Human DC can be efficiently transduced with MCMV-EGFP.
Human DC were generated from purified, immunomagnetically selected CD14+ peripheral blood monocytes, after a 7-day culture in the presence of granulocyte-macrophage colony-stimulating factor and IL-4. As expected, the iDC that were derived through the use of this protocol were characterized by their loss of cell surface CD14 expression and by their expression of high levels of HLA-DR, CD11b, and CD11c (e.g., see the data presented in Fig. ). mDC were then generated from this iDC population, by incubating the cells in medium containing 1 μg of LPS/ml for 24 h. These cells were distinguished by their increased expression of HLA-DR and CD83 (data not shown) and by their loss of phagocytic activity (e.g., see the data presented in Fig. ).
FIG. 4. Phenotypic analysis of MCMV-EGFP-infected immature human DC. Cells were infected with MCMV-EGFP (MOI of 5) and then analyzed 24 h later for expression of the indicated cell surface markers. The analysis presented is based on an initial gating of the total (more ...)
FIG. 2. MCMV infection of human monocyte-derived DC occurs independently of cellular phagocytic activity. Immature or mature human DC (iDC and mDC, respectively) were prepared, and aliquots of these cells were then either (i) directly exposed to FITC-dextran (more ...)
In order to examine the ability of MCMV to transfer exogenous DNA into primary human DC, iDC, or murine M2-10B4 cells (a positive control for virus infection) were exposed to the MCMV-EGFP recombinant virus at an MOI of 5. After 12, 24, and 48 h, the cells were examined by fluorescence microscopy in order to detect EGFP expression. As shown in Fig. , strong EGFP expression was detected in human iDC as early as 12 h after infection. This level of EGFP expression did not, however, increase substantially over time, suggesting that the virus was not replicating or spreading to new cells (see Fig. for additional information on this point). In contrast, the number of EGFP-positive cells in the M2-10B4 cell culture increased rapidly from 12 to 48 h, a finding consistent with virus replication and spread in the fully permissive M2-10B4 cells.
FIG. 1. Human monocyte-derived DC can be efficiently transduced with MCMV-EGFP. (A) Microscopic analysis of EGFP expression in immature human DC and in murine M2-10B4 cells (positive control), after exposure to MCMV-EGFP at an MOI of 5, in the presence or absence (more ...)
FIG. 3. MCMV undergoes abortive infection in human monocyte-derived DC. (A) One-step growth curve of MCMV-EGFP in both permissive M2-10B4 cells and in immature human DC. (B) Electron microscopic analysis of MCMV-EGFP-infected M2-10B4 cells (subpanel A) and human (more ...)
The experiment shown in Fig. suggests that ca. 30 to 50% of iDC from this donor could be infected with MCMV-EGFP under the conditions used (MOI of 5; samples analyzed at 12 to 48 h after virus infection). Other experiments confirmed that a dose-response relationship existed between the number of EGFP-positive DC and the MOI of MCMV-EGFP used to infect the cells (data not shown), and flow cytometric analysis confirmed the overall estimate of infection efficiency that was obtained by using the fluorescence microscopic analysis. Specifically, Fig. reveals that roughly 30% of the cells from this particular batch of human iDC were successfully transduced by the MCMV-EGFP recombinant under the conditions used (MOI = 5; time = 24 h after virus infection).
In addition to presenting an analysis of GFP expression in human DC at various times after exposure to MCMV-EGFP, the results shown in Fig. also include a fluorescence microscopic analysis of GFP expression in iDCs that were incubated with MCMV-GFP in the presence or absence of the protein synthesis inhibitor, cycloheximide. The data show that no GFP could be detected in the DC if cycloheximide was present, indicating that the presence of GFP is dependent upon new protein synthesis and not upon pinocytic or phagocytic uptake by the DC.
We attempted to examine further the possible contribution of phagocytosis to GFP expression in MCMV-EGFP exposed DC, by performing the virus infection in the presence of phagocytosis inhibitors such as cytochalasin B, cytochalasin D, or nocodazole. However, these drugs proved uniformly toxic to our DC cultures when applied at the concentrations necessary to exert a physiologic effect on phagocytosis. We therefore decided to take a different approach and to directly compare the phagocytic activity of human DC (as measured by uptake of FITC-dextran particles) with their susceptibility to infection with MCMV-EGFP. The results (Fig. ) reveal that (as expected) mDC were weakly phagocytic compared to iDC. However, the two DC populations (mDC and iDC) were essentially equal in their susceptibility to infection with MCMV-EGFP (as revealed by flow cytometric analysis of GFP expression). Thus, the phagocytic activity of human DC does not correlate with their susceptibility to infection with MCMV-EGFP or with their expression of a MCMV-vectored antigen (GFP).
The data presented in Fig. also reveal that our initial estimate of the transfection efficiency for MCMV-EGFP in human DC (30 to 50%; Fig. ) is rather conservative. In the experiment shown in Fig. , up to 85% of iDC and 70% of mDC were found to be positive for EGFP expression (as measured by flow cytometric analysis) at 24 h after exposure to MCMV-EGFP at an MOI of 5. Subsequent experiments yielded results somewhere in between these values, with most experiments giving ca. 50% transduction efficiency under standard conditions (MOI = 5, time of analysis = 24 h after infection; data not shown). This suggests that there may be some individual-to-individual or batch-to-batch variation in the susceptibility of human DC to infection with MCMV for reasons that are at present uncertain.
MCMV undergoes abortive infection in human DC.
One of the characteristic features of CMVs is their highly species-specific host range (28
). To examine whether MCMV might be undergoing a nonproductive cycle of infection in human iDC, we compared the one-step viral growth curves for MCMV-EGFP-infected human iDC versus MCMV-infected M2-10B4 cells. No virus progeny was detected in supernatants collected from MCMV-EGFP-infected human iDC, whereas MCMV titers increased steadily in the supernatant of MCMV-EGFP-infected M2-10B4 cells, reaching a peak at 3 days after exposure to the virus and declining thereafter as the cultures underwent virally induced cytopathic effects and cytolysis (Fig. ). Importantly, the transduction of human DC by MCMV was not associated with any significant loss of cellular viability relative to control cultures over the period of this experiment (data not shown).
MCMV replication in human iDC was also examined by electron microscopic (EM) examination of infected cells. EM analysis was performed both for MCMV-EGFP-infected M2-10B4 cells (as a positive control) and for MCMV-EGFP-infected human iDC (as well as for mock-infected iDC, as a negative control). These experiments were performed by using a range of viral MOIs (0.5, 1, and 5) and, 2 days later, the cells were fixed with 4% glutaraldehyde and processed for EM analysis. The results of this experiment revealed the presence of numerous virus particles, at different stages of assembly, in virally infected M2-10B4-infected cells, but not in iDC (Fig. ). In addition, the MCMV-transduced DC exhibited no signs of any virally induced cytopathic effects (not shown).
The absence of detectable MCMV replication in human iDC, combined with the efficient expression of virally vectored EGFP, suggested to us that MCMV might undergo abortive infection in human DC. To verify this, we examined the expression of viral immediate-early (immediate-early gene 1 [IE1]), early (early gene 1 [E1]) and late (gB) genes in human iDC that had been infected with MCMV-EGFP. Permissive M2-10B4 cells infected with the same virus recombinant were used a positive control for this experiment, and mock-infected M2-10B4 and iDC were used as negative controls. As shown in Fig. , immunoblot analysis of infected cell lysates with virus antigen-specific antibodies revealed that the IE1, E1, and gB proteins were all strongly expressed in the M2-10B4 cells, while only the immediate-early and early proteins could be detected in lysates from virally infected iDC (gB was not detected, even after prolonged exposure of the immunoblots; data not shown). It is also readily apparent that the level of IE1 protein expression in iDC was much reduced, in comparison to the permissive M2-10B4 cells. This suggests that MCMV replication in human iDC may be blocked both at the level of early gene expression and at the level of late gene expression. These findings also confirm that MCMV does indeed undergo abortive infection in human DC.
MCMV-EGFP modestly increases the maturation of human iDC and does not significantly alter the allostimulatory function of these cells.
Human iDC were infected with MCMV-EGFP, and the expression of selected cell surface molecules associated with DC maturation and/or DC costimulatory activity (CD11b, CD11c, CD40, CD80, CD83, CD86, and MHC class I and II) was examined by flow cytometry at 24 h after virus infection. The flow cytometric results were then analyzed by gating the DC into two populations: EGFP-positive cells (MCMV infected) and EGFP-negative cells (bystander cells in the same culture dish). Immunophenotypic analysis of the two gated cell populations is presented in Fig. (note that, in this experiment, ca. 50% of the iDC were EGFP positive; data not shown).
The data presented in Fig. show that expression of several markers was upregulated in the MCMV-infected, EGFP-positive iDC population compared to uninfected, EGFP-negative bystander cells. Among these markers were such maturation-associated molecules as CD40, CD86, and HLA-DR (see Fig. ). However, neither the infected cells nor the bystander iDCs expressed CD83 (data not shown), suggesting that the generally modest effects of MCMV infection stopped well short of inducing full DC maturation. Furthermore, similar experiments performed in mDC revealed that MCMV infection had only a minimal effect on the expression of these same cell surface proteins in mDC (data not shown).
In light of the modest maturation-enhancing effect of MCMV infection on the expression of cell surface proteins by human iDC, we wondered whether MCMV infection might exert any effect on the functional activity of human DC. To address this question, we measured the ability of MCMV-EGFP-infected iDC to promote T-cell proliferation in an allogeneic MLR. The results showed a very modest increase in cellular proliferation in the MLR assay, when MCMV-infected DC were compared to DC that were mock infected (Fig. ). This difference was found in all three of the experiments performed, but the extent of the effect observed was small enough to be statistically insignificant (see error bars, which denote the standard deviation of triplicate counts-per-minute [cpm] values).
FIG. 5. MCMV-EGFP infection has little effect on the ability of iDC to stimulate T-cell proliferation in an allogeneic MLR. iDC were infected with MCMV-EGFP (MOI = 5) or were mock infected. After 48 h, the cells were irradiated and mixed with allogeneic (more ...) MCMV-gp120-infected human DC are capable of inducing the in vitro expansion of gp120-epitope specific CD8+ T lymphocytes from naive human PBMC.
To examine the effect of MCMV infection on the ability of human DC to stimulate antigen-specific T-cell proliferation, we conducted an in vitro experiment by using the MCMV-gp120 vector. Human iDC generated from a HLA A2-positive, HIV-naive donor were infected with this virus (or mock infected), and aliquots of these cells were then pulsed with a HLA A2-restricted peptide derived from gp120 (this peptide is designated V3 and corresponds to the sequence RGPGRAFVTI [1
]). Two days later, the DC were washed, irradiated, and added to autologous PBMC at a ratio of 20:1 (PBMC to DC). After 2 weeks, some PBMC were harvested for use in tetramer staining assay, while the remaining cells were restimulated in the presence of new DC for an additional 2 weeks and then analyzed by using both tetramer staining and ELISPOT assays.
Tetrameric complexes of HLA-A2, refolded in the presence of the V3 peptide (RGPGRAFVTI), were used to perform flow cytometric analysis of the in vitro stimulated PBMC at both the 2- and 4-week time points. This analysis revealed that the frequency of V3 tetramer-reactive, CD8-positive T lymphocytes was rather modest in all experimental conditions at the 2-week time point (Fig. ).
FIG. 6. Human DC infected with an MCMV vector that encodes HIV-1 gp120 can induce the in vitro expansion of gp120 epitope-specific CD8+ T lymphocytes from a naive precursor population. (A) iDC were infected with a recombinant MCMV construct encoding HIV-1 (more ...)
However, after 4 weeks of in vitro cultivation, significantly higher frequencies of such cells could be detected in PBMC cultures that had been incubated in the presence of V3 peptide-pulsed iDC, MCMV-gp120-infected iDC, and iDC that were both V3 peptide pulsed and infected with MCMV-gp120. The highest frequency of tetramer-reactive CD8+ T cells was detected in cultures which had been expanded in the presence of iDC that were infected with MCMV-gp120 and also pulsed with the V3 peptide. Slightly lower levels of tetramer-positive CD8+ lymphocytes were detected in cultures that were expanded in the presence of iDC that were only infected with MCMV-gp120 or only pulsed with the V3 peptide but, in all cases, the frequency of tetramer-reactive cells greatly exceeded the level that was detected in cells that were expanded in the presence of untreated iDC (Fig. ).
Since the tetramer analysis did not reveal whether the in vitro-expanded, V3-specific T cells might possess functional activity specific for V3-epitope positive, autologous target cells, we performed IFN-γ ELISPOT analysis on the in vitro-expanded PBMC cultures. As shown in Fig. , this analysis revealed results that were consistent with the tetramer staining data. Thus, the frequency of V3-epitope specific, IFN-γ-secreting T cells are highest in those cultures that had been expanded in the presence of V3-pulsed and MCMV-gp120-infected iDC and much lower in cultures that were expanded in the presence of iDC that received only one of these modes of antigen exposure (Fig. ). The results suggested that there might be some synergistic action in cells stimulated with both V3-pulsed and MCMV-gp120-infected iDC.
Taken together, the results shown in Fig. show that infection with MCMV does not interfere with the ability of human DC to stimulate antigen-specific T-cell proliferation. Furthermore, this experiment also establishes that a MCMV vector can successfully elicit the functional presentation of an exogenous encoded antigen by human DC (in this case, HIV-1 gp120).