We adoptively transferred donor-derived cytomegalovirus (CMV)-specific T-cell lines into 8 stem cell transplant recipients lacking CMV-specific T-cell proliferation. All patients, of whom one was infected by a CMV strain that was genotypically ganciclovir resistant, had received unsuccessful antiviral chemotherapy for more than 4 weeks. CMVspecific lines had been prepared by repetitive stimulation with CMV antigen, which increased the percentage of CMV-specific T cells and ablated alloreactivity completely even against patients mismatched for 1 to 3 HLA antigens. After transfer of 10 7 T cells/m 2 at a median of 120 days (range, 79-479 days) after transplantation, no side effects were noticed. Despite cessation of antiviral chemotherapy, the CMV load dropped significantly in all 7 evaluable patients, with a maximal reduction after a median of 20 days (range, 5-31 days). In 2 patients with high virus load, the antiviral effect was only transient. One of these patients received a second T-cell infusion, which cleared the virus completely. At a median of 11 days after transfer, CMV-specific T-cell proliferation was demonstrated in 6 patients, and an increase in CMV-specific CD4 ؉ T cells was demonstrated in 5 patients. In 6 patients, 1.12 to 41 CMV-specific CD8 ؉ T cells/L blood were detected at a median of 13 days after transfer, with an increase in all patients lacking CMV-specific CD8 ؉ T cells prior to transfer. Hence, anti-CMV cellular therapy was successful in 5 of 7 patients, whereas in 2 of 7 patients, who received an intensified immune suppression at the time of or after T-cell therapy, only transient reductions in virus load were obtained. (Blood. 2002;99: 3916-3922)

The susceptibility of monocyte-derived immature dendritic cells (DC) to infection by various strains of human cytomegalovirus (HCMV) was analysed. Immature DC were generated by incubation of peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor for 7 days and were characterized by a CD1aM/ CD40M/CD80M/CD86M/HLA-DRM/CD14N phenotype. Viral antigen expression and production of infectious progeny virus were analysed in infected immature DC cultures. Immature DC were 80-90 % susceptible to HCMV strains that had been propagated in endothelial cell culture, whereas the infection rate was negligible with fibroblastadapted HCMV strains. Immature DC infection resulted in expression of viral immediate early, early and late genes. Productive infection was proven by the detection of infectious virus in singlestep growth curves and in infectious centre assays. It is concluded that HCMV might interfere with the host immune reaction by permissive, lytic infection of immature DC.

Summary. The hypothesis that productive infection of monocyte-derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested in this study. DCs were infected with 60-80% efficiency by HCMV strain TB40/E. Infected versus uninfected cells were analysed by fluorescence-activated cell sorting and by immunocytochemistry for surface expression of major histocompatibility complex (MHC) and co-stimulatory molecules as well as cytokine secretion during the 3 d after infection. The immunostimulatory capacity of these cells was measured by mixed leucocyte reaction. In spite of the fact that HCMV infection of DCs induced an increased release of tumour necrosis factor-a (TNF-a) and a decreased interleukin 10 (IL-10) production, expression of MHC class I and II, as well as CD40 and CD80 molecules, were downregulated on infected DCs. The mixed leucocyte reaction showed significantly reduced immunostimulatory capacity of infected DC cultures. Simultaneous detection of MHC antigens and virus antigens by double immunofluorescence revealed that downregulation occurred only on infected cells, but not on uninfected bystander cells. These findings demonstrate on a single cell level, together with the marked downregulation of MHC and co-stimulatory molecules in the presence of high TNF-a and low IL-10 levels, a direct inhibitory effect of HCMV on antigen presentation by immature DCs independent of soluble mediators.

Summary. 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 peptidespecific T cells of up to 28% and . 90% of CD8 1 T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of . 1 Â 10 9 HCMV-specific T cells was possible after 6±7 weeks when cultures were initiated with 1± 5 Â 10 6 responder cells. Thus, the approach with peptidepulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.

A central aspect of human cytomegalovirus (HCMV) pathogenesis is the interaction of the virus with different antigen-presenting cell (APC) types of the host. In principle, a number of various cell types have the potential of antigen presentation when MHC II expression is induced by appropriate stimuli. The most potent antigen presenters are monocytes/macrophages and dendritic cells (DCs), therefore called professional APCs. Interestingly, these cells seem to be targets of productive HCMV infection. The susceptibility of the monocyte/macrophage system has been analyzed intensively during the past decade. Investigation of the role of DCs during HCMV infection, however, has begun only recently.