Dendritic-cell infection by human cytomegalovirus is restricted to strains carrying functional UL131–128 genes and mediates efficient viral antigen presentation to CD8+ T cells

Giuseppe Gerna1, Elena Percivalle1, Daniele Lilleri1, Laura Lozza1, Chiara Fornara1, Gabriele Hahn2, Fausto Baldanti1 and M. Grazia Revello1

1 Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
2 Max von Pettenkofer Institut, Abteilung Virologie, LMU-München, Germany

Correspondence
Giuseppe Gerna
g.gerna{at}smatteo.pv.it


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human cytomegalovirus (HCMV) endotheliotropic clinical isolates have been reported to productively infect monocyte-derived immature dendritic cells (DC), whereas laboratory-adapted HCMV strains appear deprived of this property (Riegler et al., 2000). While confirming those results, we have been able to produce evidence that all fresh HCMV clinical isolates share both biological properties of endothelial-cell (EC) tropism (endotheliotropism) and virus transfer to leukocytes (Gerna et al., 2002a, c). We define endotheliotropism as the property of HCMV isolates to productively infect (Gerna et al., 2002c) human umbilical vein endothelial cells (HUVEC), while we define virus transfer to leukocytes as the property of HCMV isolates to be transferred to leukocytes (either polymorphonuclear leukocytes, PMNL, or monocytes) following co-culture (Revello et al., 1998) with infected cells (either endothelial or fibroblast cells). Thus, endotheliotropic and leukocyte transmissible strains will be referred to as Huv+ Leuk+. The extensive propagation in human embryonic lung fibroblasts (HELF) of clinical isolates led to the selection of non-endotheliotropic and non-leukotropic viral variants (Revello et al., 2001). These strains will be referred to as Huv Leuk.

More recently, our groups, based on the cloning in bacterial artificial chromosome (BAC) of the genome of a clinical HCMV isolate (FIX-BAC) and the systematic mutagenesis of the reconstituted virus referred to as RV-FIX (Hahn et al., 2002), have been able to identify in the UL131–128 genes the genetic region of HCMV driving both endotheliotropism and virus transfer to leukocytes (Hahn et al., 2004).

Here, we document that only viral variants or RV-FIX mutants with a Huv+ Leuk+ phenotype are also DC-tropic (DC+), following co-culture of DC with HUVEC infected with the relevant strains. DC-tropism is defined as the property of HCMV to productively infect DC. These data lead to the conclusion that UL131–128 gene products are required not only for endotheliotropism and virus transfer to leukocytes, but also for DC-tropism. In addition, a potential immunological application of these virologic findings to the study of HCMV-specific T-cell-mediated immune response is preliminarily reported by showing the HCMV-specific CD4+ and CD8+ T-cell responses to HCMV-infected DC.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Generation and culture of monocyte-derived immature DC.
Peripheral blood mononuclear cells (PBMC) were separated by Lymphoprep (Axis-Shield PoC AS) density-gradient centrifugation of peripheral blood from healthy donors. Following 3 h adherence of 1·0x107 PBMC per well in 6-well plates, non-adherent cells were removed. Medium was RPMI supplemented with 10 % fetal bovine serum (FBS). Adherent cells were gently washed once with RPMI-10 % FBS and then cultured with RPMI-10 % FBS supplemented with 800 U human recombinant granulocyte macrophage-colony-stimulating factor ml–1 (GM-CSF, Leucomax; Novartis) and 500 U human recombinant IL-4 ml–1 (R&D Systems) for 5–7 days, according to a reported procedure (Sallusto & Lanzavecchia, 1994). Following this procedure, more than 90 % adherent cells belonged to the immature DC phenotype (CD1a+, HLA-DRlow, CD14, CD83, CD86low, HLA-ABC+, CD40+).

HCMV strains, deletion mutants, natural variants and revertants.
The following two groups of HCMV strains were tested for their ability to infect and replicate productively in DC. Group A consisted of Huv+ Leuk+ HCMV strains grown in HUVEC (Hahn et al., 2004) and included: (i) four recent clinical isolates adapted to growth in HUVEC (VR1814, VR3480, VR6110 and VR6952); (ii) RV-FIX and RV-FIX deletion mutants missing single genes outside the UL131–128 locus (RV-FIX {Delta}UL45, {Delta}UL127, {Delta}UL132); and (iii) HCMV revertants (VR1814 rev, VR3480 rev, AD169 rev and Towne rev) which had reacquired both EC-tropism and leukocyte transmissibility after loss of both properties in HELF (Gerna et al., 2002b, 2003b). Group B consisted of Huv Leuk HCMV strains grown in HELF and included: (i) clinical isolates extensively propagated (Revello et al., 2001) in HELF (VR1814 and VR3480); (ii) laboratory strains (AD169 and Towne); and (iii) RV-FIX deletion mutants missing either the entire UL131–128 locus or each one of the three ORFs (Hahn et al., 2004). In detail, RV-FIX {Delta}UL132–128 (lacking the UL131–128 region including a predicted polyA signal of UL132, thus knocking out UL132–128), RV-FIX {Delta}UL131K, RV-FIX {Delta}UL130 and RV-FIX {Delta}UL128 (Hahn et al., 2004) had been shown to have lost both EC-tropism and virus transfer to leukocytes (both monocytes and PMNL).

Restriction fragment length polymorphism (RFLP) analysis.
Genomic regions UL54 (DNA Pol), UL55 (gB), UL123 (IE-1) and ULb' (UL144 to UL148) of RV-FIX at passages 14, 51 and 175 on HUVEC were amplified by PCR using the primer pairs previously reported (Revello et al., 2001; Gerna et al., 2003b) and then cleaving PCR products with two to four of the following endonucleases: HaeIII, MspI, HinP1I, AluI and BstUI (New England Biolab). RFLP patterns were compared by agarose gel electrophoresis.

DC infection by co-culture.
Immature DCs were counted and 2·0x106 cells were co-cultured overnight with HUVEC grown in a T12 flask and infected with Huv+ Leuk+ HCMV strains or mutants, at an m.o.i. of 5. Similarly, immature DCs were co-cultured with HELF infected with Huv Leuk HCMV strains or mutants. Co-cultured DCs were removed from infected cell monolayer by gentle pipetting and then washed and migrated for 4 h in a 8 µm-pore-size Transwell chamber (Costar) inserted into wells containing 10–7 M of the chemotactic peptide FMLP (Sigma). DCs were migrated to remove infected HUVEC or HELF detached from the cell monolayer. Migrated cells were washed five times and counted. In general, 3·0x105–5·0x105 DCs suspended in RPMI-10 % FBS supplemented with GM-CSF were seeded into one well of a 24-well microplate. The supernatant from the last washing (considered the 24 h harvest) and supernatants from DC cultures collected 4 and 7 days after onset of co-culture were titrated for virus infectivity. Tenfold dilutions of each harvest were centrifuged onto HELF monolayers, grown in shell vials, for 40 min at 600 g. The following day, coverslips were stained for the HCMV major immediate-early protein p72 (Gerna et al., 1990) using a pool of mAbs reactive to different epitopes of p72 (Gerna et al., 2003a). In addition, approximately 5·0x104 DCs were cytocentrifuged onto a glass slide and stained for pp65 and p72 at 3 and 24 h, and for p72 and gB at 4, 7 and 10 days p.i., using the relevant mAbs and the immunofluorescence technique (Gerna et al., 2003b; Hahn et al., 2002).

DC infection with cell-free HCMV preparations.
When DCs were infected with cell-free Huv+ Leuk+ HCMV preparations (obtained after virus adaptation to growth in HUVEC) or Huv Leuk HCMV strains (propagated in HELF), about 1·0x106 immature DCs were incubated overnight with clarified medium from infected HUVEC or HELF cultures, respectively, at an m.o.i. of 5. Cells were washed five times, counted and cultured in one well of a 24-well microplate using RPMI-10 % FBS supplemented with 800 U GM-CSF ml–1. The supernatants from the last washing (harvest of day 1) and supernatants collected at 4, 7, 10, 14 and 18 days p.i. were titrated for virus infectivity, as reported above. In parallel, at each time point, 5x104 DCs were cytocentrifuged and stained as reported above.

Co-culture of infected DC and autologous non-adhering PBMC.
DCs, following 24 h incubation (unless otherwise indicated) with Huv+ Leuk+or Huv Leuk cell-free HCMV or the relevant inactivated (by heating 5 min at 56 °C) crude viral antigen preparations, were co-cultured overnight with thawed non-adhering autologous PBMC at a ratio of 1 : 20 (5·0x104 DC : 1·0x106 non-adhering PBMC). Co-culture medium was RPMI-10 % FBS supplemented with 10 µg brefeldin A ml–1 (Sigma) to prevent release of cytokines. Cell cultures were incubated overnight at 37 °C in 5 % CO2 atmosphere.

Flow cytometry analysis.
DCs, following differentiation and prior to HCMV infection, were routinely stained with FITC-conjugated anti-CD14 (Immunotech) and PE-conjugated anti-CD1a (Caltag) mAbs, supplemented with a pool of human sera, for 30 min in ice. mAb anti-CD14 acted as a monocyte marker, and mAb anti-CD1a as a dendritic-cell marker. In parallel, cells were stained with the relevant isotype-matched control mAb. Surface marker expression was then analysed using a FACScalibur flow cytometer (Becton-Dickinson). DC phenotype following co-culture, migration and further incubation was also determined.

Following incubation with infected DC, PBMCs were washed and stained with FITC-conjugated anti-CD8 (Caltag) and TC-conjugated anti-CD4 (Caltag) mAbs in 100 µl PBS containing 5 % FBS and 5 % immunoglobulin for 30 min in ice. Cells were then washed in the same buffer and fixed for 15 min (FIX and PERM; Caltag). Following washings, cells were permeabilized and stained with PE-conjugated anti-interferon (IFN) gamma (IFN-{gamma}) mAb (Caltag) for 45 min. IFN-{gamma} induction was utilized as the measure of the immune response in CD4+ and CD8+ T cells. Then cells were resuspended in 1 % paraformaldehyde and analysed in a FACScalibur flow cytometer. HCMV-specific CD4+ and CD8+ T cells were determined on a minimal number of 3·0x104 viable CD4+ or CD8+ T cells and their frequencies were calculated by subtracting the value of the control sample incubated with mock-infected endothelial cell culture medium (consistently <=0·05 %) from the test value. To determine the total number of HCMV-specific CD4+ and CD8+ T cells, the percentages of HCMV-specific T cells positive for IFN-{gamma} were multiplied by the relevant absolute CD4+ and CD8+ T cell count, which was determined by a direct immunofluorescent flow cytometry method (Ortho Diagnostic Systems), as reported (Gerna et al., 2001).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HCMV productive infection in DC following co-culture with infected HUVEC
Prior to co-culture, HUVEC were infected with Huv+ Leuk+ HCMV strains and HELF were infected with Huv Leuk HCMV strains (not growing in HUVEC). Following co-culture with productively infected HUVEC, all viruses with the Huv+ Leuk+ phenotype (isolates VR1814, VR3480, VR6110, VR6952; RV-FIX and the RV-FIX deletion mutants {Delta}UL45, {Delta}UL127, {Delta}UL132; and the revertants of Huv Leuk viruses VR1814 rev, VR3480 rev, AD169 rev and Towne rev) were able to productively infect DC (Table 1). As shown in Fig. 1, all Huv+ Leuk+ viruses yielded infectious virus in the medium at about the same level 96 h after DC infection. Virus yield slightly decreased at 168 h p.i. Analysis of infected DC surface molecules, as compared to mock-infected DC, showed a CD1a+ and CD14 phenotype. The same phenotype was maintained following co-culture, migration and further incubation (data not shown). Following adaptation to growth in HUVEC, infectious virus started to be released into the medium at a quantifiable titre after 20 to 30 passages. This means that, when testing HCMV strains or variants at earlier passages, only co-culture with infected HUVEC could be used to infect DC.


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Table 1. DC-tropism of HCMV clinical isolates and their natural variants, deletion mutants and laboratory strains

 


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Fig. 1. Virus yields in dendritic cells co-cultured with HUVEC productively infected with Huv+ Leuk+ strains or with HELF productively infected with Huv Leuk HCMV strains. (a) Huv+ Leuk+ VR1814 (left) and VR6952 (right), and the relevant HELF-grown Huv Leuk variants. (b) Huv+ Leuk+ and Huv Leuk RV-FIX deletion mutants. (c) Huv Leuk laboratory strains AD169 (left) and Towne (right), and the relevant Huv+ Leuk+ revertants (rev).

 
On the other hand, following co-culture with productively infected HELF, all viruses with the Huv Leuk phenotype (clinical isolates extensively propagated in HELF, i.e. VR1814 and VR3480; the reference laboratory strains AD169 and Towne; and RV-FIX deletion mutants {Delta}UL132–128, {Delta}UL131, {Delta}UL130 and {Delta}UL128) were unable to infect DC. (Table 1, Fig. 1)

Immunofluorescent images showing the expression of immediate-early and late HCMV antigens in DC infected by a recent clinical isolate (VR1814 Huv+ Leuk+), one strain long-term propagated on HELF (VR1814 Huv Leuk), RV-FIX (generated using the genetic background of VR1814), RV-FIX{Delta}UL132–128 and RV-FIX{Delta}UL132 are shown in Fig. 2.



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Fig. 2. DC infection by Huv+ Leuk+ (VR1814, RV-FIX, RV-FIX{Delta}UL132) following co-culture with infected HUVEC, and Huv Leuk (RV-FIX{Delta}UL132–128, VR1814) HCMV strains following co-culture with infected HELF. Immunostaining of HCMV-infected DC for immediate-early p72 antigen 24 h p.i. is shown in (a), (c), (e), (g) and (h). In addition, combined immunostaining for p72 and glycoprotein B (gB) at 96 h p.i. is shown in (b), (d) and (f).

 
Infectivity released to the medium of DC cultures infected with Huv+ Leuk+ cell-free virus preparations
At the time when experiments were performed, only VR1814 and RV-FIX had been highly passaged in HUVEC, releasing quantifiable infectious virus to the medium. However, VR1814 had been passaged 80 times (VR1814/HUVEC-80), while RV-FIX was propagated only 50 times (VR1814/HUVEC-50). These virus preparations were used to infect DC at an m.o.i. of 5. Following overnight incubation for virus adsorption and penetration, DC medium was harvested every 3–4 days and tested for virus infectivity using the shell vial assay and HELF cells (Fig. 3a). Virus titre rapidly increased until 4 days p.i., then continued increasing, although more slowly, until 10 days p.i. Subsequently, virus yield decreased until 21 days p.i. The trend shown by the two viruses was similar, even though the peak titre of VR1814 was more than one log10 higher than that of RV-FIX.



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Fig. 3. (a) Kinetics of viral growth in DC infected with cell-free virus preparations from a clinical isolate (VR1814) and the relevant BAC-cloned reconstituted virus RVFIX (Hahn et al., 2002), following extensive propagation in HUVEC. (b) Monocyte-derived DCs following in vitro differentiation in the presence of GM-CSF and IL-4 for 5 days and, then, infection with cell-free RVFIX. Nuclear immunostaining for pp65 and p72 at 3 h (i and iii) and 24 h (ii and iv) p.i., respectively. Subsequently, nuclear immunostaining for p72 and cytoplasmic immunostaining for gB is shown at days 4 (v), 7 (vi) and 10 (vii). No staining is detected in AD169-infected DC (viii). (c) Percentage of HCMV-activated T cells following infection of DC with Huv+ Leuk+ VR 1814 for different times.

 
This difference in titre was most likely due to the difference in the passage number between the two viruses, as the two viruses subsequently showed overlapping titres (data not shown). In addition, RFLP analysis of PCR-amplified UL54, UL55, UL123 and ULb' genomic regions (some of the most differentiating regions among HCMV strains) showed identical patterns of DNA fragment migration between an initial passage (14) of RV-FIX and two long-term passages (51 and 175, respectively) on HUVEC (data not shown).

The kinetics of the viral protein expression during virus replication is shown in Fig. 3(b) (i)–(viii). The numbers of DC positive for both pp65 (i and iii) and p72 (ii and iv) slightly increased from 3 h (i and ii) to 24 h (iii and iv) p.i. At 96 h and 168 h p.i., gB was abundantly expressed in the cytoplasm of DC, while cytopathic effect was progressing (v and vi). At 10 days p.i. DC started undergoing lysis (vii). No viral protein was expressed when DCs were incubated with Huv Leuk virus strains or mutants (viii).

Huv+ Leuk+ HCMV strains generate syncytia in HUVEC at high m.o.i.
When VR1814 infected HUVEC monolayers at high m.o.i. (around 10), syncytial formations started appearing shortly after infection (1–3 h), reaching conspicuous number and size at 24 h p.i. (Fig. 4a). VR1814 rev behaved similarly. On the contrary, no syncytial effect was observed in HUVEC expressing the UL131–128 gene region (Hahn et al., 2004) when infected with Huv Leuk VR1814. Huv Leuk virus strains used at the same m.o.i. did not yield syncytial formations (Fig. 4b).



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Fig. 4. (a) Multiple syncytia with pp65-positive nuclei are generated in HUVEC following infection with Huv+ Leuk+ VR1814 at high m.o.i. Immunostaining with a pool of pp65-specific monoclonal antibodies. (b) HUVEC culture infected with Huv Leuk VR1814 showing neither syncytia nor nuclear pp65 staining.

 
HCMV-infected DC as a stimulus for the determination of HCMV-specific CD4+- and CD8+-mediated immune responses
When DCs infected with cell-free VR1814 Huv+ Leuk+ were used 2–96 h p.i. to stimulate PBMC from HCMV-seropositive subjects, it was observed that the peak of CD4+ immune response was reached between 2 and 24 h p.i., thus anticipating the peak of the CD8+-mediated immune response, which was reached between 24 and 48 h p.i. (Fig. 3c). At subsequent times, the level of the immune response progressively decreased. On this basis, the infection time of 24 h was selected as the optimal time for determination of both the CD4+- and CD8+-mediated immune responses.

A representative example of the FACS analysis leading to quantitative determination of CD4+ and CD8+ HCMV-specific IFN-{gamma} producing T cells is given in Fig. 5(a) and (b). The median frequency of HCMV-specific CD4+ and CD8+ IFN-{gamma} producing T cells in 21 HCMV-seropositive and six HCMV-seronegative healthy blood donors following 16–18 h activation by autologous DC infected 24 h in advance with HUVEC-adapted cell-free VR1814 virus strain, is reported in Fig. 5(c). The percentage of IFN-{gamma} producing T cells showed a wide range of variability in different subjects for both CD4+ and CD8+ HCMV-specific response. However, a clear-cut separation was observed between immunocompetent HCMV-seropositive and HCMV-seronegative individuals. In fact, while the number of CD4+ and CD8+ IFN-{gamma} producing T cells was negligible (consistently <0·20 cells µl–1) in non-immune subjects, it reached median levels of 5·13 (0·67–35·57) and 2·90 (0·49–12·36) cells µl–1, respectively, in immune persons. Therefore, subjects with >=0·4 µl–1 blood HCMV-specific CD4+ or CD8+ T cells were considered as ‘responders' to the HCMV stimulus for the relevant T-cell subset, whereas subjects with <0·2 µl–1 blood HCMV-specific CD4+ or CD8+ T cells (mean value±2 SD of six HCMV seronegative donors) were considered as ‘non-responders’. Finally, subjects with an intermediate value were considered as ‘equivocal responders' (Fig. 5c, grey zone).



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Fig. 5. FACS analysis of CD4+ and CD8+ IFN-{gamma} producing T cells. PBMC, after stimulation with mock-infected (a) or HCMV-infected DC (b), were stained in the cytokine flow cytometry assay. CD4+ and CD8+ T cells (y axis) were gated from viable PBMC and analysed for IFN-{gamma} expression (x axis). Results of one representative experiment. (c) CD4+ and CD8+ T cell-mediated HCMV-specific immune responses in HCMV-seronegative and seropositive healthy subjects. Thick horizontal bars indicate median values. The grey area indicates the grey zone separating positive from negative response to HCMV stimuli or immune from non-immune subjects (equivocal results).

 
When the same HCMV-immune subjects showing a strong CD4+ and CD8+ T-cell immune response following the stimulus induced by DC infected with Huv+ Leuk+ VR1814 were tested using autologous DC incubated with the Huv Leuk virus strains or mutants, only the CD4+ immune response was elicited (Table 2). Again, when the same subjects were tested following incubation of DC with inactivated crude viral antigen, the CD4+ response was induced, but not CD8+.


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Table 2. Frequency of HCMV-specific IFN-{gamma} producing CD4+ and CD8+ T cells following activation by DC incubated with either infectious virus or inactivated crude viral antigen preparations

BD, blood donor.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Results of the present study lead to the following conclusions: (i) the UL131–128 genes of HCMV genome are required for DC infection, as they were required for EC-tropism and virus transfer to leukocytes (Hahn et al., 2004); (ii) RV-FIX{Delta}UL131, {Delta}UL130 and {Delta}UL128 are functionally equivalent to the UL132–128 knockout mutant; (iii) DC infection may occur either following co-culture with HUVEC infected with endotheliotropic HCMV strains or by means of cell-free endotheliotropic HCMV strains released into the medium of infected HUVEC cultures; and (iv) 24 h infected DC appear to be a suitable stimulus for the determination of both CD4+- and CD8+-mediated HCMV-specific immune responses.

We have recently documented that UL131–128 genes represent genetic determinants of both EC-tropism and HCMV transfer to leukocytes (Hahn et al., 2004). In fact, all recent clinical HCMV isolates are provided with both biological properties, whereas all deletion mutants missing the entire locus or the single ORFs of the locus, as well as the natural variants carrying mutations in the same locus, are deprived of both properties. In addition, it was shown that tropism competence can be re-acquired in Huv Leuk natural variants following re-adaptation to growth in HUVEC and reversion of UL131–128 mutations naturally acquired (Hahn et al., 2004). Finally, loss of EC-tropism and leukocyte transfer could be complemented in HUVEC transduced with the UL131–128 individual genes from Huv+ Leuk+ VR1814 and infected with the relevant Huv Leuk viral variants (Hahn et al., 2004). However, a UL24-deletion mutant of Towne was recently found to be defective in growth in microvascular endothelial cells (Dunn et al., 2003) with respect to the parental Towne-BAC. These data seem to suggest that other determinants of EC-tropism may exist, which are functionally only active in specific cell types. In contrast with these data, we found Towne not infectious for HUVEC unless a 2 nt insertion in UL130 mutation was reverted (Hahn et al., 2004). This discrepancy may be due to the different specificity of different endothelial cell systems or may be associated with an unpredicted reverse mutation in UL130 of the Towne strain cloned as BAC by Dunn et al. (2003). However, as a general consideration, the finding that RV-FIX exhibits supernatant-associated DC-tropism only after extensive adaptation to growth in HUVEC strongly suggests that additional genetic alterations have occurred during this adaptation, which are responsible for cell-free DC-tropism of HCMV strains.

In this report, we demonstrate that all HCMV strains or natural variants or deletion mutants tropism competent for HUVEC and leukocytes are also able to productively infect DC. Since release of viable virus from infected HUVEC occurs only after 20–30 passages, infection of DC by virus strains or variants at earlier passages can only take place by co-culture of DC with infected HUVEC. Infection of a high proportion of DC by cell-free virus released from infected HUVEC can only be achieved with an m.o.i. of 1–10, as in the case of the VR1814 and RV-FIX of this study.

Since the DC used in this study are monocyte-derived and it has been previously shown that monocytes (Waldman et al., 1995) as well as PMNL (Gerna et al., 2000) and HUVEC interact closely and bidirectionally by transferring and receiving viral and cellular material, it is not surprising that DC somewhat behave as leukocytes of the myeloid lineage with respect to endothelial cells. Three steps are needed for completion of this interaction: attraction, adhesion and fusion. Attraction is mediated by cellular and viral chemokines. In this respect, the UL128 gene product is indicated as a potential CC chemokine (Akter et al., 2003). Then adhesion could occur through interaction between Mac-1 (CD11b/CD18) and ICAM-1, and between DC-specific ICAM grabbing nonintegrin (DC-SIGN) and ICAM-2 and ICAM-3 (Geijtenbeek et al., 2000a, b). Finally, the fusion event should be mediated by the intervention of the as-yet-unidentified fusogenic factor encoded by the UL131–128 locus. This mechanism should play its major role when infection occurs by co-culture of DC and endothelial cells. However, when cell-free virus infects DC, following extensive adaptation to HUVEC, the mechanism of infection could be similar, if UL131–128 gene products are present in the viral envelope. The recently advocated role of DC-SIGN as the HCMV receptor that ultimately determines entry and replication of HCMV in permissive cells is not sufficient to justify virus entry into human fibroblasts, the most permissive cells, where DC-SIGN is not expressed (Halary et al., 2002). Thus, DC-SIGN might be an important cellular co-factor mediating interactions of HCMV with DC and endothelial cells. These interactions can be initiated by virus attachment to heparan sulfate proteoglycans (Compton et al., 1993). Subsequently, such an attachment may become stronger through virus interaction with annexin II, which binds to gB (Pietropaolo & Compton, 1997) or a 92·5 kDa protein which binds to gH (Baldwin et al., 2000) or, finally, DC-SIGN binding to gB. However, the final fusion event for entry of HCMV into leukocytes, DC and endothelial cells requires the intervention of a fusogenic factor, which could reasonably be represented by the UL131–128 gene products. The amino acid sequence of the UL131, UL130 and UL128 products qualifies them as secretory proteins (Akter et al., 2003; Hahn et al., 2004), which could either physically or functionally interact with each other to trigger microfusion between adhering membranes of contiguous cells or even between viral envelope and cell membrane. Whether one of these proteins may be bound to viral envelope remains to be determined.

Our herein reported experimental findings indicate that infection of HUVEC with EC tropic HCMV strains at a high m.o.i. causes a striking syncytial effect within a few minutes or hours, which is totally absent when infection is attempted with a non-EC tropic virus strain inoculated at the same high m.o.i. (as determined on HELF). In addition, rapid penetration of the major component (pp65) of dense bodies (DB) from Huv+ Leuk+ viruses into the nucleus of HUVEC and DC was observed, whereas this finding was not observed when HUVEC (or DC) were incubated with DB containing virus preparations from Huv Leuk HCMV strains. Thus, both the syncytial effect and the nuclear targeting of pp65 within endothelial cells by Huv+ Leuk+ HCMV strains seem to suggest a fusion effect of both the virion and DB envelope with the cell membrane. This effect was not observed in UL131–128 deletion mutants and in Huv Leuk viruses. Earlier work by Sinzger et al. (2000) reported that the block of AD169 (Huv Leuk) infection in HUVEC was a post-entry step involving translocation of viral DNA to the nucleus. Whether the lack of penetration of pp65 into the nucleus of HUVEC (and DC) may also be a post-entry event is not clear at the moment. Partial accumulation of pp65 inside the cytoplasm of DC as well as other cells was reported using purified DB preparations from AD169 infected human fibroblasts, but not following DC infection by an endotheliotropic HCMV strain (Pepperl-Klindworth et al., 2003). However, using our virus preparations consisting of infected cell culture medium, we could not observe an accumulation of pp65 inside the cytoplasm of either HUVEC or DC, following incubation with Huv Leuk viruses. Finally, it must be kept in mind that the cell differentiation stage is critical for virus infection, as shown by the finding that monocytes cannot be productively infected unless previously differentiated into either macrophages (Ibanez et al., 1991; Lathey & Spector, 1991; Taylor-Wiedeman et al., 1991; Minton et al., 1994) or immature DC (Riegler et al., 2000).

The use of DC infected with a wild-type virus appears to be a potent stimulator of both CD4+- and CD8+-mediated immune responses, if DC are co-cultured with PBMC within 24 h p.i. Subsequently, the antigenic stimulus is reduced, possibly due to the decreased expression of major histocompatibility complex (MHC) class I and II as well as co-stimulatory molecules and impaired pro-inflammatory cytokine production, thus resulting in impaired antigen presentation function (Andrews et al., 2001; Raftery et al., 2001; Moutaftsi et al., 2002). The study of the kinetics of the cell-mediated immune response following co-culture of PBMC with DC at different times from infection shows that CD4+ T-cell response has already occurred by 2 h p.i. and is close to the peak between 6 and 24 h p.i., whereas the CD8+-mediated immune response is delayed, reaching its peak at 24 h p.i. and then remaining stable between 24 and 48 h p.i.

The different kinetics of the CD4+- and CD8+-specific immune response allow us to speculate on the role of different viral proteins mostly contributing to induction of the T-cell immune response. The finding that the CD4+ anticipates the CD8+ T-cell response shortly after infection of DC may be due to the rapid penetration and processing of pp65 from DB with formation of pp65 peptides-MHC class II complexes, which are immediately transported and exposed on the cell membrane (Sallusto et al., 1995). Thus, presence in cytoplasmic vesicles of preformed MHC class II molecules and penetration of enveloped DB inside cytoplasmic vacuoles by phagocytosis may explain the earliness of the CD4+-mediated immune response. On the other hand, following penetration of DB with a mechanism shared by virions, pp65-derived peptides may be complexed to MHC class I molecules stimulating a CD8+ immune response. However, this process takes longer, since synthesis of MHC class I molecules is delayed (Rescigno et al., 1998). In addition, at least in these experimental conditions, DC must be infected with endotheliotropic virus strains to generate the highest CD8+ T-cell response and peptides from de novo synthesized viral antigens other than pp65, such as IE1/2, may be exposed on the DC membrane during productive HCMV infection, thus increasing the overall level of the immune response. On the other hand, CD4+ T-cell activation is not substantially affected by the ability of the virus to infect DC. In this respect, it was also found that infection of DC with a DC-tropic virus blocked in its infectivity by neutralizing antibody strongly reduced only the CD8+ and not the CD4+ T-cell activation (data not shown).

The contribution of each of the major immunodominant proteins of HCMV to the cell-mediated immune response will be dissected in the near future by infecting DC with vaccinia virus recombinants individually carrying pp65, IE-1, gB and pp150 genes (Diamond et al., 1997; Gyulai et al., 2000). Thus, the role played by each viral protein in eliciting CD4+ and CD8+ HCMV-specific immunity will be investigated in detail. However, the DC-based assay reported in this study allows a relatively rapid determination of both CD4+ and CD8+ T-cell immune responses using the same preparation of autologous infected DC. This may represent a major advance in the determination and follow-up of cell-mediated immune response of immunocompetent and immunocompromised patients. In fact, cytokine flow cytometry and a single blood sample can reliably evaluate both arms of the cell-mediated immune response.


   ACKNOWLEDGEMENTS
 
We thank Linda D'Arrigo for revision of the English. We are indebted to the technical staff of the Servizio di Virologia. We are grateful to Francesco F. Fagnoni and Rita Maccario for helpful discussion. This work was partially supported by Ministero della Salute, Ricerca Corrente IRCCS Policlinico San Matteo (grant no. 80425), Ricerca Finalizzata 2003 (Convenzione 109) and by a grant of the Wilhelm Sander-Stiftung (G. H.).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 27 July 2004; accepted 14 October 2004.