逆病毒载体介导双耐药基因在脐血CD34^＋细胞中的表达和抗性研究

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.     

逆转录病毒载体介导MGMT和MDR1基因增强人脐血CD34＋细胞对联合化疗抗性的研究

To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) and multidrug resistance gene (MDR1) increase resistance to 1,3-Bis(2-Chloroethy1)-1-Nitrosourea (BCNU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric performation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then transfected repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34+ cells was approximately 92% and recover rate was 75%, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 x 10(5) cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4.5 to 7.8-fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.     

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells

Purpose: The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Methods: Clone 96 (CI96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Results: Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. Conclusions: We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.Abbreviations Ab Antibody - CTL Cytolytic T lymphocyte - mAb Monoclonal antibody - TCR T-cell receptor     

Treatment of recurrent glioma with intracavitary alloreactive cytotoxic T lymphocytes and interleukin-2

 For a single-dose toxicity assessment, five patients with recurrent malignant glioma (ages 29–46 years) were treated with intracavitary alloreactive cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2). The trial tested the hypothesis that alloreactive CTL, sensitized to the major histocompatibility complex (MHC) proteins of the patient, offer selective, targeted killing of glioma cells that express MHC. Patient lymphocytes, which also express MHC, were irradiated and placed into CellMax artificial capillary systems with lymphocytes from MHC-disparate donors and CTL developed over a 2- to 3-week period with a low concentration of IL-2. The CTL largely expressed CD3 and CD11a/CD8 markers and lysed targets displaying patient MHC. CTL were implanted into the tumor bed at surgery and a catheter was used for subsequent infusions. Patients received one to five treatment cycles every other month; one cycle generally consisted of two or three CTL infusates administered within a 1- to 2-week period. Different unrelated donors were used for each cycle. Treatment was well tolerated; transient toxicity at grades 1–3 was recorded by NCI Common Toxicity Scale criteria. Two glioblastoma patients have died; one from tumor recurrence locally and the other from recurrence at a site distant from the treatment. Two of the five patients completed five cycles; one anaplastic oligodendroglioma patient shows no evidence of tumor 30 months from the start of immune therapy and an anaplastic astrocytoma patient shows stable disease 28 months after initiation of therapy. One anaplastic oligodendroglioma patient, who dropped the protocol during her second treatment cycle, has no evidence of tumor 28 months after recurrence. Received: 21 May 1997 / Accepted: 17 July 1997     

CD44v10 expression in the mouse and functional activity in delayed type hypersensitivity

We have described recently that expression of CD44 exon v10 (CD44v10) is down-regulated upon metastasis of squamous cell carcinoma, whereas it is up-regulated in skin metastases of malignant melanoma. The striking regulation of CD44v10 prompted us to generate a murine CD44v10-specific monoclonal antibody to define expression and possible functions of this particular CD44 variant isoform. In the mouse, expression of exon v10 was restricted to basal layers of the epidermis and squamous epithelium of the oral cavity, the esophagus, the omasum, glandular epithelium of the submandibular and the uterine gland, as well as subpopulations of bone marrow cells and activated lymphocytes. Expression started late during development, e.g., was not observed before day 16 of gestation and there was no evidence for developmental regulation of CD44v10 expression. Functional in vivo studies revealed that anti-CD44v10 had no effect on wound healing but inhibited edema and granuloma formation in delayed type hypersensitivity (DTH). Furthermore, lymphocyte-monocyte interactions could be inhibited by anti-CD44v10. Because a CD44v10 transfected tumour line did not show any distinct pattern of cell-matrix or cell-cell adhesion, the data point toward an involvement of CD44v10 in cell migration, possibly by acting as a target structure for cytokines/chemokines provided by the contacted partner cell. J. Cell. Physiol. 171:305–317, 1997. © 1997 Wiley-Liss, Inc.     

Poor transduction efficiency of human hematopoietic progenitor cells by a high-titer amphotropic retrovirus producer cell clone.

The transduction efficiency of human bone marrow CD34+ cells with supernatants from the retrovirus producer cell clone PA317/LGSN 16 was only one-fifth of that with supernatants from GP+ envAm12/LGSN 15, even though both producers had similar infection titers on 3T3 cells. PA317/LGSN 16-conditioned medium inhibited the proliferation of the bone marrow CD34+ cells, and this inhibitory effect was partially blocked by anti-transforming growth factor beta antibodies. These studies suggest that cytokine secretion plays a role in the suppression of retrovirus transduction of human CD34+ cells.     

Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor. II. Characterization of anti-idiotypic CTL lines and clones.

In an earlier communication we showed that idiotypic immunoglobulin (Id+ Ig) of a B cell hybrid, 2C3, can induce cytotoxic T lymphocytes (CTL) in the spleens of mice that are hyperimmunized with the irradiated tumor cells. To understand the extent of heterogeneity in the splenic CTL population, stable anti-idiotypic CTL lines and clones were established from 2C3-primed splenocytes. One representative CTL line A102 which exhibited the phenotype of CD3+, CD4-, and CD8+, has been maintained in long-term culture for more than 18 months. Cytotoxic specificity of A102 was determined by cold target inhibition assay using a panel of syngeneic and allogeneic B cell tumors. The CTL line A102 was highly cytotoxic to 2C3, only weakly to other syngeneic tumors, but not at all to allogeneic B cell tumor CH12. Furthermore, CTL-mediated cytolysis was significantly abrogated by blocking 2C3 cells with anti-idiotypic monoclonal and polyclonal antibodies. These results clearly show that 2C3 Id represents the immunodominant epitope(s) recognized by the CTL line A102. To isolate a highly Id-specific effector population, A102 was repeatedly subcloned by limiting dilution. One such clone 102.F5 exhibited considerable specificity toward Id+ 2C3 while another clone 102.E10 showed no such specificity in a competitive cytotoxicity assay. This was further confirmed by the inhibition studies with anti-Id mAb. Thus, hyperimmunization with irradiated 2C3 cells evokes a spectrum of anti-2C3 cytotoxic effector cells, of which a major population is reactive to the idiotypic determinants associated with 2C3 Ig.     

Decreased CXCR3+ CD8 T cells in advanced human immunodeficiency virus infection suggest that a homing defect contributes to cytotoxic T-lymphocyte dysfunction

To exert their cytotoxic function, cytotoxic T-lymphocytes (CTL) must be recruited into infected lymphoid tissue where the majority of human immunodeficiency virus (HIV) replication occurs. Normally, effector T cells exit lymph nodes (LNs) and home to peripheral sites of infection. How HIV-specific CTL migrate into lymphoid tissue from which they are normally excluded is unknown. We investigated which chemokines and receptors mediate this reverse homing and whether impairment of this homing could contribute to CTL dysfunction as HIV infection progresses. Analysis of CTL chemokine receptor expression in the blood and LNs of untreated HIV-infected individuals with stable, chronic infection or advanced disease demonstrated that LNs were enriched for CXCR3(+) CD8 T cells in all subjects, suggesting a key role for this receptor in CTL homing to infected lymphoid tissue. Compared to subjects with chronic infection, however, subjects with advanced disease had fewer CXCR3(+) CD8 T cells in blood and LNs. CXCR3 expression on bulk and HIV-specific CD8 T cells correlated positively with CD4 count and negatively with viral load. In advanced infection, there was an accumulation of HIV-specific CD8 T cells at the effector memory stage; however, decreased numbers of CXCR3(+) CD8 T cells were seen across all maturation subsets. Plasma CXCL9 and CXCL10 were elevated in both infected groups in comparison to the levels in uninfected controls, whereas lower mRNA levels of CXCR3 ligands and CD8 in LNs were seen in advanced infection. These data suggest that both CXCR3(+) CD8 T cells and LN CXCR3 ligands decrease as HIV infection progresses, resulting in reduced homing of CTL into LNs and contributing to immune dysfunction.     

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNγ-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2⁺/gp100⁺ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.Niels Schaft and Jan Dörrie contributed equally     

Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis

CD44 is the major hyaluronan cell surface receptor and functions as an adhesion molecule in many different cell types, including human breast epithelial cells. The coexpression of certain CD44 variants (CD44v), such as CD44v (v10/ex14), with CD44s (standard form) appears to be closely associated with human breast tumor metastasis. In this study we have established a stable transfection of CD44v (v10/ex14) cDNA into nontumorigenic human breast epithelial cells (HBL100) which contain endogenous CD44s. Our results indicate that coexpression of both CD44v (v10/ex14) and CD44s alters the following important biological properties of these cells: 1) there is a significant reduction in hyaluronic acid (HA)-mediated cell adhesion; 2) there is an increased migration capability in collagen-matrix gel; and 3) these cells constitutively produce certain angiogenic factors and effectively promote tumorigenesis in athymic nude mice. These findings suggest that coexpression of CD44v (v10/ex14) and CD44s may trigger the onset of cell transformation required for breast cancer development. J. Cell. Physiol. 171:152–160, 1997. © 1997 Wiley-Liss, Inc.     

Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.     

Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response

Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1(157-165)-specific CD8(+) cells, detected ex vivo with an A2/H-2K(b) chimeric class I tetramer. These NY-ESO-1(157-165)-specific CD8(+) cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1(157-165)-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.     

Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

Telomere length is abnormally short in the CD8(+) T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8(+) cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8(+) cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.     

A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.

A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways.     

Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.     

Infection of human hematopoietic progenitor cells using a retroviral vector with a xenotropic pseudotype

In an effort to determine if viral envelope type influences the infectivity of human hematopoietic progenitor cells with retroviral vectors, we have pseudotyped the retroviral vector N2, which confers G418-resistance, in either an amphotropic or xenotropic envelope. Vector titres obtained by the pseudotype procedure were nearly two orders of magnitude lower than the titer obtained when N2 was packaged using the amphotropic PA317 packaging cell line. Despite its low titer, xenotropically pseudotyped N2 generated G418-resistant hematopoietic colonies at levels approaching those observed after bone marrow was infected using vector packaged using PA317 cells. These results suggest that manipulations of vector envelope may lead to improvements in the level of infection of human hematopoietic stem cells.     

Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.     

Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells

The development of rapid, efficient, and safe methods for generating Ag-specific T cells is necessary for the clinical application of adoptive immunotherapy. We show that B cells stimulated with CD40 ligand and IL-4 (CD40-B cells) can be efficiently transduced with retroviral vectors encoding a model Ag, CMV tegument protein pp65 gene, and maintain high levels of costimulatory molecules after gene transfer. CTL lines specific for pp65 were readily generated in all four healthy CMV-seropositive donors by stimulating autologous CD8(+) T cells with these transduced CD40-B cells, both of which were derived from 10 ml peripheral blood. ELISPOT assays revealed that the CTL lines used multiple HLA alleles as restricting elements. Thus, CD40-B cells transduced retrovirally with Ag-encoding cDNA can be potent APC and facilitate to generate Ag-specific CTL in vitro.     

Enrichment of an antigen-specific T cell response by retrovirally transduced human dendritic cells.

The superior ability of dendritic cells (DC) in triggering antigen-specific T cell responses makes these cells attractive tools for the generation of antitumor or antiviral immunity. We report here an efficient retroviral transduction system for the introduction of antigens into DC. A retroviral vector encoding several CTL epitopes in a string-of-beads fashion in combination with the marker gene green fluorescence protein (GFP) was generated. Polyepitope transduced EBV-LCL could be isolated on the basis of GFP expression and were found to be sensitive to lysis by antigen-specific cytotoxic T cells, demonstrating that antigens encoded by the retroviral construct were stably expressed, processed, and presented in the context of HLA class I molecules. CD34(+) cells isolated from G-CSF mobilized peripheral blood were transduced with high efficiency (40-60%) with this retroviral construct. These cells could be considerably expanded in vitro and differentiated into mature DC without loss of the transduced antigen. DC transduced with the polyepitope constructs were able to mount a CTL response against an influenza epitope in the context of HLA-A2, demonstrating the antigen-specific CTL priming capacity of retrovirally transduced DC. Staining of the T cells with tetramers of HLA-A2 and the influenza virus peptide demonstrated a marked antigen-specific CTL enrichment after 2 in vitro stimulations using DC transduced with the polyepitope. However, additional in vitro stimulations of the T cells with transduced DC did not result in a further enrichment of tetramer staining cells.