Dendritic cell-induced apoptosis of human cytomegalovirus-infected fibroblasts promotes cross-presentation of pp65 to CD8+ T cells.

An efficient host response to human cytomegalovirus (HCMV) infection may depend on rapid sensing of the infection by the innate immune response prior to deployment of viral immunosubversive functions. Control of HCMV dissemination could be ensured by apoptosis of cells immediately following infection. In the present report, it is demonstrated that changes in the ratio of c-FLIP to FLICE contributed to early sensitivity of HCMV-infected MRC5 fibroblasts to tumour necrosis factor alpha (TNF-alpha), providing an innate response to infection. Dendritic cells (DCs) co-cultured with HCMV-infected MRC5 cells acquired the ability to secrete TNF-alpha in an amount sufficient to kill infected fibroblasts. Blockage of TNF-alpha binding to its receptor on MRC5 cells with soluble TNF-R reduced the number of dead, HCMV-infected fibroblasts ingested by DCs, thus highlighting the impact of the apoptotic state of infected cells for efficient loading of DCs. Those DCs loaded with antigens available early in infection, such as input virion-associated pp65, could then engage antigen processing for cross-presentation to specific CD8(+) T cells. Cross-presentation was impaired when MRC5 cells were treated with the pan-caspase inhibitor ZVAD before co-culture with DCs. Altogether, our data suggest that the innate killing capacity of DCs at the early stage of infection plays a role in the activation of anti-HCMV CD8(+) T cells.

p73-dependent apoptosis through death receptor: impairment by human cytomegalovirus infection.

The discovery of p73, a p53-related protein with various isotypes resulting from different promoter usage or splicing events, provided new insights into regulation of neurogenesis and tumorigenesis. Among p73 isoforms described thus far, TA-truncated molecules (DeltaN) appeared as key proteins according to their antagonistic activity against transcription factor activity of p53 family members. We previously showed that infection by human cytomegalovirus (HCMV) induced drug resistance and altered p53- and p73-dependent apoptosis of infected cells through accumulation of DeltaN-p73alpha. In accordance with the ability of p53 to induce apoptosis through death receptors, we asked whether p73 activation could compensate for p53 deficiency. We showed that p73 transcriptional activity sensitized cells to apoptosis through death receptors in a caspase-dependent pathway. Expression of the death-inducing signaling complex (DISC) proteins was unchanged, whereas p73 activation through either cisplatin treatment or ectopic overexpression induced up-regulation of Fas transcription and expression at cell surface. According to its ability to flood cells with DeltaN-p73alpha, HCMV inhibited p73-dependent Fas-mediated apoptosis, gaining an additional trick to favor its survival in the host cell. Owing to the involvement of p53- and p73-dependent death receptor signaling in development of the central nervous system, immune surveillance of neural cells, and sensitivity of tumors to drugs, our previous and present data prompt us to consider stabilization of DeltaN-p73alpha by HCMV as a possible mechanism in impairment of embryogenesis and in tumorigenesis.

Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Impaired killing of HCMV-infected retinal pigment epithelial cells by anti-pp65 CD8(+) cytotoxic T cells.

PURPOSE: Host defense against infection by human cytomegalovirus (HCMV) is ensured in great part by cytotoxic CD8(+) T lymphocytes (CTLs) directed against the tegument protein pp65. The hyperimmediate release of incoming pp65 into the major histocompatibility complex (MHC) class I pathway after fusion of the virus with the cell membrane provides a very early mechanism of defense. In retinal pigment epithelial (RPE) cells HCMV is known to enter through endocytosis. This study was conducted to determine whether this means of penetration into the cells would allow the virus to elude immune surveillance. METHODS: Infection of RPE cells with HCMV AD169 was performed for 6 hours, 48 hours, and 8 days. Expression of intracellular pp65 in RPE cells and in the astrocytoma reference cell line U373MG was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. Killing of both HCMV-infected cell lines by HLA-A2-restricted CD8(+) CTLs directed against pp65 was monitored by (51)Cr-release assays. RESULTS: RPE cells were not lysed by CTLs directed against incoming pp65, contrary to U373MG. Moreover, both cell lines were not killed by anti-pp65 CTLs later after infection, because of the MHC class-I-downregulating effect of HCMV unique short (US2-11) proteins. CONCLUSIONS: In RPE cells, both HCMV entry through endocytosis and the immunosuppressive effect of US proteins could allow the virus to evade immune surveillance at any stage of infection, which could promote viral spreading within the retina.