UL40-mediated NK evasion during productive infection with human cytomegalovirus

Human cytomegalovirus (HCMV) exploits a range of strategies to evade and modulate the immune response. Its capacity to down-regulate MHC I expression was anticipated to render infected cells vulnerable to natural killer (NK) attack. Kinetic analysis revealed that during productive infection, HCMV strain AD169 first enhanced and then inhibited lysis of primary skin fibroblasts by a CD94/NKG2A(+)NKG2D(+)ILT2(+) NK line. The inhibition of cytotoxicity against strain AD169-infected fibroblasts was abolished by prior treatment of targets or effectors with anti-MHC I and anti-CD94 monoclonal antibodies, respectively, implying a CD94/HLA-E-dependent mechanism. An HCMV strain AD169, UL40 deletion mutant could not inhibit CD94/NKG2A(+) NK killing against skin fibroblasts. The contribution of UL40 to evasion of primary NK cells then was tested in a system where targets and effectors were MHC-matched. Primary NK cells activated with IFNalpha as well as cultured primary NK cell lines showed increased killing against DeltaUL40-infected fibroblasts compared with AD169-infected targets. This effect was abrogated by depletion of CD94(+) cells. These findings demonstrate that HCMV encodes a mechanism of evasion specifically targeted against a proportion of CD94(+) NK cells and show that this system functions during a productive infection.     

NKp46 and DNAM-1 NK-cell receptors drive the response to human cytomegalovirus-infected myeloid dendritic cells overcoming viral immune evasion strategies

Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs, reflecting the efficiency of immune evasion mechanisms, and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast, DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection, although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors, which were partially reduced on HCMV infection; yet, only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts, human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs, which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs, stressing the importance of the dynamics of viral immune evasion mechanisms.     

Imprint of human cytomegalovirus infection on the NK cell receptor repertoire

Expression of the activating CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E was analyzed in peripheral blood lymphocytes (PBLs) from healthy adult blood donors; the expression of other natural killer (NK) cell receptors (ie, CD94/NKG2A, KIR, CD85j, CD161, NKp46, NKp30, and NKG2D) was also studied. Human cytomegalovirus (HCMV) infection as well as the HLA-E and killer immunoglobulin-like receptor (KIR) genotypes were considered as potentially relevant variables associated with CD94/NKG2C expression. The proportion of NKG2C(+) lymphocytes varied within a wide range (<0.1% to 22.1%), and a significant correlation (r = 0.83; P < .001) between NKG2C(+) NK and T cells was noticed. The HLA-E genotype and the number of activating KIR genes of the donors were not significantly related to the percentage of NKG2C(+) lymphocytes. By contrast, a positive serology for HCMV, but not for other herpesviruses (ie, Epstein-Barr and herpes simplex), turned out to be strongly associated (P < .001) with increased proportions of NKG2C(+) NK and T cells. Remarkably, the CD94/NKG2C(+) population expressed lower levels of natural cytotoxicity receptors (NCRs) (ie, NKp30, NKp46) and included higher proportions of KIR(+) and CD85j(+) cells than CD94/NKG2A(+) cells. Altogether, these data support that HCMV infection selectively shapes the natural killer cell receptor (NKR) repertoire of NK and T cells from healthy carrier individuals.     

Expansion of a unique CD57⁺NKG2Chi natural killer cell subset during acute human cytomegalovirus infection

During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94-NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56(dim)CD16(+) NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94-NKG2C receptor and CD57 in CMV(+) donors. These CD57(+)NKG2C(hi) NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57(+)NKG2C(hi) NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57(+)NKG2C(hi) NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C(+) NK cells proliferated, became NKG2C(hi), and finally acquired CD57. Thus, we propose that CD57 might provide a marker of "memory" NK cells that have been expanded in response to infection.     

Human cytomegalovirus infection is associated with increased proportions of NK cells that express the CD94/NKG2C receptor in aviremic HIV-1-positive patients

In healthy blood donors, serological positivity for human cytomegalovirus (HCMV) is associated with an increased proportion of NK cells bearing the CD94/NKG2C NK cell receptor (NKR). The expression of the activating CD94/NKG2C NKR and of the inhibitory CD94/NKG2A NKR was studied in a cohort of 45 aviremic human immunodeficiency virus type 1 (HIV-1)-positive patients receiving highly active antiretroviral therapy. The proportions of NKG2C+ NK cells were significantly increased in HIV-1-positive patients (mean +/- SD, 25.9% +/- 23.0%), compared with those in 31 healthy individuals (mean +/- SD, 16.1% +/- 20.7%). Yet, the association vanished when HCMV serological status was considered in a multivariate regression model. These results support the conclusion that changes in the NKR repertoire in HIV1-positive patients are related to a concomitant HCMV infection.     

婴儿巨细胞病毒感染与自然杀伤细胞C型凝集素受体的表达

目的 探讨自然杀伤(NK)细胞C型凝集素受体表达与婴儿人巨细胞病毒(HCMV)感染的关系.方法 2006年1月至2008年6月HCMV感染患儿79例和母乳性黄疸对照患儿39例.根据外周血中性粒细胞pp65抗原血症结果,分为活动性HCMV感染组48例和非活动性HCMV感染组31例,并对活动性HCMV感染组的48例婴儿进行更昔洛韦治疗2周.应用流式细胞术检测外周血NK细胞C型凝集素受体NKG2A、NKG2C、NKG2D表达.三组数据比较采用Kruskal-Wallis独立样本非参数检验,两组之间比较采用Mann-Whiteney配对样本非参数检验.结果 NK细胞抑制性受体NKG2A表达在活动性HCMV感染组、非活动性HCMV感染组和对照组之间差异无统计学意义(x2=3.95,P＞0.05);NK细胞活化性受体NKG2C、NKG2D表达在活动性HCMV感染组、非活动性HCMV感染组和对照组之间差异有统计学意义(x2=24.91,P＜0.01;x2=47.80,P＜0.01).NK细胞活化性受体NKG2C、NKG2D表达在HCMV感染组较对照组明显升高(Z=-4.72,P＜0.01;Z=-5.15,P＜0.01).NK细胞活化性受体NKG2D表达在活动性HCMV感染组较非活动性HCMV感染组明显升高(Z=-5.08,P＜0.01).更昔洛韦治疗后NK细胞活化性受体NKG2D表达降低(Z=-1.34,P=0.07).结论 在调节NK细胞功能和婴儿抗HCMV免疫方面,NKG2C和NKG2D表达可能起重要作用.     

Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus?

Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR(+)NKG2A(-) signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C(+) NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56(-)CD16(+)p75/AIRM1(-) NK-cell subset (mostly KIR(+)NKG2A(-)) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation.     

Selective expansion of intraepithelial lymphocytes expressing the HLA-E-specific natural killer receptor CD94 in celiac disease

BACKGROUND & AIMS: Celiac disease is a gluten-induced enteropathy characterized by the presence of gliadin-specific CD4(+) T cells in the lamina propria and by a prominent intraepithelial T-cell infiltration of unknown mechanism. The aim of this study was to characterize the subset(s) of intraepithelial lymphocytes (IELs) expanding during active celiac disease to provide insights into the mechanisms involved in their expansion. METHODS: Flow-cytometric analysis of isolated IELs and/or immunohistochemical staining of frozen sections were performed in 51 celiac patients and 50 controls with a panel of monoclonal antibodies against T-cell and natural killer (NK) receptors. In addition, in vitro studies were performed to identify candidate stimuli for NK receptor expression. RESULTS: In normal intestine, different proportions of IELs, which were mainly T cells, expressed the NK receptors CD94/NKG2, NKR-P1A, KIR2D/3D, NKp46, Pen5, or CD56. During the active phase of celiac disease, the frequency of CD94(+) IELs, which were mostly alphabeta T cells, was conspicuously increased over controls. In contrast, the expression of other NK markers was not modified. Furthermore, expression of CD94 could be selectively induced in vitro by T-cell receptor activation and/or interleukin 15, a cytokine produced by intestinal epithelial cells. CONCLUSIONS: The gut epithelium favors the development of T cells that express NK receptors. In active celiac disease, there is a specific and selective increase of IELs expressing CD94, the HLA-E-specific NK receptor that may be related to T-cell receptor activation and/or interleukin 15 secretion.     

Slow receptor acquisition by NK cells regenerated in vivo from transplanted fetal liver or adult bone marrow stem cells

Adult mouse natural killer (NK) cells express two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, whereas fetal and neonatal NK cells express only CD94/NKG2. After birth, Ly49(+) NK cells slowly increase and CD94/NKG2(+) NK cells decrease. The aim of this study was to determine whether murine NK cells develop differently from transplants of fetal liver and adult marrow stem cells and whether the adult marrow microenvironment is critical for NK receptor maturation. Enriched populations of stem cells were transplanted into adult mice, and the kinetics of NK receptor acquisition was examined. NK cells from osteopetrotic Csf1(op)/Csf1(op) mice, in which hematopoiesis within the marrow is severely limited, were also analyzed.NK cells regenerated from both fetal and adult stem cells initially resembled neonatal NK cells in their slow acquisition of Ly49 over several weeks, although the adult stem cell-derived NK cells matured approximately 10 days sooner. NK cells from adult Csf1(op)/Csf1(op) mice expressed normal levels of Ly49. Maturation of the NK receptor repertoire is a slow process regardless of their stem cell origin or reduced marrow space caused by osteopetrosis.     

Calcineurin-dependent negative regulation of CD94/NKG2A expression on naive CD8+ T cells

Immune responses lead to expression of immunoregulatory molecules on T cells, including natural killer (NK) receptors, such as CD94/NKG2A on CD8(+) T cells; these receptors restrain CD8(+) responses, thereby preventing T-cell exhaustion in chronic infections and limiting immunopathology. Here, we examined the requirements for inducing CD94/NKG2A on T cells responding to antigen. In vitro, moderate induction of CD94/NKG2A expression occurred after exposure of naive CD8(+) (but not CD4(+)) cells to CD3 ligation or specific peptide. Surprisingly, expression was inhibited by CD28/B7 costimulation. Such inhibition applied only to CD94/NKG2A and not other NK receptors (NKG2D) and was mediated by IL-2. Inhibition by IL-2 occurred via a NFAT cell-independent component of the calcineurin pathway, and CD94/NKG2A induction was markedly enhanced in the presence of calcineurin blockers, such as FK506 or using calcineurin-deficient T cells, both in vitro and in vivo. In addition to CD28-dependent inhibition by IL-2, CD94/NKG2A expression was impaired by several other cytokines (IL-4, IL-23, and transforming growth factor-β) but enhanced by others (IL-6, IL-10, and IL-21). The complex interplay between these various stimuli may account for the variable expression of CD94/NKG2A during responses to different pathogens in vivo.     

Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells

The stages of human natural killer (NK) cell differentiation are not well established. Culturing CD34(+) progenitors with interleukin 7 (IL-7), IL-15, stem cell factor (SCF), FLT-3L, and murine fetal liver cell line (EL08.1D2), we identified 2 nonoverlapping subsets of differentiating CD56(+) cells based on CD117 and CD94 (CD117(high)CD94(-) and CD117(low/-)CD94(+) cells). Both populations expressed CD161 and NKp44, but differed with respect to NKp30, NKp46, NKG2A, NKG2C, NKG2D, CD8, CD16, and KIR. Only the CD117(low/-) CD94(+) population displayed cytotoxicity and interferon-gamma production. Both populations arose from a single CD34(+)CD38(-) Lin(-) cell and their percentages changed over time in a reciprocal fashion, with CD117(high)CD94(-) cells predominating early and decreasing due to an increase of the CD117(low/-)CD94(+) population. These 2 subsets represent distinct stages of NKcell differentiation, since purified CD117(high) CD94(-) cells give rise to CD117(low/-)CD94(+) cells. The stromal cell line (EL08.1D2) facilitated the transition from CD117(high)CD94(-) to CD117(low/-)CD94(+) via an intermediate phenotype (CD117(low)CD94(low/-)). EL08.1D2 also maintained the mature phenotype, preventing the reversion of CD117(low/-)CD94(+) cells to the intermediate (CD117(low)CD94(low/-)) phenotype. An analogous population of CD56(+)CD117(high)CD94(-) cells was found in cord blood. The identified stages of NK-cell differentiation provide evidence for coordinated acquisition of HLA-specific inhibitory receptors (ie, CD94/NKG2A) and function in developing human NK cells.     

Switch from inhibitory to activating NKG2 receptor expression in HIV-1 infection: lack of reversion with highly active antiretroviral therapy

BACKGROUND: HIV-1 infection is characterized by increase in inhibitory receptors and loss of activating receptors on natural killer (NK) cells, resulting in loss of cell activity. Exceptionally, for an inhibitory receptor, the proportion of NK cells bearing CD94-NKG2A decreases during HIV-1 infection. It is not understood whether HIV-1 itself or other concomitant infections drive these changes. OBJECTIVES: To investigate the relationship between HIV-1 viraemia and changes in C-type lectin-like receptor expression in NK cells and to investigate the effect of highly active antiretroviral therapy (HAART) on these changes. METHODS: Three cohorts of patients were studied: (1) before, during and after treatment interruption in aviraemic and viraemic patients receiving HAART (n = 15); (2) HIV-1-positive treatment-naive individuals (n = 13); and (3) HIV-1-positive individuals receiving successful HAART for a minimum of 1 year without interruption (n = 11). Flow cytometry was used to study the expression of NKG2A before and after treatment interruption and to define expanded populations of NK cells in untreated and treated HIV-1-positive individuals. Assays were performed in vitro to assess the cytotoxicity of the expanded populations. RESULTS: Increases in plasma HIV-1 RNA during treatment interruption in aviraemic HAART-treated individuals did not influence the proportion of NK cells carrying the complex CD94-NKG2A. Loss of NKG2A NK cells corresponded to the dramatic expansion of a distinct population of cells expressing a functional activating CD94-NKG2C receptor with skewed expression of killer cell immunoglobulin-like receptor family and natural cytotoxicity receptors. CONCLUSION: Changes in the NK cell repertoire during HIV-1 infection were not a result of HIV-1 viraemia alone but resembled those associated with concomitant infections.     

NK-cell reconstitution after haploidentical hematopoietic stem-cell transplantations: immaturity of NK cells and inhibitory effect of NKG2A override GvL effect

Natural killer (NK) cell alloreactivity is reported to mediate strong GvL (graft versus leukemia) effect in patients after haploidentical stem-cell transplantation (SCT) for acute myeloid leukemia (AML). Because subsequent immune reconstitution remains a major concern, we studied NK-cell recovery in 10 patients with AML who received haplomismatched SC transplants, among whom no GvL effect was observed, despite the mismatched immunoglobulin-like receptor (KIR) ligand in the GvH direction for 8 of 10 patients. NK cells generated after SCT exhibited an immature phenotype: the cytotoxic CD3- CD56(dim) subset was small, expression of KIRs and NKp30 was reduced, while CD94/NKG2A expression was increased. This phenotype was associated to in vitro lower levels of cytotoxicity against a K562 cell line and against primary mismatched AML blasts than donor samples. This impaired lysis was correlated with CD94/NKG2A expression in NK cells. Blockading CD94/NKG2A restored lysis against the AML blasts, which all expressed HLA-E, the ligand for CD94/NKG2A. Our present study allows a better understanding of the NK-cell differentiation after SCT. These results revealed that the NK cells generated after haplomismatched SCT are blocked at an immature state characterized by specific phenotypic features and impaired functioning, having potential impact for immune responsiveness and transplantation outcome.     

Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin-beta receptor expression

A crucial step in murine natural killer (NK) cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49 receptor expression are still largely undetermined. It has been shown that interaction between lymphotoxin alpha1beta2 (LTalpha1beta2) and LTbeta receptor (LTbetaR), expressed on lymphoid progenitor cells and nonlymphoid bone marrow stromal cells, respectively, is important for both quantitative and functional NK cell development. Therefore, we have investigated the role of LT-LTbetaR-mediated signaling in Ly49 and CD94/NKG2 receptor acquisition. We show that the NK receptor repertoire of LTbetaR-/- mice can only be partially analyzed because of the residual 129/Ola mouse genetic background, due to a physical linkage of the LTbetaR locus and the loci encoding the Ly49 and CD94/NKG2 receptors. Therefore, we transferred wild-type B6 lymphoid-committed progenitor cells into LTbetaR-/- mice, which differentiated into NK cells with a normal NK cell receptor repertoire. Also, administration of LTbetaR-immunoglobulin (Ig), which acts as a soluble receptor for LTalpha1beta2, resulted in reduced NK cell percentages but did not influence the Ly49 and CD94/NKG2 receptor acquisition on remaining NK cells. These results indicate that LTbetaR-mediated signals are not required for Ly49 and CD94/NKG2 receptor acquisition.     

Immunological changes in different patient populations with chronic hepatitis C virus infection

AIM: To investigate killer inhibitory and activating receptor expression by natural killer(NK), natural killer T-like(NKT-like) and CD8+ T lymphocytes in patients with chronic hepatitis C virus(HCV) infection with elevated and with persistently normal alanine aminotransferase(PNALT).METHODS: The percentage of peripheral blood Treg cells, KIR2DL3, ILT-2, KIR3DL1, CD160, NKG2 D, NKG2 C expressing NK, T and NKT-like cells, cytokine production and NK cytotoxicity were determined by flow cytometry. Twenty-one patients with chronic HCV infection with elevated alanine aminotransferase, 11 HCV carriers with persistently normal alanine aminotransferase and 15 healthy volunteers were enrolled. RESULTS: No significant differences were observed in the percentage of total T, NK or NKT-like cells between study groups. Comparing the activating and inhibitoryreceptor expression by NK cells obtained from HCV carriers with PNALT and chronic HCV hepatitis patients with elevated alanine aminotransferase, NKG2 D activating receptor expression was the only receptor showing a significant difference. NKG2 D expression of NK cells was significantly lower in patients with elevated alanine aminotransferase. The expression of CD160, NKG2 D and NKG2 C activating receptor by CD8+ T cells were significantly lower in patients with chronic HCV hepatitis than in healthy controls and in HCV carriers with PNALT. Plasma TGF-β1 levels inversely correlated with NKG2 D expression by NK cells. In vitro TGF-β1 treatment inhibited NK cells cytotoxic activity and downregulated NKG2 D expression. CD8+ T cells from HCV carriers with PNALT showed significantly elevated expression of CD160, NKG2 D and NKG2 C activating receptors compared to chronic HCV patients with elevated alanine aminotransferase. Enhanced expression of inhibitory KIR2DL3 receptor, and decreased ILT-2 expression on NK cells were also found in chronic hepatitis C patients compared to healthy controls.CONCLUSION: Our study demonstrated a complex dysregulation of activating and inhibitory receptor expression, such as decreased NKG2 D and CD160 activating receptor expression and increased KIR2DL3 inhibitory receptor expression by NK and cytotoxic T cells and may provide further mechanism contributing to defective cellular immune functions in chronic hepatitis C. Increased NKG2 D receptor expression in HCV patients with persistently normal ALT suggests an important pathway for sustaining NK and CD8 T cell function and a protective role against disease progression.     

Human cytomegalovirus sequences expressed in latently infected individuals promote a latent infection in vitro

Latency enables human cytomegalovirus (HCMV) to persist in the hematopoietic cells of infected individuals indefinitely and prevents clearance of the pathogen. Despite its critical importance to the viral infectious cycle, viral mechanisms that contribute to latency have not been identified. We compared the ability of low-passage clinical and laboratory-adapted strains of HCMV to establish a latent infection in primary human CD34(+) cells. The low-passage strains, Toledo and FIX, established an infection with the hallmarks of latency, whereas the laboratory strains, AD169 and Towne, replicated producing progeny virus. We hypothesized that ULb' region of the genome, which is unique to low-passage strains, may encode a latency-promoting activity. We created and analyzed recombinant viruses lacking segments or individual open reading frames (ORFs) in the ULb' region. One 5-kb segment, and more specifically the UL138 ORF, was required for HCMV to establish and/or maintain a latent infection in hematopoietic progenitor cells infected in vitro. This is the first functional demonstration of a virus-coded sequence required for HCMV latency. Importantly, UL138 RNA was expressed in CD34(+) cells and monocytes from HCMV-seropositive, healthy individuals. UL138 might be a target for antivirals against latent virus.     

Ex vivo generation of human cytomegalovirus-specific cytotoxic T cells by peptide-pulsed dendritic cells

Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T-cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblasts as stimulator cells confines this approach to HCMV-seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. The CTLs recognized HCMV-infected autologous fibroblasts. No lysis was observed with either non-infected autologous or HLA-mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide-specific T cells of up to 28% and > 90% of CD8(+) T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 x 10(9) HCMV-specific T cells was possible after 6--7 weeks when cultures were initiated with 1--5 x 10(6) responder cells. Thus, the approach with peptide-pulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.