAP12 to protein tyrosine kinase (PTK) activation pathways 4 and may trigger or costimulate T cell proliferation and effector functions 1315. By contrast, the human NKG2D killer lectin-like receptor (KLR) is linked by DAP10 to a phosphoinositide 3-kinase (PI3 K) activation pathway 16, 17; NKG2D functions as a costimulatory molecule in T cells 18, 19 but may activate NK cells and IL-15-stimulated intraepithelial T lymphocytes in a TCR-independent manner 20, 21.

Human NKG2D interacts with several stress-inducible ligands including MICA, MICB and a family of proteins termed “UL16-binding proteins” (ULBP) that are expressed by some normal tissues, tumour cells and virus-infected cells 16, 19, 2225. The KLR activates NK cells and costimulates the response of CD8+ CTL against human cytomegalovirus (HCMV)-infected targets 18, 26; the identification of immune evasion mechanisms that interfere with surface expression of NKG2D ligands underline its importance in the antiviral response. The UL16 glycoprotein inhibits surface expression of MICB, ULBP1, ULBP2 22, 2729 and also interacts with RAET1G 24. The gpUL142 HCMV molecule has been recently reported to down-regulate MICA 30.

Human NKG2D was originally identified on NK cells, TCRγδ+ cells and TCRαβ+CD8+ T lymphocytes, but CD4+NKG2D+ T cells have been described in rheumatoid arthritis (RA) and some cancer patients 31, 32. Goronzy and Weyand 33 hypothesized that CD4+ T lymphocytes expressing NKG2D and aNKR represent senescent effector-memory T cells that may contribute to the pathogenesis of RA and other chronic inflammatory disorders 15. NKG2D might exacerbate RA progression by reacting with its ligands abnormally expressed by the inflamed synovium 31, 33. On the other hand, stimulation by soluble MIC molecules (sMIC) has been proposed to account for the increased frequencies of CD4+NKG2D+ T cells producing Fas ligand in patients bearing MIC+ tumours 32. Recently, CD4+NKG2D+ T cells have been identified in patients with human T cell lymphotropic virus type I (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) 34.

In the present study, we provide the first evidence supporting the notion that a subset of HCMV-specific CD4+ T cells displays NKG2D. Remarkably, expression of the KLR was not systematically associated with expression of other NKR (i.e. ILT2, KIR and CD94/NKG2). Engagement of NKG2D costimulated TCR-dependent proliferation and cytokine production, indicating that the KLR may contribute to the response of a subset of HCMV-specific CD4+ T lymphocytes against infected HLA class II+ cells bearing NKG2D ligands.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements

Expression of NKG2D on HCMV-stimulated CD4+ T cells

Human NKG2D is commonly displayed by NK cells as well as TCRγδ+ and TCRαβ+CD8+ T lymphocytes, but it has also been detected in TCRαβ+CD4+ cells from RA and cancer patients 31, 32. In the present study, we analysed the expression of NKG2D in HCMV-specific CD4+ cells. In agreement with previous reports 35, T cell proliferation was detected when peripheral blood mononuclear cells (PBMC) from healthy HCMV-seropositive (HCMV+) blood donors (n=9) were cultured in the presence of the virus. Cells recovered from HCMV-stimulated samples were predominantly CD4+ T lymphocytes (84±7%), which displayed an oligoclonal pattern of TCR rearrangement (data not shown), consistent with the expansion of T cells specific for viral antigens 35; no response was observed in HCMV-seronegative (HCMV) individuals (n=3) (data not shown).

As compared to fresh PBMC and to control samples cultured in the absence of the virus, substantial numbers of HCMV-stimulated CD4+ lymphocytes displayed NKG2D (Fig. 1); the proportion of CD4+ T lymphocytes expressing NKG2D on day 10 of stimulation varied widely in different HCMV+ donors (n=9; mean ± SD, 21±15%; range, 1–75%) and were rare (⩽4%) in the absence of HCMV stimulation. The expansion of NKG2D+ cells was comparable upon incubation with different HCMV strains (i.e. Towne and AD169) and was undetectable in samples from HCMV-seronegative donors (not shown). Similar results were obtained using purified or UV-inactivated virus preparations (not shown).

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Figure 1. Expansion of CD4+NKG2D+ T cells in HCMV-stimulated PBMC. PBMC from a healthy HCMV+ donor (#1) were cultured for 10 days either alone (untreated) or in the presence of the virus (AD169). Two-colour flow cytometry analysis was carried out staining fresh (day 0) and cultured (day 10) samples with anti-CD4 mAb in combination with BAT221 anti-NKG2D, HP-F1 anti-ILT2, HP-3B1 anti-CD94 mAb or with a mixture of anti-KIR mAb (HP-3E4, CH-L, DX9, 5133).

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Distribution of ILT2, KIR and CD94/NKG2 NKR on HCMV-stimulated CD4+ T cells

Flow cytometry analysis carried out in parallel with a panel of mAb specific for different NKR indicated that ILT2+ and KIR+ cells were also increased among HCMV-stimulated CD4+ T cells (Fig. 1); by contrast, few CD4+CD94/NKG2+ cells were detectable (Fig. 1 and data not shown). Remarkably, the expression of NKG2D, ILT2 and KIR did not systematically coincide. In fact, when expression of NKG2D and the ILT2 inhibitory receptor were compared on HCMV-stimulated CD4+ cells, distinct distribution patterns were observed. In most cases (n=6), CD4+ILT2+ and CD4+NKG2D+ cells were detected (Fig. 2A, donor #2), and three-colour flow cytometry analysis revealed the existence of a subset coexpressing both molecules (Fig. 3A). However, in some individuals only CD4+ cells selectively bearing either NKG2D (n=1) or ILT2 (n=2) were expanded (Fig. 2A, donors #3 and #4). When samples from several representative donors (n=5) were reanalysed 4–10 months later, the distribution patterns observed in the first study were reproduced in every case, regardless of the variability in the proportions of recovered NKG2D+ or ILT2+ cells; the phenotypes of HCMV-stimulated CD4+ cells from two individuals (#2 and #3) studied at different time points are shown for comparison (Fig. 2A, B).

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Figure 2. Dissociated expression of NKG2D, ILT2 and KIR in HCMV-stimulated CD4+ T cells. Experiments were carried out as described in Fig. 1, comparing the expression of NKG2D, ILT2 and KIR in HCMV-stimulated PBMC from HCMV+ donors (n=9). (A) Data correspond to samples from three individuals (#2, #3 and #4) representative of the different distribution patterns of NKG2D and ILT2 observed. (B) HCMV-stimulated PBMC samples from donors #2 and #3, analysed in assays carried out 6 months later, illustrate the dissociated expression of NKG2D and KIR.

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Figure 3. Expression of ILT2, perforin and CD28 in HCMV-stimulated CD4+NKG2D+ T cells. Three-colour immunofluorescence and flow cytometry analysis of HCMV-stimulated cells was carried out as described in the Materials and methods, gating on CD4+ cells. (A) Cells were sequentially stained with anti-NKG2D (ON72) and allophycocyanin (APC)-conjugated goat anti-mouse Ig, followed by biotin-labelled anti-ILT2 (HP-F1), streptavidin-FITC and CD4-PE. (B) Cells stained with anti-NKG2D and CD4-PE were sequentially fixed, permeabilised and incubated with anti-perforin-FITC. Data correspond to an experiment representative of three performed with different donors. (C) Cells stained by indirect immunofluorescence with anti-NKG2D were subsequently labelled with anti-CD4-FITC and anti-CD28-PE. Data correspond to samples from two donors illustrating the different distribution patterns observed.

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The complex diversity of KIR haplotypes and, particularly, the inability of the available mAb to discriminate homologous activating and inhibitory KIR 1 did not allow precise analysis of the relationship between individual members of this receptor family and NKG2D. Yet, two-colour analysis of CD4+ cells using a mixture of different anti-KIR mAb as described 36 also ruled out coordinated expression of NKG2D with these receptors (Fig. 2B).

The dissociated distribution of NKG2D, KIR and ILT2 in HCMV-specific CD4+ cells indicates that their expression is differentially regulated. NKG2D was reported to be induced under the influence of cytokines (i.e. IL-15) and TCR-dependent stimulation 31, 37; furthermore, it has been proposed that NKG2D engagement by soluble MIC molecules promotes the expansion of CD4+NKG2D+ cells in cancer patients bearing MIC+ tumours 32. KIR and ILT2 were shown to be displayed by effector-memory T cells 6, 8, yet the mechanisms regulating their expression remain poorly defined. CD8+ T cells bearing inhibitory NKR have been reported to be increased in HIV-infected patients 38. ILT2+ T cells have also been observed to be augmented during HCMV infection in transplant patients 39 as well as in HCMV+ blood donors 36. ILT2 was detected in HCMV-specific CD8+ T cells 9, 10 and in CD4+ T lymphocytes responding to Mycobacterium tuberculosis antigens 40. Our results provide a first indication that ILT2 may also be expressed by HCMV-specific CD4+ T cells. ILT2 interacts with different HLA class I molecules and with the UL18 HCMV glycoprotein 41, 42. The regulatory function of ILT2 in the response of CD4+ T cells against HCMV-infected cells deserves further attention.

CD4+NKG2D+ T cells are CD28 or CD28dim cytotoxic T lymphocytes

Additional phenotypic studies using three-colour flow cytometry analysis revealed that CD4+NKG2D+ lymphocytes express perforin (Fig. 3B) and granzyme B (data not shown), thus corresponding to a described subset of HCMV-specific CD4+ CTL 43; it is noteworthy that both perforin and granzyme B were also detected in CD4+NKG2D cells (Fig. 3B; data not shown). Considering the costimulatory function of NKG2D in CD8+ cells, we compared its distribution to that of CD28. Both molecules appeared dissociated in HCMV-stimulated CD4+ lymphocytes (Fig. 3C), but a NKG2D+CD28dull subset was detectable in some samples (Fig. 3C). These observations suggest that HCMV-specific CD4+ T cells, previously shown to display an effector-memory phenotype 44, 45, may switch the use of costimulatory receptors, becoming CD28 NKG2D+.

NKG2D functions as a costimulatory receptor in a subset of HCMV-specific CD4+ T cells

To verify whether NKG2D+ cells were specific for HCMV antigens, CD4+NKG2D+ and CD4+NKG2D subsets were sorted and restimulated with the virus in the presence of irradiated (40 Gy) autologous PBMC. A specific proliferative response and IFN-γ production were detected in both populations (Fig. 4), thus indicating that CD4+NKG2D+ cells represent only a fraction of HCMV-specific T lymphocytes, as predicted by the phenotypic analyses described above. Attempts to grow CD4+NKG2D+ T cell clones were unsuccessful, suggesting that their proliferative capacity is limited, in line with previous studies showing that HCMV-specific T cells undergo replicative exhaustion 46.

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Figure 4. Proliferation and IFN-γ production by CD4+NKG2D+ and CD4+NKG2D T cell subsets in response to HCMV stimulation. HCMV-stimulated cells were sequentially stained by indirect immunofluorescence with anti-NKG2D and FITC-tagged rabbit anti-mouse Ig, followed by CD4-PE. CD4+NKG2D+ and CD4+NKG2D cell subsets were sorted and incubated in 96-well plates (105 cells/well) together with autologous irradiated PBL (2 × 105/well) in the presence or absence of HCMV. (A) Cultures were labelled with 3HTdR at 48 h and harvested 18 h later. (B) IFN-γ was measured by ELISA in supernatants harvested after 48 h. Similar results were obtained in two different experiments.

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To assess the function of NKG2D, HCMV-stimulated CD4+ T cells were incubated with suboptimal concentrations of anti-CD3 mAb in the presence or absence of anti-NKG2D mAb. Under these conditions, engagement of the KLR synergized with TCR-dependent signals, efficiently triggering proliferation and cytokine production (Fig. 5). These results support the notion that NKG2D functions as a prototypic costimulatory receptor in HCMV-specific CD4+ cells. In line with this, the KLR has recently been shown to be coupled to the DAP10 adapter in CD4+NKG2D+ cells derived from cancer patients 32.

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Figure 5. NKG2D costimulates TCR-dependent proliferation and cytokine production in CD4+ T cells. HCMV-stimulated T cells (day 10) stained with CD4-PE were sorted. CD4+ T cells were incubated in 96-well plates (105 cells/well) in the presence of the indicated plate-bound antibodies (x-axis), as described in the Materials and methods. (A) Cultures were labelled with 3HTdR at 48 h and harvested 18 h later. (B–C) IFN-γ and TNF-α were analysed by ELISA in supernatants harvested at 48 h. Data correspond to samples from two donors containing 25% (donor #1) and 45% (donor #5) CD4+NKG2D+ cells that were independently analysed in different experiments; cells from a third individual that did not display NKG2D were tested as a control (NKG2D).

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The design of an in vitro experimental system suitable to directly study the costimulatory role of NKG2D and other NKR (i.e. ILT2) in the response of CD4+ T cells to HCMV-infected cells is warranted, though some technical limitations must be overcome. It is noteworthy that fibroblasts, which are commonly used to analyse the response to HCMV, do not express HLA class II molecules, and other susceptible cell types (i.e. endothelial and hemopoietic cells) are far less permissive to in vitro infection 47.

Collectively, our data suggest that CD4+NKG2D+ cells expanding in HCMV-stimulated cultures correspond to virus-specific memory T cells that have acquired NKG2D while losing CD28. By switching the use of costimulatory molecules, CD4+ cells primed by professional antigen-presenting cells (APC) might efficiently respond to other HLA class II+ virus-infected cell types. Though most studies have focused on the response of CD8+ CTL to HCMV antigens 48, CD4+ T cells specific for epitopes of viral proteins such as pp65 and IE1 have been identified 4951. Recently, the gB HCMV glycoprotein was shown to be processed via the endosomal pathway by non-professional APC, being efficiently presented by HLA class II molecules to CD4+ T cells 52. HLA class II expression in different HCMV-infected cell types may be either constitutive (i.e. macrophages) or inducible by proinflammatory cytokines, and it is impaired by some virus molecules 53. On the other hand, NKG2D ligands are displayed in HCMV-infected cells, and immune evasion mechanisms that interfere with their expression indirectly reflect the importance of the KLR in the anti-viral response 18, 22, 2729.

CD4+NKG2D+ T cells were originally described in RA patients 31. Goronzy and Weyand hypothesized that immunosenescence contributes to the development of RA and proposed that expression of NKG2D and aNKR (i.e. KIR2DS2) may lower the activation threshold of senescent CD4+CD28 T lymphocytes, favouring their participation in the pathogenesis of the disease. NKG2D might exacerbate RA upon interaction with its ligands expressed by the inflamed synovium 31, 33. A role for NKR+ T cells in the development of other chronic inflammatory disorders such as atherosclerosis has also been suggested 15. It is noteworthy that HCMV infection is considered to be a major contributor to the immunosenescence process 54. According to our observations, the possibility that the increased numbers of CD4+NKG2D+ T cells found in RA and cancer patients 32 may represent HCMV-specific T cells should be envisaged. Frequent subclinical reactivation of the virus, favoured by the immune dysfunction and/or immunosuppressive therapy in these patients, might account for the increased proportions of CD4+NKG2D+ T cells. On the other hand, the possibility that this T lymphocyte subset may also expand in response to different antigens, including other microbial pathogens, is not ruled out. Regardless of their primary antigenic specificity, NKG2D would enhance the response of potentially autoreactive CD4+ T cells against non-infected tissues, where expression of its ligands may be either constitutive or inducible by a variety of stimuli 26, 55, 56.

NKG2D has been detected in murine NK cells, CD8+ T lymphocytes and macrophages, and it has been shown to participate in the immune response against murine cytomegalovirus (MCMV) 19, 57 as well as in the pathogenesis of experimental autoimmune type I diabetes 58. The expression of NKG2D in murine effector-memory CD4+ CTL should be carefully reassessed.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements

Our results provide first evidence supporting the notion that human NKG2D functions as a costimulatory molecule in a subset of HCMV-specific CD4+ T lymphocytes and thus may contribute to their response against HLA class II+ virus-infected cell types displaying NKG2D ligands. The possibility that the increased numbers of CD4+NKG2D+ T cells found under some pathological conditions may be primarily specific for HCMV or other microbial pathogens should be envisaged. Further studies are required to explore the putative role of CD4+NKG2D+ cells in the pathogenesis of chronic inflammatory disorders. In this regard, the dissociated expression of NKG2D and inhibitory NKR (i.e. ILT2, KIR) in CD4+ T lymphocytes deserves special attention, as it might increase the risk of autoimmune reactions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements

Subjects

Heparinized blood samples were obtained from healthy adult individuals. Written informed consent was obtained from every donor, and the study protocol was approved by the Ethics Committee (CEIC-Institut Municipal d'Assistencia Sanitaria). As described 36, standard clinical diagnostic tests were used to analyse serum samples from blood donors for circulating IgG antibodies against CMV (Abbot Laboratories, Abbot Park, IL); nine HCMV-seropositive (HCMV+) and three seronegative (HCMV) donors were studied.

HCMV preparations

As described 59, AD169 and Towne strains of HCMV were propagated in HFF or MRC-5 fibroblast cell lines following standard procedures. Viral titres were determined by standard plaque assays on MRC-5 cells. The AD169 strain of virus was inactivated under UV light as described 59. The purified AD169 strain of HCMV was obtained from ABI Advanced Biotechnologies Inc. (Columbia, MD).

Lymphocyte cultures

Culture medium was RPMI 1640 with Glutamax-I and 25 mM Hepes (Gibco, UK), supplemented with 10% v/v heat-inactivated fetal calf serum (FCS), penicillin (100 U/mL) and streptomycin (10 μg/mL), referred to as complete medium. PBMC obtained from heparinized blood by Ficoll-Hypaque gradient centrifugation (Lymphoprep, Axis-Shield PoC AS, Oslo, Norway) were incubated in 24-well plates (2 × 106 cells/well) in complete medium supplemented with 10 U/mL human recombinant interleukin-2 (hrIL-2; Proleukin, Chiron, Emeryville, CA) either alone or in the presence of cell-free HCMV (2 × 105 PFU/well). Cell cultures were maintained at 37°C in a 5% CO2 humid atmosphere for 10–12 days; every 3 days 50% of the supernatant was replaced with fresh medium supplemented with IL-2; when high cell density was attained, cell cultures were split.

Antibodies

HP-3E4 anti-KIR2DL1/S1/S3, HP-3B1 anti-CD94, HP-F1 anti-CD85j mAb and BAT221 anti-NKG2D were generated in our laboratories 23, 36. 5.133 anti-KIR3DL1/L2 and KIR2DS4 was provided by Dr. Marco Colonna. Z199 anti-NKG2A and ON72 anti-NKG2D mAb were provided by Dr. Alessandro Moretta (University of Genova, Italy). Dx9 anti-KIR3DL1 mAb was provided by Dr. Lewis Lanier (UCSF, San Francisco, CA). CH-L anti-KIR2DL2/S2/L3 was provided by Dr. Silvano Ferrini (University of Genova, Italy). 3A1 anti-CD7 has been described 60, and anti-NKG2C (MAB1381) was from R&D Systems (Minneapolis, MN). Indirect immunofluorescence analysis was carried out with PE- or FITC-tagged F(ab’)2 rabbit anti-mouse Ig antibodies (Dakopatts, Glostrup, Denmark) or allophycocyanin (APC)-labelled goat anti-mouse Ig (BD Biosciences Pharmingen). The following fluorochrome-tagged mAb were used for multicolour staining: CD4-PE, CD4-FITC, CD28-PE, anti-human Perforin-FITC and FITC-conjugated mouse anti-human Granzyme B (BD Biosciences Pharmingen). HP-F1 was labelled with biotin using EZ-Link Sulfo-NHS-Biotin (Pierce) according to the manufacturer's instructions and was used in combination with streptavidin-FITC (BD Biosciences Pharmingen).

Immunofluorescence and flow cytometry analysis

Multicolour immunofluorescence and flow cytometry analysis was performed as described 36. Briefly, cells were pretreated with saturating concentrations of human aggregated Ig to block FcR and were then incubated with the individual unlabeled mAb. After washing, samples were labelled either with FITC-tagged F(ab’)2 rabbit anti-mouse Ig antibody (Dako) or allophycocyanin (APC)-labelled goat anti-mouse (BD Biosciences Pharmingen). In some experiments, cells were incubated with biotin-labelled HP-F1 followed by streptavidin-FITC (BD Biosciences Pharmingen). Subsequently, samples were washed and incubated with PE- or FITC-conjugated antibodies (BD Biosciences Pharmingen). Flow cytometry analysis was carried out as described (FACScan, Becton Dickinson, Mountain View, CA).

For multicolour intracellular staining, the BD Cytofix/Cytoperm Kit was used (BD Biosciences Pharmingen). Briefly, cells were stained with ON72 anti-NKG2D and allophycocyanin (APC)-labelled goat anti-mouse Ig, followed by CD4-PE. Samples were fixed, permeabilised following the manufacturer's instructions and stained with anti-human Perforin-FITC or FITC-conjugated anti-human Granzyme B mAb (BD Biosciences Pharmingen). Cells stained with CD4-PE alone or in combination with BAT221 anti-NKG2D and FITC-tagged F(ab’)2 rabbit anti-mouse Ig, as described above, were sorted under sterile conditions (FACSVantage, Becton Dickinson, Mountain View, CA) and used in functional assays.

Cytokine production and cell proliferation assays

NKG2D+CD4+ and NKG2DCD4+ HCMV-stimulated cells were sorted (FACSVantage, BD), resuspended in complete medium and cultured in 96-well plates (105 cells/well) with irradiated (40 Gy) autologous PBMC (2 × 105 cells/well), either alone or in the presence of HCMV (104 PFU/well). In other experiments, CD4+ cells were sorted and stimulated with mAb preadsorbed to culture plates as described 13. Briefly, 96-well plates were coated with sheep anti-mouse Ig (10 μg/mL) overnight at 4°C, washed with PBS and incubated with specific antibodies for 3 h at room temperature. The following mAb were used: 3A1 anti-CD7 (10 μg/mL), SpvT3b anti-CD3 (2 ng/mL) and ON72 anti-NKG2D (50 μL hybridoma supernatant). After washing plates, CD4+ sorted cells (105/well) were added in complete medium; each experimental condition was set up in triplicate. In every case, supernatants were harvested after 48 h, and production of IFN-γ and TNF-α was analysed by ELISA (Human IFNγ Module Set and Human TNFα Module Set; Bender MedSystems, San Bruno CA). As described 13, 3[H]-Thymidine (3HTdR) was added (1 μCi/well) at 48 h, and cultures were further incubated for 18 h at 37°C; cells were harvested, and 3HTdR incorporation was measured in a β-counter (LKB Wallac, Turku, Finland).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements

This work was supported by grants from the Ministerio de Educación y Ciencia (SAF2004–07632 and SAF2005–05633). A. S-B- is supported by a fellowship from DURSI (Generalitat de Catalunya). We are grateful to Dr. Oscar Fornas for advice in flow cytometry analysis as well as to Gemma Heredia and Carmen Vela for technical support.

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