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Keywords:

  • Effector-memory T cells;
  • HLA-E;
  • Human CMV;
  • NK-CTL 

Abstract

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

HLA-E-restricted T cells represent a minor cytolytic T lymphocyte (CTL) population characterized by the surface expression of HLA class I-specific inhibitory receptors and by the capability of killing a large panel of allogeneic target cells (therefore named NK-CTL). Here we show that this subset of T cells is present in a sizeable fraction in the peripheral blood of human cytomegalovirus (HCMV)-seropositive healthy individuals. We provide evidence that NK-CTL recognize in an HLA-E-restricted fashion a naturally processed CMV-derived peptide in the transporter associated with antigen processing (TAP)-2–/– UL40+ RMA-S cell transfectants. Moreover, we show that they recognize and kill HCMV-infected target cells. NK-CTL are characterized by the CD8βdull CD45RA+CD28CD27CCR7CD56+ surface phenotype, thus suggesting that they belong to the effector-memory cell compartment. Consistent with the effector-memory phenotype, they promptly produce IFN-γ, but not IL-2, upon interaction with the specific HCMV UL40-derived peptide. Our data suggest that HLA-E-restricted CTL may represent an additional effector cell type involved in defenses against HCMV, a virus which escapes the control exerted by conventional CTL or NK cells.

Abbreviations:
HCMV:

human CMV 

MICA:

MHC class I-related chain A 

TAP:

transporter associated with antigen processing 

ULBP:

UL16-binding protein 

US:

unique short

Introduction

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

Human cytomegalovirus (HCMV) has evolved several strategies to evade the immune system of the infected host. In particular, HCMV codes for several proteins that down-modulate cell surface expression of classical HLA class I molecules (i.e. HLA-A, -B, and -C) on infected cells 1, thus preventing, at least in part, the presentation of antigenic peptides to conventional TCRαβ+ CTL. Although down-regulation of MHC class I expression is associated with protection against antigen-specific CTL, it also may enhance NK cell-mediated killing of HCMV-infected cells. Indeed, both in mice and in humans, NK cells, which sense the lack of HLA class I surface expression on target cells, play a relevant role against CMV infections. Along this line, it has been demonstrated in humans that the absence of NK cells increases the susceptibility to several herpes virus infections, including CMV 2. However, HCMV also evolved strategies to counteract the susceptibility to NK cells. Thus, HCMV appears particularly efficient in blocking the effector function of NK cells since it can inhibit the activating NK receptor function 3, 4 or enhance that of inhibitory NK cell receptors 5, 6.

In humans, defense against CMV could be mediated by other effector cell types. In this context, we have recently shown that a previously identified CD3+CD8+TCRαβ+ T cell population, characterized by the surface expression of HLA class I-specific inhibitory receptors 7 and by the capability of killing a large panel of allogeneic tumor cell lines (therefore named NK-CTL), recognizes peptides in an HLA-E-restricted fashion 810. HLA-E is an oligomorphic MHC class Ib molecule capable of binding a limited number of peptides 11. The first identified were conserved peptides derived from the leader sequence of various HLA class I alleles 12; binding of these peptides enables surface expression of HLA-E molecules in most normal cells 13. Subsequently, several HLA-E-binding peptides have been identified that were derived from viruses, including CMV 5, 1416.

Remarkably, one of such CMV-derived peptides (i.e. the AD-169 UL4015–23 VMAPRTLIL peptide) is identical to a peptide common to the leader sequence of various HLA-Cw alleles 5. Available in high amounts during CMV infection, it binds to HLA-E molecules and enables their expression at the cell surface. It is of note that the VMAPRTLIL peptide does not require transporter associated with antigen processing (TAP)-mediated transport from cytosol to rough endoplasmic reticulum. Accordingly, its presentation at the cell surface in association with HLA-E is not affected by the CMV unique short 6 (US6) gene product which blocks TAP function 5, 15. Thus, in CMV-infected cells while the classical HLA class I molecules are sharply down-regulated as a consequence of different molecular mechanisms, HLA-E is expressed in normal amounts or even overexpressed at the cell surface 5, 6, 15.

Under these conditions, HLA-E-restricted NK-CTL displaying specificity for the UL40-derived VMAPRTLIL peptide could play an additional role in defenses against CMV, at least in certain individuals 17. Actually, among CMV-seropositive individuals, NK-CTL were only detectable in those who did not contain in their HLA class I haplotype any HLA-Cw allele carrying the VMAPRTLIL peptide 17. We have recently shown that NK-CTL recognize the UL4015–23 epitope both on peptide-pulsed murine TAP-2–/–- cell transfectants (RMA-S_EM) and on a UL40+ cell line (293T/HLA-E+/UL40+) 17. However, no evidence exists so far that they are able to recognize cells infected with CMV. Our present data provide the first direct demonstration that NK-CTL lines can efficiently kill CMV-infected cells. By the use of appropriate HLA-E tetramers, we further demonstrate that NK-CTL are present in sizeable fractions in freshly isolated PBL of selected CMV-seropositive healthy individuals, display an effector-memory phenotype, and produce IFN-γ, but not IL-2, upon VMAPRTLIL peptide recognition.

Results

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

NK-CTL recognize in an HLA-E-restricted fashion a naturally processed CMV-derived peptide in TAP-2–/– cell transfectants

It is well known that several early genes of HCMV encode proteins that interfere at various stages and by different mechanisms with the MHC class I expression, thus preventing the peptide presentation to CTL. For example, HCMV, both in early and late phases of infection, encodes for US6 glycoprotein that inhibits TAP function 1. However, it has been shown that the UL40-derived VMAPRTLIL peptide can be processed and associated with HLA-E molecules in a TAP-independent manner 5, 15. Therefore, we investigated whether NK-CTL were capable of recognizing the VMAPRTLIL peptide in the context of HLA-E molecules on murine TAP-2–/– RMA-S cell transfectants that expressed HLA-E, human β2-microglobulin and UL40 (RMA-S_EM_UL40). NK-CTL derived from four different donors were tested for their ability to lyse RMA-S cell transfectants. As shown in Fig. 1, RMA-S_EM_UL40 cells were lysed efficiently. Pulsing of target cells with the exogenous synthetic VMAPRTLIL peptide did not result in further increase of lysis. Importantly, lysis was inhibited by the W6/32 mAb (recognizing different HLA class I molecules including HLA-E) added to the cytolytic assay. Fig. 1 also shows that RMA-S_EM cells (i.e. not transfected with UL40) were not lysed unless they had been pre-incubated with saturating concentrations (105 nM) of the AD169-UL40-derived peptide (VMAPRTLIL).

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Figure 1. UL40-expressing cells are lysed by HLA-E-specific NK-CTL in the absence of functional TAP molecules. The TAP-2–/– RMA-S cells expressing HLA-E plus human β2-microglubulin (RMA-S_EM) or HLA-E plus human β2-microglobulin and UL40 (RMA-S_EM_UL40), which had been pre-incubated or not with the VMAPRTLIL peptide, were labeled with 51Cr and analyzed for susceptibility to lysis by NK-CTL derived from donors KK, GG, GF and DP at an E/T ratio of 20:1. Inhibition of lysis of RMA-S_EM_UL40 transfectants was observed in the presence of the anti-HLA class I mAb W6/32. The results are representative of four independent experiments.

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NK-CTL recognition of virus-infected target cells

In order to directly assess the capability of NK-CTL to recognize and kill HCMV-infected target cells, NK-CTL derived from the representative donors KK and GG were used as effector cells in cytolytic assays against fibroblasts infected with the HCMV strain AD169. Since autologous fibroblasts were not available, we used allogeneic fibroblasts selected for their resistance to NK-CTL-mediated lysis. In particular, we selected the WI38 human skin fibroblast cell line that did not contain in its HLA class I haplotype (HLA-A*02, -A*68, -B*08, -B*58, -Cw*07) any HLA-Cw allele carrying the VMAPRTLIL peptide 9, 17.

Fig. 2A shows that NK-CTL displayed a poor cytolytic activity against uninfected fibroblasts at different time intervals. In contrast, the same target cells that had been infected with the HCMV strain AD169 were efficiently lysed. Note that lysis was maintained as long as 8 days after infection. These results are in agreement with the previous observation that UL40 transcription occurs both in the early and in the late phases of CMV infection 18. Although not shown, cell lysis was not further increased upon mAb-mediated masking of HLA class I-specific inhibitory receptors (KIR2D and LIR1) expressed by the NK-CTL of the two donors. This is not surprising since, as shown in Fig. 2B, upon CMV infection WI38 fibroblasts markedly down-regulated the surface expression of HLA class I molecules (i.e. the ligands for HLA class I-specific inhibitory NK receptors).

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Figure 2. Human fibroblasts infected with HCMV are lysed by NK-CTL derived from donors KK and GG. (A) The WI38 fibroblastic cell line infected with HCMV strain AD169 was lysed by NK-CTL up to day 8 after infection (black bars). Uninfected WI38 cells were not lysed (grey bars). (B) CMV-infected human fibroblasts (here analyzed at day 2 and day 5 after infection) were characterized by a strong down-regulation of HLA class I surface expression, detected with mAb A6.136 (open profiles). Shaded histograms are Ig isotype control stainings. (C) Inhibitory effect of the anti-TCR pan αβ mAb on the lysis of CMV-infected WI38 cells. (D) The cytolytic activity of NK-CTL against WI38 infected cells was analyzed in the presence or in the absence of the anti-NKG2D mAb BAT221. Masking of HLA class I molecules was performed by the mAb W6/32. NK-CTL analyzed in cytolytic assays were derived from donors KK and GG. An E/T ratio of 20:1 was used. The results are representative of four independent experiments.

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Fig. 2C shows that NK-CTL-mediated lysis of WI38 infected target cells was TCR-mediated since it could be inhibited by TCR blocking with an anti-TCRαβ mAb. However, since masking of TCR did not completely inhibit cytolytic activity, we further investigated the involvement of the NKG2D molecule, which functions as triggering receptor in NK cells 19, 20. Remarkably, NKG2D is consistently expressed by NK-CTL as well. Moreover, the WI38 cell line after infection with the HCMV strain AD169 up-regulated the surface expression of MHC class I-related chain A (MICA) and UL16-binding protein 3 (ULBP3) molecules (i.e. the NKG2D ligands) (not shown). As shown in Fig. 2D, lysis of WI38 infected cells was partially inhibited by the addition to the cytolytic assay of either an anti-HLA class I mAb (W6/32) or an anti-NKG2D mAb (BAT221), and virtually abrogated when mAb specific for the two molecules were used in combination. Taken together, our data indicate that HLA-E-restricted NK-CTL recognize HCMV-infected cells, thus providing an additional source of effector cells in the control of CMV infections.

Frequency and phenotypic characterization of HLA-E-restricted, HCMV-specific CD8+ T cells isolated from peripheral blood of HCMV-seropositive healthy donors

Having provided evidence that in vitro expanded NK-CTL lines can kill HCMV-infected cells, we further investigated whether cells displaying this functional capability were present in vivo in freshly isolated PBL. In order to directly assess the frequency of VMAPRTLIL-specific NK-CTL, we used appropriate HLA-E tetramers. In particular, allele HLA-E*0101 tetrameric complexes were obtained by refolding this HLA class Ib molecule with either VMAPRTLIL or VMAPRTLVL peptide and conjugated with streptavidin-PE. Since NK-CTL isolated from some donors are characterized by low surface density of CD94/NKG2A receptor, whose specific ligand is the HLA-E itself 21, PBMC were pre-incubated with mAb specific for the CD94 molecule so that binding of HLA-E tetramers was confined to NK-CTL expressing an HLA-E-specific TCR. As shown in Fig. 3, in donor KK (haplotype: HLA-A*02, -A*32; -B*44; -Cw*07), HLA-EVMAPRTLIL tetramers stained a sizeable fraction of CD8β+ cells. Half of these HLA-E tetramer-positive cells were both TCRVβ16- and KIR2D-negative (Fig. 3B, D). However, upon mAb-mediated masking of CD94, HLA-EVMAPRTLIL tetramers only bound to CD8β+ cells expressing both TCRVβ16 and KIR2D (Fig. 3C, E). It is of note that the percentage of HLA-EVMAPRTLIL tetramer-positive cells was 1.1% of CD8β+ T cells (corresponding to approximately 0.4% of unfractioned PBL).

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Figure 3. Frequency in the peripheral blood of HLA-E-restricted, HCMV-specific CD8+ T cells. Three-color immunofluorescence analysis was performed by staining PBMC, derived from HCMV-seropositive donors, with HLA-EVMAPRTLIL tetramers and anti-CD8β mAb, in combination with anti-TCRVβ16 or anti-KIR2D mAb. Cells were analyzed by FCM after gating on CD8β+ T cells (A). HLA-E tetramer staining was performed either in the absence (B, D) or in the presence of anti-CD94 mAb (C, E). Data refer to samples obtained from donor KK. Numbers in the panels refer to the percentages of stained cells. Similar results were obtained with two other donors of NK-CTL.

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We have previously shown that the TCR of NK-CTL from donor KK recognizes with high avidity RMA-S_EM cells pulsed with the VMAPRTLIL, but not with the VMAPRTLVL peptide, which equally bind to HLA-E 17. Thus, we investigated whether HLA-E tetramers refolded with the VMAPRTLVL peptide could bind to NK-CTL upon mAb-mediated masking of CD94/NKG2A receptors. As expected, no HLA-EVMAPRTLVL staining of CD8β+ cells could be detected upon CD94 mAb-mediated masking (not shown). Similar data were obtained in two additional donors characterized by the presence in the peripheral blood of NK-CTL displaying the same HLA-E-restricted VMAPRTLIL specificity (see 17).

We further analyzed the HLA-EVMAPRTLIL tetramer-positive NK-CTL population for various informative markers. In particular, Fig. 4 shows that these cells were characterized by the surface expression of CD45RA and CD56 (Fig. 4A, B). CD8β was expressed at low density (Fig. 4C). On the contrary, HLA-EVMAPRTLIL tetramer-positive NK-CTL did not express CD45RO, CD27, CD28, CCR7 (Fig. 4D–G) and CD25 (not shown). Intracellular staining revealed that HLA-EVMAPRTLIL tetramer-positive NK-CTL expressed both perforin (Fig. 4H) and granzyme A and B (not shown). A similar phenotypic profile could be detected in NK-CTL isolated from the other donors.

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Figure 4. Phenotypic analysis of freshly isolated NK-CTL. Two-color immunofluorescence analysis was performed by staining PBMC, isolated from the HCMV-seropositive healthy donor KK, with HLA-EVMAPRTLIL tetramers in combination with mAb specific for different surface or intracellular markers. Before staining, cells had been incubated with an anti-CD94 mAb to prevent binding of HLA-E tetramers to this receptor. Numbers in the panels indicate the percentages of stained cells. Similar results were obtained with two other NK-CTL donors analyzed.

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Taken together, these data would indicate that NK-CTL are highly differentiated effector-memory T cells 22, 23. On the other hand, the lack of expression of CD25 suggests that NK-CTL have not experienced recent activation 24.

HLA-E-restricted, HCMV-specific T cells freshly isolated from peripheral blood rapidly produce IFN-γ upon peptide stimulation

While tetramer staining of T cells enables to determine the frequency and the phenotype of peptide-specific CTL, it does not provide information on their function. Thus, we analyzed the capability of freshly isolated NK-CTL to produce IFN-γ upon specific peptide stimulation. In this context, it is well established that antigen-experienced CD8+CD28CD27CD45RA+ T cells promptly produce both IFN-γ and TNF-α, but not IL-2, upon antigen stimulation 23. Fig. 5 shows that, upon stimulation of PBMC from donor KK with the VMAPRTLIL peptide, IFN-γ-producing cells were confined to TCRVβ16+ T cells that expressed KIR2D (Fig. 5A, B). Remarkably, upon stimulation with staphylococcal enterotoxin B (SEB, a superantigen which binds to several TCRVβ segments but not to TCRVβ16 expressed by NK-CTL of donor KK), IFN-γ-producing cells were confined to lymphocytes lacking both TCRVβ16 and KIR2D (Fig. 5C, D). On the other hand, stimulation with the VMAPRTLVL peptide failed to induce IFN-γ production (Fig. 5E). Although not shown, VMAPRTLIL-responding T cells failed to produce IL-2, consistent with the concept that they belong to the effector-memory compartment.

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Figure 5. IFN-γ production by freshly isolated NK-CTL upon stimulation with the VMAPRTLIL peptide. PBMC from NK-CTL donor KK were stimulated with the VMAPRTLIL peptide (A, B), SEB (C, D) or the VMAPRTLVL peptide (E). After 4 h, cells were analyzed by two-color immunofluorescence using anti-IFN-γ and anti-TCRVβ16 mAb or by three-color immunofluorescence using anti-IFN-γ, anti-KIR2D and anti-TCRαβ mAb. IFN-γ production by KIR2D+ T cells (B, D) was analyzed after gating on TCRαβ+ T cells. Similar results were obtained with two other NK-CTL donors analyzed.

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Discussion

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

In the present paper we provide direct evidence that HLA-E-restricted CTL can recognize and kill CMV-infected cells, thus representing an additional type of effector cells playing a role in defense against a virus which can escape recognition by CTL restricted by classical HLA class I molecules. We also provide evidence that these cells are present in vivo, display an effector-memory phenotype, and can readily respond to specific stimulation by HLA-E-binding CMV-derived peptide. NK-CTL were originally identified as a TCRαβ+ CD8+ T cell population that expressed HLA class I-specific inhibitory receptors including KIR, LIR1 and CD94 7. They were identified in the peripheral blood from some donors as a T cell subset characterized by the homogeneous expression of a given TCRVβ (different in different donors) and a memory phenotype 25. Subsequently, it has been shown that at least a fraction of this T cell subset was HLA-E-restricted. These data provided the first evidence that HLA-E functions as a restriction element for TCRαβ-mediated recognition 8. In view of the limited polymorphism of HLA-E as well as the limited number of HLA-E-binding peptides, NK-CTL displayed a broad capability of recognizing HLA-E-expressing allogeneic cells 9, 17. On the other hand, autologous cells could only be lysed when pulsed with non-self HLA-E-binding peptides including CMV-derived ones 17, 26.

HCMV has evolved an impressive variety of strategies to escape CTL responses by interfering at different stages with the MHC class Ia-mediated presentation of peptides to CTL. Different CMV-encoded viral proteins belonging to the US family inhibit the MHC class I expression both in humans and in mice by acting at different time intervals after infection and by exploiting different molecular mechanisms 27. In particular, the HCMV gene products US2 and US11 redirect nascent HLA class I chains back into the cytosol, resulting in their degradation, whereas US3 retains HLA class I complexes in the endoplasmic reticulum; finally, both in early and late phases of infection HCMV encodes for US6 that inhibits the TAP-mediated delivery of antigenic peptides to the endoplasmic reticulum 1. As a consequence, conventional CTL may result, at least in part, inefficient in counteracting CMV infection. Other cell-mediated effector mechanisms may result crucial for effective host defenses against CMV. Indeed, CMV-infected cells become more susceptible to NK-mediated lysis both because of down-regulation of HLA class I molecules and up-regulation of ligands for triggering NK receptors (including MICA and ULBP) 2831.

However, it has been reported that some CMV-encoded proteins can also down-modulate NK cell responses 1. In particular, a potential target for HCMV to control NK cell-mediated lysis is the inhibitory receptor CD94/NKG2A. The only ligand known so far for CD94/NKG2A is HLA-E, which is expressed on virtually all cells 13, 32. In HCMV-infected cells, HLA class I alleles (source of HLA-E-binding peptides) are lacking, while TAP molecules are functionally blocked by US6. Under these conditions, HCMV itself, through the expression of UL40 protein, can supply peptides which bind HLA-E in a TAP-independent fashion. This results in surface expression of HLA-E at even higher concentrations than in uninfected cells 5, 6, 15. Along this line, the up-regulation of HLA-E in CMV-infected cells can be viewed as a mechanism of viral escape from NK cells expressing CD94/NKG2A.

Our present study provides evidence that NK-CTL may represent an additional effector cell type in the control of this virus, which establishes lifelong persistent infection characterized by alternative periods of latency and reactivation in infected hosts. Indeed, NK-CTL are able to kill infected cells despite the sharp HLA class Ia down-regulation, by recognizing the UL40-derived VMAPRTLIL peptide presented in the context of HLA-E. Furthermore, the NKG2D-activating receptor expressed by NK-CTL can recognize on HCMV-infected target cells both MICA and ULBP3 molecules, thus providing a costimulatory signal amplifying the TCR-mediated response 28. It should be stressed that NK-CTL are present only in the peripheral blood of certain HCMV-seropositive healthy individuals carrying an appropriate HLA class I haplotype (i.e. not containing any HLA-Cw allele bearing the VMAPRTLIL peptide) 17. It is possible to speculate that these individuals are more resistant to severe CMV infection and/or reactivation occurring under certain pathological conditions.

As reported in various studies, in HCMV-seropositive healthy individuals, in particular in the elderly, there is a general increase in size of virus-specific T lymphocyte populations characterized by the CD27CD28CD45RA+CCR7 surface phenotype and the intracellular expression of cytotoxic granules containing perforin and granzymes 3335. Remarkably, this phenotype is identical to that of freshly isolated NK-CTL. Functional analysis reveals that, upon activation with the appropriate VMAPRTLIL peptide, NK-CTL are able to promptly produce IFN-γ, but not IL-2, in the absence of prior restimulation in vitro. All the various phenotypic and functional data illustrated above, together with the fact that NK-CTL represent a oligo-/monoclonal expansion characterized by the expression of HLA class I-specific inhibitory receptors, support the notion that they are effector-memory T cells possibly resulting from a chronic, antigen-driven stimulation 36. Since effector-memory cells are known to derive from the central memory compartment, one may ask why HLA-EVMAPRTLIL tetramers failed to stain cells belonging also to this subset. A likely explanation is that such cells are present in extremely low numbers in the peripheral blood and/or are confined to other lymphoid compartments (e.g. LN and spleen), where they might interact with CMV-infected cells.

Materials and methods

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

Monoclonal antibodies and tetramers

The following mAb were used in this study: GL183 (IgG1, anti-KIR2DL2/S2/L3), Y249 (IgM, anti-KIR2DL2/S2/L3), EB6 (IgG1, anti-KIR2DL1/S1), XA141 (IgM, anti-KIR2DL1/S1), F278 (IgG1, anti-ILT2/LIR1), XA185 (IgG1, anti-CD94), Y9 (IgM, anti-CD94), Z270 (IgG1, anti-NKG2A), W6/32 (IgG2a, anti-HLA class I), A6.136 (IgM, anti-HLA class I), BAT-221 (IgG1, anti-NKG2D), KD1 (IgG2a, anti-CD16), BAB281 (IgG1, anti-NKp46), A13 (IgG1, anti-Vδ1), BB3 (IgG1, anti-Vδ2) and BAM195 (IgG1, anti-MICA) were produced in our laboratory. Leu-2a (IgG1, anti-CD8), Leu-3a (IgG1, anti-CD4) and anti-CD28 (IgG1) mAb were purchased from BD Pharmingen. Anti-TCRVβ16 (IgG1), anti-TCRVβ22 (IgG1), anti-TCRVβ9 (IgG2a) and anti-TCRVβ5.1 (IgG1) mAb were purchased from Immunotech (Marseille, France). The M551 (IgG1, anti-ULBP3) mAb was kindly provided by Amgen (Seattle, WA). FITC- and PE-conjugated anti-isotype goat anti-mouse mAb were purchased from Southern Biotechnology (Birmingham, AL). The reactivity of mAb with cell populations was assessed by indirect immunofluorescence and cytofluorimetric analysis as previously described 37. Briefly, 105 cells were stained with the corresponding mAb followed by appropriate FITC- or PE-conjugated anti-isotype specific goat anti-mouse second reagents.

For analysis of surface marker expression on freshly isolated PBMC the following fluorescent-labeled conjugated mAb were used in different combinations: Anti-TCR pan αβ-PC5 (R-PE covalently linked to cyanin 5.1, IgG2b), anti-CD8β-PC5 (IgG2a) (Immunotech), anti-CD25-PE (IgG1) anti-CD27-FITC (IgG1), anti-CD28-FITC (IgG1), anti-CD45RO-FITC (IgG2a), anti-CD45RA-FITC (IgG2b), anti-CCR7-PE (IgG2a), anti-CD56-PE (IgG1) (BD Pharmingen). For intracellular staining the following mAb were used anti-IFN-γ-PE (IgG1), anti-granzyme A-PE (IgG1) (BD Pharmingen), anti-granzyme B-PE (IgG1; Caltag Laboratories, Burlingame, CA) and anti-perforin (IgG2b; Ancell, Bayport, MN). Soluble HLA-E tetramersPE were generated nearly as described 38. HLA-E*0101 and β2-microglobulin proteins were refolded either with VMAPRTLIL or VMAPRTLVL synthetic peptides.

Isolation of PBMC and flow cytometric analysis

PBMC were isolated from peripheral blood of normal donors by a Ficoll-Hypaque density gradient, as previously described 37. PBMC were resuspended in RPMI 1640 (Seromed, Berlin, Germany) supplemented with 10% FCS (Sigma-Aldrich, St. Louis, MO) and 1% antibiotic mixture (5 mg/mL penicillin, 5 mg/mL streptomycin stock solution). Cells were then washed in PBS containing 0.5% w/v BSA (PBA; Sigma-Aldrich). To enumerate VMAPRTLIL- or VMAPRTLVL-specific T cells, a total of 106 PBMC were incubated with an appropriate concentration of HLA-E tetramersPE (50 nM) for 40 min at room temperature, protected from light. Then fluorescent-labeled conjugated mAb were added and incubated for 30 min at 4°C, protected from light. Cells were then washed in PBA and analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences). XA185 mAb (10 µg/mL) was used in blocking experiments.

Intracellular perforin and cytokine staining

For intracellular perforin staining, 106 PBMC were incubated with HLA-E tetramersPE washed once with PBA, then fixed with 4% formaldehyde solution, and subsequently permeabilized by washing with 0.1% saponine. Cells were then incubated with anti-perforin mAb followed by appropriate FITC-conjugated anti-isotype specific goat anti-mouse second reagent. For cytokine staining, PBMC were resuspended in RPMI 1640 supplemented with 5% of human serum AB (MP Biomedicals, Bruxelles, Belgium) at the concentration of 107/mL and stimulated with either the VMAPRTLIL or the VMAPRTLVL peptide (10 μg/mL) in the presence of brefeldin A for 4 h at 37°C. In addition, as positive control PBMC were incubated with SEB (1.5 μg/mL; Sigma-Aldrich) and anti-CD28 mAb (1 μg/mL). Cells were washed, and then the same intracellular staining procedure as described above was performed using anti-IFN-γ-PE mAb.

Isolation, culture and cloning of NK-CTL

PBMC were depleted of CD4+, CD16+, NKp46+, Vδ1+, Vδ2+ cells by negative selection using appropriate mAb and magnetic beads coated with anti-mouse IgG (Dynal, Oslo, Norway). The culture medium used was RPMI 1640 (Seromed) supplemented with 10% FCS (Sigma-Aldrich), 1% antibiotic mixture (5 mg/mL penicillin, 5 mg/mL streptomycin stock solution) and human recombinant IL-2 was kindly provided by Chiron Corp. Ficoll-Hypaque (Histopaque 1077) was purchased from Sigma-Aldrich. Mixed lymphocyte cultures (MLC) were set up as described 39 by culturing CD8+ T cells in round-bottom microwells (105 cells per well) in the presence of irradiated (5 000 rad) allogeneic PBMC (2×105 cells). At day 3, 25 U/mL of IL-2 was added to cultures. At day 8, cells were either restimulated or cloned under limiting dilution conditions 39. At day 14, the specificity of the resulting NK-CTL expanded after MLC was evaluated in a cytolytic assay and immunofluorescence analysis was performed.

Virus infection

The HCMV strain AD169 was purchased from ATCC. In all of the experiments, WI38 fibroblasts were grown at 80% of confluence and then infected with HCMV strain AD169 at a multiplicity of infection of 5. After adsorption of the virus for 1 h at 37°C, the inoculum was removed and fresh medium was added. After 48 h the percentage of HCMV-infected cells was determined using the Light DiagnositcTM CMV pp65 Antigenemia Immunofluorescence Assay (Chemicon International, CA) and FCM. At day 2, 5 or 8 after infection cells were harvested using trypsin-EDTA and used for cytotoxicity experiments. The expression of HLA class I (detected by the A6.136 mAb), MICA (detected by the BAM195 mAb), and ULPB3 (detected by the M155 mAb) molecules on target cells, either uninfected or after viral infection, was evaluated using indirect immunofluorescence and FCM.

Cytolytic activity

NK-CTL were tested for cytolytic activity in a 4-h 51Cr-release assay in the presence or in the absence of mAb. The concentration of the mAb used for the masking experiments was 10 μg/mL. The E/T ratios used are indicated in the Fig. legends. Target cells used in these experiments were represented by the murine TAP-2–/– T cell lymphoma RMA-S cell line cotransfected with either human β2-microglobulin and HLA-E*01033 allele (RMAS_EM; kindly provided by J. E. Coligan, NIAID, NIH, Rockville, MD), or human β2-microglobulin, HLA-E*01033 allele and AD169-derived UL40 (RMAS_EM_UL40; kindly provided by E. H. Weiss, Institut für Anthropologie und Humangenetik, Munich, Germany), and the human skin fibroblast cell line WI38 either uninfected or after infection with the HCMV strain AD169. All these cell lines were also tested in cytolytic assays after overnight incubation at 37°C with the UL40-derived VMAPRTLIL peptide (200 μM).

Acknowledgements

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

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.); Istituto Superiore di Sanità (I.S.S.); Ministero della Salute; Ministero dell'Istruzione, dell'Università e della Ricerca (M.I.U.R); Fondazione Compagnia di San Paolo, Torino, Italy; and European Union FP6, LSHB-CT-2004–503319-AlloStem (The European Commission is not liable for any use that may be made of the information contained). Paola Mazzarino is recipient of a fellowship awarded by Fondazione Italiana per la Ricerca sul Cancro (F.I.R.C.)

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