Human cytomegalovirus (HCMV) genetic determinants of endothelial-cell tropism and virus transfer to leukocytes (both polymorphonuclear and monocyte) have been recently identified in the UL131-128 genes. Here it is documented that the same genetic determinants of HCMV are responsible for monocyte-derived dendritic-cell (DC) tropism, i.e. all endotheliotropic and leukotropic strains of HCMV are also DC-tropic (or dendrotropic). In fact, all recent clinical HCMV isolates and deletion mutants sparing the UL131-128 locus as well as the endotheliotropic revertants AD169 and Towne were able to productively infect DC following co-culture with infected endothelial cells. On the contrary, the same clinical isolates extensively propagated in human fibroblasts, the UL131-128 deletion mutants and the reference laboratory strains were not. Peak extracellular virus titres in DC were reached 4-7 days post-infection (p.i.). Viral proteins pp65 and p72 were detected 1-3 h p.i., involving the great majority of DC 24 h p.i., while gB was abundantly detected 96 h p.i., when a cytopathic effect first appeared. Infection of DC with cell-free virus released into the medium could only be achieved with HCMV strains extensively adapted to growth in endothelial cells, reaching the peak titres 10 days p.i. DC infected for 24 h with cell-free virus and incubated for 16 h with autologous peripheral blood mononuclear cells were found to act as a potent stimulator of both HCMV-specific CD4 + -and CD8 + -mediated immune responses, as determined by cytokine flow cytometry. DC incubated with inactivated crude whole viral antigen preparations were only capable of eliciting a significant CD4 + -mediated immune response.

Absolute and human cytomegalovirus (HCMV)-specific CD4+ and CD8 + T-cell counts were monitored in 38 solid organ (20 heart, 9 lung and 9 kidney) transplant recipients during the first year after transplantation by a novel assay based on T-cell stimulation with HCMV-infected autologous dendritic cells. According to the pattern of T-cell restoration occurring either within the first month after transplantation or later, patients were classified as either early (n = 21) or late responders (n = 17). HCMV-specific CD4 + and CD8+ T-cell counts were consistently lower in late compared to early responders from baseline through 6 months after transplantation. In addition, in late responders, while HCMV infection preceded immune restoration, HCMV-specific CD4 + restoration was significantly delayed with respect to CD8 + T-cell restoration. The number of HCMV-specific CD4 + and CD8+ T-cells detected prior to transplantation significantly correlated with time to T-cell immunity restoration, in that higher HCMV-specific T-cell counts predicted earlier immune restoration. Clinically, the great majority of early responders (18/21, 85.7%) underwent self-resolving HCMV infections (p = 0.004), whereas the great majority of late responders (13/17, 76.5%) were affected by HCMV infections requiring antiviral treatment (p = <0.0001). Simultaneous monitoring of HCMV infection and HCMV-specific T-cell immunity predicts T-cell-mediated control of HCMV infection.

The development of adaptive T cell immunity after primary HCMV infection appears to be a complex and slow process until a memory T cell response develops. The T cell immune response appears to influence vertical HCMV transmission.

We investigated immune reconstitution against human cytomegalovirus (HCMV) in 57 hematopoietic stem cell transplant (HSCT) recipients, aged 1 to 24 years, through a novel method combining T-cell stimulation by HCMV-infected autologous dendritic cells with simultaneous cytometric quantification of HCMVspecific, IFN␥-producing CD4 ؉ and CD8 ؉ T cells. Lymphoproliferative response (LPR) to HCMV antigens was also determined. Patients were stratified into 2 groups according to HCMV serostatus, comprising 39 HCMV-seropositive (R ؉ ) and 18 HCMV-seronegative (R ؊ ) patients who received a transplant from a seropositive donor. Recovery of both HCMVspecific CD4 ؉ and CD8 ؉ T-cell immunity occurred in all 39 R ؉ patients within 6 months and in 6 (33%) of 18 R ؊ patients within 12 months. In R ؉ patients, the median numbers of HCMV-specific CD8 ؉ and CD4 ؉ T cells were significantly higher than those of healthy controls, starting from days ؉60 and ؉180, respectively. In R ؊ patients, the median numbers of HCMV-specific T cells were consistently lower than in R ؉ patients. LPR was delayed compared with reconstitution of IFN␥-producing T cells. Patients with delayed specific immune reconstitution experienced recurrent episodes of HCMV infection. HCMV seropositivity of young HSCT recipients is the major factor responsible for HCMV-specific immune reconstitution, irrespective of donor serostatus, and measurement of HCMVspecific T cells appears useful for correct management of HCMV infection. (Blood.