Healthy CMV-seropositive Donors Research Articles

Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.

TNFR2 Is Expressed Preferentially By Late Differentiated CD8 T-Cells and Can be Triggered By TNFR2-Specific Ligand to Induce Cell Death of Recently Activated Antigen-Specific T Cells: A Possible Role of TNFR2 in T-Cell Deflation

Recent evidence suggests that Tumor Necrosis Factor (TNF) can selectively kill antigen-specific autoreactive CD8+ T-cells through engagement with TNF Receptor 2 (TNFR2) (1). Within the immune system, TNFR2 expression is restricted to subsets of T-cells, a profile which is in marked contrast to the ubiquitous pattern of expression of TNFR1. However, the spectrum and physiological significance of TNFR2 expression by CD8+ T-cell subpopulations is unknown.In this study we analysed the expression of TNFR2 by CD8 T-cell subsets isolated from normal healthy donors by flow cytometry. In addition, in order to understand the physiological significance of TNFR2 expression on recently activated T cells, we further studied expression on CMV-specific CD8 T-cells which expanded in stem cell transplant patients in response to episodes of CMV reactivation. The expression of TNFR2 was compared to that of other common gamma chain receptors including IL2R and IL7R, and to the expression of a receptor for inflammatory cytokine IL6.TNFR2 expression was found to increase during differentiation of CD8+ T cells. In particular, TNFR2 expression was seen on 6.5% of naïve, 14.6% of central memory, 37.9% of effector memory and 45.2% of CD45RA-revertant effector memory (TEMRA) CD8+ T cells. In contrast, common gamma chain cytokine receptor expression was skewed towards less differentiated T-cell subsets. For example, IL-7R was expressed by 63% of central memory populations but only 18.4% of the TEMRA subset. Comparable expression of IL2R was 12.1% on TCM and 2% on TEMRA. Of interest, IL-6 receptor expression was predominantly expressed by naïve CD8 T-cells (69.5%). In support of these results, we went on to show that expression of TNFR2 was inducible on primary T cells following activation with anti-CD3 and IL-2 in vitro. Healthy CMV seropositive donors had a larger median number of CD8+ T cells expressing TNFR2 (53%) in comparison to CMV seronegative donors (15%), (p<0.0001), consistent with the known accumulation of differentiated T-cells within CMV seropositive individuals.The expression of TNFR2 was then examined on CMV-specific CD8 T-cells which were undergoing acute expansion in response to viremia in six haemopoietic stem cell transplant patients. The expansion of CMV-specific CD8 T-cells was accompanied by an increase in the intensity of TNFR2 expression which later decreased during the retraction of antigen-specific T-cells during resolution of viremia. In order to explore the functional significance of TNFR2 expression, T-cells isolated from healthy donors were treated with recombinant TNFR2-specific ligand. This induced cell loss ranging from 13% to 60% of all CD8 T-cells in relation to untreated control cells, with selective depletion of the TNFR2+ population. A similar proportion of CMV-specific T-cells from transplant patients were eliminated by ex vivo stimulation of TNFR2.In conclusion our work shows that TNFR2 expression increases during differentiation of CD8+ T cells. In addition, we were able to utilize virus-specific T cells from SCT patients to show that expression is increased during the acute response to stimulation with antigen. We also provide evidence that TNFR2 activation can lead to the partial elimination of antigen-specific CMV-specific T-cells and it may thus play an important role in the ‘deflation’ of a pathogen-specific T-cell immune response following resolution of infection. These data suggest that TNFR2 expression may act as a ligand to signal activation-induced cell death in late differentiated populations of CD8+ T cells. Further investigations are required to assess the molecular pathways of TNFR2 signalling that are activated following receptor ligation in vivoand whether or not these are disrupted in disorders associated with chronic CD8+ T cell lymphproliferation.(1) L. Ban et al, PNAS 2008, 105: 3644 DisclosuresNo relevant conflicts of interest to declare.

Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.

BackgroundThe adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.MethodsTo obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ+ T cells exceeded 0.03% of CD3+ lymphocytes as determined by IFN-γ cytokine secretion assay.ResultsStarting with low concentration of IFN-γ+ T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ+CD3+ T cells (1.42 × 106, 0.05 × 106, and 1.15 × 106) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8+ than CD4+ memory T cells, both of which were effectively enriched to a total of 81.0% CD8+IFN-γ+ and 38.4% CD4+IFN-γ+ T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.ConclusionsThe generation of antiviral CD4+ and CD8+ T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0336-5) contains supplementary material, which is available to authorized users.

Comparison of the Effect of Standard and Novel Immunosuppressive Drugs on CMV-Specific T-Cell Cytokine Profiling

Data on how different immunosuppressive drugs affect cytomegalovirus (CMV)-specific T-cell responses may help guide more rational modification of immunosuppression in patients with CMV replication. We assessed the in vitro effects of individual standard and novel immunosuppressive drugs on a broad range of CMV-specific T-cell responses. Peripheral blood mononuclear cells from healthy CMV-seropositive donors were preincubated with serial dilutions of tacrolimus, mycophenolate (MPA), sirolimus, tofacitinib, and belatacept. CMV-pp65 or CMV-pp72 peptide pools were used for stimulation. CMV-specific cytokine (Th1 and Th2) and chemokine responses were determined (a total of 5400 measurements). P<0.01 was set as significant. After CMV stimulation, dose-dependent suppression of Th1, Th2, and chemokines was seen, but significant differences between drugs were present. For example, tacrolimus was more potent in inhibiting CMV-specific Th1 cytokines versus Th2, whereas MPA preferentially inhibited Th2 cytokines. In a comparison of the relative potency of each drug at different dosing ranges, tacrolimus had the strongest Th1 inhibitory effect (median inhibition of interferon-γ at 97.5%; P=0.004-0.008) followed by sirolimus (median inhibition at 82.4%). The remaining agents (MPA, belatacept, and tofacitinib) had less apparent dose-dependent effects on interferon-γ (belatacept median inhibition at 21.5%; P=0.004 vs. tacrolimus). Immunosuppression-specific and dose-dependent reductions in CMV-specific cytokine release were observed with significant differences in Th1 versus Th2 profiles and in relative potency of the drugs.

Allo-Reactive Potential of Multi-Virus Specific Cytotoxic T Cell Lines Recognizing Adenovirus Hexon, Cytomegalovirus pp65 and Epstein Barr Virus.

Abstract 5121 IntroductionCytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are major pathogens after allogeneic hematopoietic stem cell transplant. The current antiviral therapy is associated with significant myelosuppression, impaired hematopoietic reconstitution and consequently, higher rates of other infections. Adoptive immunotherapy utilizing allogeneic donor-derived virus specific cytotoxic T cell lines (CTL) has been shown to effectively treat refractory viral infections. Although such lines carry the theoretical risk of allo-reactivity and the resultant graft versus host disease, this complication has not been widely reported in recipients of single virus specific cell lines. To improve the efficiency of this therapeutic modality we have developed an approach to generate a single cell line that recognizes all three of these clinically relevant viruses while also having greatly reduced allo-reactive potential. MethodsPeripheral blood mononuclear cells (PBMC) from 3 healthy CMV seropositive donors were primed 3 times with autologous monocyte-derived dendritic cells pulsed with overlapping pools of pentadecapeptides spanning the antigen coding region of highly conserved regions of the Ad hexon protein and/or pools spanning the entire CMV pp65 protein. The lines were expanded for minimally one additional week using peptide loaded EBV transformed B cells (BLCL) as antigen presenting cells (APC). CMV, Ad and EBV anti-viral activity was monitored weekly by chromium release assay and the specificity of the lines was controlled by varying the peptide pools used for subsequent priming. The IFN-gamma ELISPOT assay was used to identify single peptides and to determine HLA restriction. The frequency and subset of single and pool peptide-reactive cells was determined using IFN-gamma intracellular cytokine production by flow cytometry. Fresh PBMC and established lines were additionally primed with fully allogeneic stimulators and response measured using intracellular cytokine production. ResultsEach of the 3 lines had robust proliferation and CTL activity against CMV and Ad peptide-pulsed targets. The lines consisted of 44.5-82.4% CD3+CD8+cells and 18.1-56.7% CD3+CD4+cells. CMV induced IFN-gamma response in 4.3-76.6% of CD3+CD8+cells and 0.4-47.5% of CD3+CD4+cells. Ad induced 1.4-43.9% of CD3+CD8+cells and 6.9-45.7% of CD3+CD4+cells to produce IFN-gamma. The 3 lines recognized 2-4 single CMV peptides and 2-6 single Ad peptides. The reactivity against these viral peptides was HLA restricted. At the time of testing none of the lines showed a strong EBV response. However, EBV reactivity could be detected with 1-2 additional primings, indicating that these precursors were present. Compared to fresh PBMC from the same donors, there was a 34.0±5.6 fold decrease in the frequency of alloreactive CD3+CD8+cells demonstrated in the intracellular cytokine assay. ConclusionsGeneration of multi-viral specific CTL in a single culture is feasible so long as antigen stimulation is controlled and this approach will greatly reduce the time, cost and number of cells required to restore viral immunity for patients receiving allogeneic stem cell transplant. The risk of allo-reactivity in these lines is greatly reduced compared to fresh PBMC. This makes adoptive cell therapy more practical, easier to implement and safer. DisclosuresNo relevant conflicts of interest to declare.

Cytokine Production Signatures Are Intrinsically Associated with Human T Cell Maturation Stages.

Our recent published studies have suggested that impaired immune reconstitution after allogeneic stem cell transplantation is associated with a greater proportion of circulating late memory T cells, defined phenotypically. To characterize the relationship between immunophenotypic markers of T cell maturation and functional attributes of T cells, we optimized an 8-color, 10-parameter cytokine flow cytometry (CFC) approach and studied T cells from healthy donors. T cells were exposed to stimuli that both bypass (PMA:Ionomycin, P:I) and signal through the T cell receptor (Staph enterotoxin B, SEB; and CMV pp65 peptide pools) and stained with CD45RA and CD27 to demarcate naïve (N, CD45RA+CD27+), and three progressively mature memory subsets: M1 (CD45RA−CD27+), M2 (CD45RA−CD27−), M3 (CD45RA+CD27−) CD4+ and CD8+ T cells. We assessed the 15 possible combinations of cells producing IL-2, IFNγ, TNFα, and MIP1β alone or in combination within maturation subsets. When we initially studied the production of individual cytokines, we found that the bulk of IL-2 production was produced by activated N and M1 cells in both CD4 and CD8 lineages. In contrast, IFNγ and MIP1β were produced by later maturation stages (M2 and M3) of CD4+ and CD8+ T cells. In contrast to the polarized production of individual cytokines at the extremes of the maturation spectrum, early and middle memory cells (M1 and M2) cells produced heterogeneous combinations of cytokines (e.g, IL-2+IFNγ+ and TNFα+MIP-1β+ cells). We also found that IL-2/IFNγ co-producing cells, shown to be particularly important for the control of chronic viral pathogens, exist mainly in the M1 and M2 stages, and not the M3 stage. The above results were consistent with both P:I and SEB stimulation, and across several healthy subjects tested. Our cross-sectional results were confirmed by in vitro differentiation experiments, wherein we sorted naive (CD45RA+CD27+) CD4+ and CD8+ T cells and demonstrated that their function evolved as expected following stimulation with PHA and IL-2, which resulted in differentiation into M1 and M2 cells in culture. Finally, we stimulated PBMC from healthy CMV-seropositive donors with a CMV pp65 peptide mixtures and examined maturation and cytokine production. Consistent with prior observations, most CMV-specific T cells were M2 and M3 cells. Surprisingly, the most abundant functional subsets consisted of cells producing either MIP1β alone or MIP1β and other cytokines. Consistent with our results following polyclonal stimulation, we found that IL-2/IFNγ co-producing CMV specific T cells existed in M1 and M2, but not in the M3 stage. These results demonstrate that:1.Functional cytokine signature is strongly associated with T cell maturation stage;2.Nearly all IL-2 production occurs in N, M1 and M2 cells;3.M3 cells produce little IL-2, but substantial amounts of MIP1β;4.IL-2/IFNγ co-production is rare in M3 cells, but exist in M1 and M2 cells, perhaps suggesting why late stage skewing of memory T cells may lead to functional T cell impairment in vivo; and5.that MIP1β is the most abundant cytokine produced by CMV-specific T cells.Overall, our results demonstrate that phenotypically defined maturation stages in both CD4+ and CD8+ T cell lineages are strongly associated with functional signatures irrespective of stimulus type, and that multidimensional analyses of human T cells may be beneficial when assessing human T cells in the clinical setting.

Autologous Designer Antigen-presenting Cells by Gene Modification of T Lymphocyte Blasts With IL-7 and IL-12

An effective immune response to antigen requires professional antigen-presenting cell (APC), which not only present antigen, but also provide costimulation and cytokines (eg, IL-12) that drive T cell differentiation down the appropriate effector pathway (Tc1/TH1). For T cell-based immunotherapy protocols, the availability of large numbers of autologous professional APC is a major limitation because professional APC do not proliferate in vitro. T cells themselves can proliferate exponentially in vitro and have the ability to present antigen. They can also express costimulatory molecules after activation. Therefore, we hypothesized that if activated T cells were genetically modified to express proinflammatory cytokines required to polarize T cells toward a Tc1 response, they could fulfill the requirements for an abundant, autologous APC. To test this potential, T cells were activated by CD3/CD28 antibodies and pulsed with model HLA-A2+ peptides derived from CMVpp65, MAGE-3, and MART-1. Activated T-APC readily reactivated CD8 pp65 memory T cells from healthy CMV seropositive donors; however, the activation of MAGE-3 and MART-1-specific CD8 T cells required both IL-7 and IL-12, which could be provided either exogenously or by genetic modification of the T-APC. Responder T cells could be expanded to large numbers with subsequent stimulations using activated, peptide-pulsed T-APC and IL-2. Tumor antigen-specific T cell lines killed both peptide-pulsed target cells and tumor cell lines. Thus, T cells provide a platform for the generation of autologous APC that can be customized to express both antigens and therapeutic molecules for the induction of antigen-specific T cell immunity.

Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells.

Cytomegalovirus (CMV) infection has been linked to inflammation-related disease processes in the human host, including vascular diseases and chronic transplant rejection. The mechanisms through which CMV affects the pathogenesis of these diseases are for the most part unknown. To study the contributing role of the host immune response to CMV in these chronic inflammatory processes, we examined endothelial cell interactions with peripheral blood mononuclear cells (PBMC). Endothelial cultures were monitored for levels of fractalkine induction as a marker for initiating the host inflammatory response. Our results demonstrate that in the presence of CMV antigen PBMC from normal healthy CMV-seropositive donors produce soluble factors that induce fractalkine in endothelial cells. This was not observed in parallel assays with PBMC from seronegative donors. Examination of subset populations within the PBMC further revealed that CMV antigen-stimulated CD4(+) T cells were the source of the factors, gamma interferon and tumor necrosis factor alpha, driving fractalkine induction. Direct contact between CD4(+) cells and the endothelial monolayers is required for this fractalkine induction, where the endothelial cells appear to provide antigen presentation functions. These findings indicate that CMV may represent one member of a class of persistent pathogens where the antigen-specific T-cell response can result in the induction of fractalkine, leading to chronic inflammation and endothelial cell injury.

562. Human T-Cells Modified as Antigen Presenting Cells Are Superior to Professional APC in Stimulating T-Cell Responses

Adoptive transfer of T-cells expressing herpes simplex thymidine kinase (HSV-TK) induces an immune response against the transgene in HIV patients and in allogeneic stem cell transplant recipients' resulting in eradication of transferred cells. The elimination of transferred cells is mediated by HSV-TK-specific CD8+ T-cells. As a vigorous T-cell response to the modified T-cells has been detected in clinical trials we reason that genetically modified human T-cells may represent a novel class of antigen presenting cells for eliciting CD8+ T-cell responses. In the current study human T-cells are oncoretrovirally transduced with a model antigen, CMV tegument pp65 protein. The ability of these genetically modified cells to function as antigen presenting cells (T-cellAPC) was investigated. Both CD4+ and CD8+ T-cells genetically modified with CMV pp65 gene can stimulate CMV-specific T cells as determined by IFN-γ production of activated T-cells. Furthermore, the potential of T-cellAPC to induce and expand CMV pp65-specific CTLs was addressed and compared to CD40-B–mediated CTL expansion. Both CD40-B and T-cellAPC induced and expanded simultaneously antigen-specific CTLs in vitro to more than one epitope with different HLA restriction alleles from healthy CMV seropositive donors. After only one round of seven days in vitro stimulation with T-cellAPC, CMV pp65-restricted T-cells were expanded up to 97-fold as determined with tetramer staining. In contrast, CTL cultures stimulated with autologous CD40-BAPC expanded only up to 41-fold. The observed difference in expansion potential mediated either with T-cellAPC or CD40-BAPC could result in phenotypic functional differences in the Ag-specific CTL populations. To address this issue the expression of co stimulation and chemokine receptors on tetramer positive cells was determined. Despite differential expression profile of costimulatory ligands on T-cellAPC and CD40-BAPC, both expanded CTL populations are of effector/memory phenotype and efficiently kill CMV-infected fibroblasts. Thus, T-cells transduced with oncoretroviral vectors to express full-length antigens are potent APC and can potentially be utilized as a new vaccine strategy in vivo against viral pathogens and cancer.

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.

Human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy maintain activated CD8+ T cell subsets as a strong adaptive immune response to cytomegalovirus.

CD8(+) T lymphocyte function specific for human cytomegalovirus (CMV) was evaluated in 14 patients infected with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy (HAART) and 26 CMV-seropositive donors without HIV infection. Fifty-seven percent of the HIV-infected group had CMV-specific cytolytic activity in freshly isolated peripheral blood mononuclear cells (PBMC) against targets expressing CMV pp65. Both interferon (IFN)-gamma secretion by CD8(+) T cells and the frequency of human leukocyte antigen (HLA)-tetramer-positive T cells in HLA-A*0201-positive HIV-infected subjects correlated with CMV-specific cytolysis. In contrast, PBMC from healthy CMV-seropositive donors did not have either measurable CMV-specific cytolysis or secretion of IFN-gamma without in vitro stimulation. The T helper response to CMV antigens was vigorous in healthy CMV-seropositive donors but low in the cohort of HIV-infected patients. Potent CD8(+) cytotoxic T lymphocyte responses to CMV in HIV-infected patients receiving HAART is the converse of what is found in healthy CMV-seropositive subjects and may be the predominant adaptive immune response against CMV in HIV-infected patients.

Simultaneous Ex Vivo Expansion of Cytomegalovirus and Epstein-Barr Virus–Specific Cytotoxic T Lymphocytes Using B-Lymphoblastoid Cell Lines Expressing Cytomegalovirus pp65

AbstractCytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8+ CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.