Epstein-Barr virus infection of Langerhans cell precursors as a mechanism of oral epithelial entry, persistence, and reactivation

Abstract

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with many malignant and nonmalignant human diseases. Life-long latent EBV persistence occurs in blood-borne B lymphocytes, while EBV intermittently productively replicates in mucosal epithelia. Although several models have previously been proposed, the mechanism of EBV transition between these two reservoirs of infection has not been determined. In this study, we present the first evidence demonstrating that EBV latently infects a unique subset of blood-borne mononuclear cells that are direct precursors to Langerhans cells and that EBV both latently and productively infects oral epithelium-resident cells that are likely Langerhans cells. These data form the basis of a proposed new model of EBV transition from blood to oral epithelium in which EBV-infected Langerhans cell precursors serve to transport EBV to the oral epithelium as they migrate and differentiate into oral Langerhans cells. This new model contributes fresh insight into the natural history of EBV infection and the pathogenesis of EBV-associated epithelial disease.

FIG. 1.

Differentiation of blood-derived pre-LC into LC during prolonged culture. Examination by light microscopy revealed that most of the CD1a⁺ pre-LC newly isolated from blood were small round cells (black arrows). After 3 days, the number of small round cells decreased and larger cell clusters with short dendrite-like projections (white arrow) appeared. By 6 weeks, most cell clusters elaborated a large spherical structure of interlocking dendritic projections surrounding the central core. These structures were stable for more than 10 months of culture and did not increase in number over time, suggesting a lack of cell division. Fluorescent immunostaining and imaging with a laser scanning confocal microscope demonstrated expression of both CD1a and CD207/Langerin in these cell clusters, confirming their differentiation into the LC phenotype. Staining with isotype control fluorescent antibodies was negative. DIC, Nomarski differential interference contrast.

FIG. 2.

Limiting-dilution PCR of the EBV BALF1 gene in pre-LC isolated from blood. (A) For control cells, variable numbers of cells of the Namalwa Burkitt's lymphoma cell line were diluted into a constant number of cells of the Raji Burkitt's lymphoma cell line. Each Namalwa cell carries 2 integrated copies of the EBV genome (two copies of the BALF1 target sequence), whereas each Raji cell carries approximately 50 copies of an EBV genome with the BALF1 gene target sequence naturally deleted. After extraction of the DNA from the admixed cells, PCR amplification was performed in nine identical reaction tubes at each level of dilution containing the indicated number of copies of the BALF1 gene target present along with the DNA of 10⁵ Raji cell equivalents. These results demonstrate that the PCR amplification assay is both sensitive and specific to a single copy of target sequence. (+) Control = B958 lymphoblastoid cell line DNA; (−) Control = no DNA. (B) Newly isolated pre-LC (representative subject example). The DNA from a total of 125,900 CD1a⁺ cells (Table 7, AIDS subject 15) was extracted, distributed among nine identical reaction tubes at each level of dilution containing the indicated number of cell equivalents, and amplified by PCR for the BALF1 gene target sequence. (+) Control = B958 lymphoblastoid cell line DNA; (−) Control = no DNA.

FIG. 3.

EBV M-RT-PCR of pre-LC isolated from blood. CD1a⁺ pre-LC were isolated from the blood of eight healthy subjects and six with AIDS, each with fewer than 200 CD4⁺ cells/ml of blood (mean, 70; range, 4 to 164). Cells were studied as newly isolated, after culture for 3 days, or after culture for 3 days and treatment with chemical inducers of EBV replication for 2 more days. RNA extracted from the cells was studied by EBV M-RT-PCR amplification and specific probe hybridization. (+) Control = B958 lymphoblastoid cell line DNA and Akata Burkitt's lymphoma cell line DNA; (−) Control = no DNA. (A) Newly isolated pre-LC. EBER-1 expression was demonstrated in healthy subjects 3, 4, 5, 6, 7, and 10 and in AIDS subjects 11, 12, 14, 15, and 16. With a band in the reverse transcriptase-negative (RT −) reaction mixture, it is uncertain if the result for subject 6 represents true EBER-1 expression or detection of viral genomic DNA sequences in the RNA preparation. Despite a band in the reverse transcriptase-negative reaction mixture for subject 13, the absence of a band in the corresponding reverse transcriptase-positive (RT +) reaction mixture was interpreted as an absence of EBER expression for this subject. (B) Cultured pre-LC. EBER-1 expression was demonstrated in all three subjects, including subject 8, in whom EBER-1 expression was not detected in newly isolated pre-LC. (C) Control cells. The EBV-positive Namalwa Burkitt's lymphoma cell line expresses the least EBER-1 of the known EBV-positive cell lines. The sensitivity of detection of the EBV M-RT-PCR assay for EBER-1 expression was demonstrated to be a single Namalwa cell diluted into 10⁵ cells of the EBV-negative RHEK-1 cell line. (D) Newly isolated pre-LC. BZLF1 expression was demonstrated in healthy subject 4. (E) Cultured pre-LC. New weak BZLF1 expression was demonstrated in all three healthy subjects in whom BZLF1 expression was not detected in newly isolated pre-LC. (F) Cultured and induced pre-LC. Newly induced strong BZLF1 expression was demonstrated in all three healthy subjects in whom BZLF1 expression was not detected in newly isolated pre-LC.

FIG. 4.

EBV EBER-FISH of pre-LC isolated from blood. Control cells and purified CD1a⁺ pre-LC isolated from the blood of healthy subjects were examined by in situ hybridization for latency-associated EBER transcription. Cells were imaged by fluorescent laser scanning confocal microscopy. DIC, Nomarski differential interference contrast. (A) EBV-positive Raji Burkitt's lymphoma cells express high levels of EBER (up to 10⁵ or 10⁶ transcripts per cell). Nuclear saturation of fluorescence was consistently detected. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than Raji cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells. (C) EBV-negative BJAB Burkitt's lymphoma cells did not show EBER hybridization. (D) EBV-negative RHEK-1 epithelial cells did not show EBER hybridization. (E) Nuclear EBER expression was detected in a newly isolated CD1a⁺ pre-LC. The numerous black circles represent antibody-conjugated magnetic microbeads that are still attached to most of the CD1a⁺ cells, including the EBER-positive cell. (F) Nuclear EBER expression was detected in a CD1a⁺ pre-LC that was cultured in vitro for 3 days before EBER in situ hybridization.

FIG. 4.

EBV EBER-FISH of pre-LC isolated from blood. Control cells and purified CD1a⁺ pre-LC isolated from the blood of healthy subjects were examined by in situ hybridization for latency-associated EBER transcription. Cells were imaged by fluorescent laser scanning confocal microscopy. DIC, Nomarski differential interference contrast. (A) EBV-positive Raji Burkitt's lymphoma cells express high levels of EBER (up to 10⁵ or 10⁶ transcripts per cell). Nuclear saturation of fluorescence was consistently detected. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than Raji cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells. (C) EBV-negative BJAB Burkitt's lymphoma cells did not show EBER hybridization. (D) EBV-negative RHEK-1 epithelial cells did not show EBER hybridization. (E) Nuclear EBER expression was detected in a newly isolated CD1a⁺ pre-LC. The numerous black circles represent antibody-conjugated magnetic microbeads that are still attached to most of the CD1a⁺ cells, including the EBER-positive cell. (F) Nuclear EBER expression was detected in a CD1a⁺ pre-LC that was cultured in vitro for 3 days before EBER in situ hybridization.

FIG. 4.

EBV EBER-FISH of pre-LC isolated from blood. Control cells and purified CD1a⁺ pre-LC isolated from the blood of healthy subjects were examined by in situ hybridization for latency-associated EBER transcription. Cells were imaged by fluorescent laser scanning confocal microscopy. DIC, Nomarski differential interference contrast. (A) EBV-positive Raji Burkitt's lymphoma cells express high levels of EBER (up to 10⁵ or 10⁶ transcripts per cell). Nuclear saturation of fluorescence was consistently detected. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than Raji cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells. (C) EBV-negative BJAB Burkitt's lymphoma cells did not show EBER hybridization. (D) EBV-negative RHEK-1 epithelial cells did not show EBER hybridization. (E) Nuclear EBER expression was detected in a newly isolated CD1a⁺ pre-LC. The numerous black circles represent antibody-conjugated magnetic microbeads that are still attached to most of the CD1a⁺ cells, including the EBER-positive cell. (F) Nuclear EBER expression was detected in a CD1a⁺ pre-LC that was cultured in vitro for 3 days before EBER in situ hybridization.

FIG. 5.

EBV M-RT-PCR of LC isolated from oral epithelium. CD1a⁺ LC were isolated from cells obtained by brush biopsy of grossly normal oral mucosal epithelia of seven healthy subjects. Half of each oral LC specimen was studied as newly isolated cells, and the other half was studied after culture of the cells in the presence of chemical inducers of EBV replication for 3 days. RNA extracted from the cells was studied by EBV M-RT-PCR amplification and specific probe hybridization. (+) Control = B958 lymphoblastoid cell line DNA and Akata Burkitt's lymphoma cell line DNA; (−) Control = no DNA. (A) Newly isolated oral LC. EBER-1, BZLF1, EBNA-1-Fp/Qp, and EBNA-1-Cp/Wp expression was demonstrated in subjects 10 and 20. LMP-1 expression was demonstrated in subject 10. LMP-2A and gp220 expression was not detected. RT +, with reverse transcriptase; RT −, without reverse transcriptase. (B) Cultured and induced oral LC. Newly induced BZLF1 expression was demonstrated in subjects 4, 5, 17, 18, and 21 and was especially strong in subject 5. (Note that the PCR product of subject 10 leaked from the well of the gel, explaining the apparent lack of BZLF1 hybridization for the induced cells of that subject, and that BZLF1 expression was also previously detected in subject 20 prior to induction.) Newly induced LMP-2A and EBNA-1-Cp/Wp expression was demonstrated in subjects 5 and 18. Newly induced strong gp220 expression was demonstrated in subject 5. LMP-1 and EBNA-1-Fp/Qp expression was not detected.

FIG. 6.

EBV EBER/BHLF1-FISH of oral epithelial tissue. Control cells and oral surgical biopsy tissue sections from HIV-positive subjects were examined by in situ hybridization for latency-associated EBER transcription and replication-associated BHLF1 transcription. Tissues were imaged by fluorescence laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. (A) EBV-positive B958 lymphoblastoid cells express high levels of EBER in most cells, and nuclear saturation of fluorescence was consistently detected. Approximately 1 to 5% of B958 cells also express replicative genes, including the early replicative gene BHLF1. Two different patterns of nuclear fluorescence were seen with BHLF1 expression, diffuse and punctate. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than B958 cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells, but BHLF1 expression was not detected in these cells, which harbor only latent EBV infection. (C) EBV-negative RHEK-1 epithelial cells did not hybridize to either EBER or BHLF1. (D) Hybridization with both the EBER and BHLF1 probes was seen in a band-like pattern in the upper spinous layer of oral hairy leukoplakia, consistent with the known localization of productive EBV replication in the oral epithelium. (E, F, G, and H) Nuclear EBER probe hybridization was always associated with nuclear cohybridization of the BHLF1 probe in the upper spinous layer of oral hairy leukoplakia. Nuclear chromatin margination was present, and the most intense EBER probe hybridization strongly colocalized with the BHLF1 probe in the punctate hybridization pattern. This phenomenon of EBER probe hybridization in the upper spinous layer of oral hairy leukoplakia does not represent latent EBV infection but instead is consistent with EBER probe cross-hybridization to EBER gene sequences present in single-stranded EBV DNA synthesized in the nuclei of these cells during productive EBV replication, as previously described for EBER in situ hybridization in oral hairy leukoplakia (28). In the cells with the strongest nuclear EBER hybridization, additional weaker EBER hybridization was often seen in the cytoplasm and likely represents EBER probe cross-hybridization to EBER gene sequences present in artifactually denatured double-stranded EBV DNA in maturing virions being prepared for release from the cells. Furthermore, the cells immediately below and immediately above the EBER-BHLF1 cohybridizing cells often showed nuclear hybridization with only the BHLF1 probe. This result is consistent with early gene expression both preceding and persisting after viral DNA synthesis in the differentiation-dependent cascade of replicative EBV gene expression in oral epithelium, as previously described in oral hairy leukoplakia (40, 52). (I, J, and K) Three tissue sections (panel I, normal tongue epithelium without EBV replication; panels J and K, tongue epithelium with oral hairy leukoplakia) each demonstrated a solitary EBER-expressing cell located in or immediately above the basal layer. The locations of the epithelial basement membrane and basal layer are illustrated by the white lines and circles. The tissue section in panel K represents a cut through the mucosal rete ridges (white circles) in a plane that is perpendicular to the plane represented by the tissue sections in panels I and J. In all three cases, the EBER probe localization was confirmed to be intranuclear in a single cell by computer-generated three-dimensional reconstruction of the cell with a sequential series of 0.6-μm-deep confocal microscopy images. This expression of EBER in the absence of BHLF1 indicates the presence of latent EBV infection in each of these three solitary cells, similar to that demonstrated in the Namalwa cell line (panel B). (L) A tissue section of tongue epithelium with oral hairy leukoplakia demonstrated a solitary EBER- and BHLF1-coexpressing cell in the basal or lower spinous epithelial layer. The location of the epithelial basement membrane is not evident in this photomicrograph, but the cell appears to be located at the top of a rete ridge and was distinctly distant from the EBV replication in the upper spinous epithelial layer. The punctate nuclear colocalization of BHLF1 with the more diffuse EBER and the absence of EBER in the cell cytoplasm together suggest that this cell represents EBV reactivation of early replicative gene expression in a previously latently infected cell, similar to that demonstrated in the B958 cell line (panel A).

FIG. 6.

EBV EBER/BHLF1-FISH of oral epithelial tissue. Control cells and oral surgical biopsy tissue sections from HIV-positive subjects were examined by in situ hybridization for latency-associated EBER transcription and replication-associated BHLF1 transcription. Tissues were imaged by fluorescence laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. (A) EBV-positive B958 lymphoblastoid cells express high levels of EBER in most cells, and nuclear saturation of fluorescence was consistently detected. Approximately 1 to 5% of B958 cells also express replicative genes, including the early replicative gene BHLF1. Two different patterns of nuclear fluorescence were seen with BHLF1 expression, diffuse and punctate. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than B958 cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells, but BHLF1 expression was not detected in these cells, which harbor only latent EBV infection. (C) EBV-negative RHEK-1 epithelial cells did not hybridize to either EBER or BHLF1. (D) Hybridization with both the EBER and BHLF1 probes was seen in a band-like pattern in the upper spinous layer of oral hairy leukoplakia, consistent with the known localization of productive EBV replication in the oral epithelium. (E, F, G, and H) Nuclear EBER probe hybridization was always associated with nuclear cohybridization of the BHLF1 probe in the upper spinous layer of oral hairy leukoplakia. Nuclear chromatin margination was present, and the most intense EBER probe hybridization strongly colocalized with the BHLF1 probe in the punctate hybridization pattern. This phenomenon of EBER probe hybridization in the upper spinous layer of oral hairy leukoplakia does not represent latent EBV infection but instead is consistent with EBER probe cross-hybridization to EBER gene sequences present in single-stranded EBV DNA synthesized in the nuclei of these cells during productive EBV replication, as previously described for EBER in situ hybridization in oral hairy leukoplakia (28). In the cells with the strongest nuclear EBER hybridization, additional weaker EBER hybridization was often seen in the cytoplasm and likely represents EBER probe cross-hybridization to EBER gene sequences present in artifactually denatured double-stranded EBV DNA in maturing virions being prepared for release from the cells. Furthermore, the cells immediately below and immediately above the EBER-BHLF1 cohybridizing cells often showed nuclear hybridization with only the BHLF1 probe. This result is consistent with early gene expression both preceding and persisting after viral DNA synthesis in the differentiation-dependent cascade of replicative EBV gene expression in oral epithelium, as previously described in oral hairy leukoplakia (40, 52). (I, J, and K) Three tissue sections (panel I, normal tongue epithelium without EBV replication; panels J and K, tongue epithelium with oral hairy leukoplakia) each demonstrated a solitary EBER-expressing cell located in or immediately above the basal layer. The locations of the epithelial basement membrane and basal layer are illustrated by the white lines and circles. The tissue section in panel K represents a cut through the mucosal rete ridges (white circles) in a plane that is perpendicular to the plane represented by the tissue sections in panels I and J. In all three cases, the EBER probe localization was confirmed to be intranuclear in a single cell by computer-generated three-dimensional reconstruction of the cell with a sequential series of 0.6-μm-deep confocal microscopy images. This expression of EBER in the absence of BHLF1 indicates the presence of latent EBV infection in each of these three solitary cells, similar to that demonstrated in the Namalwa cell line (panel B). (L) A tissue section of tongue epithelium with oral hairy leukoplakia demonstrated a solitary EBER- and BHLF1-coexpressing cell in the basal or lower spinous epithelial layer. The location of the epithelial basement membrane is not evident in this photomicrograph, but the cell appears to be located at the top of a rete ridge and was distinctly distant from the EBV replication in the upper spinous epithelial layer. The punctate nuclear colocalization of BHLF1 with the more diffuse EBER and the absence of EBER in the cell cytoplasm together suggest that this cell represents EBV reactivation of early replicative gene expression in a previously latently infected cell, similar to that demonstrated in the B958 cell line (panel A).

FIG. 6.

EBV EBER/BHLF1-FISH of oral epithelial tissue. Control cells and oral surgical biopsy tissue sections from HIV-positive subjects were examined by in situ hybridization for latency-associated EBER transcription and replication-associated BHLF1 transcription. Tissues were imaged by fluorescence laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. (A) EBV-positive B958 lymphoblastoid cells express high levels of EBER in most cells, and nuclear saturation of fluorescence was consistently detected. Approximately 1 to 5% of B958 cells also express replicative genes, including the early replicative gene BHLF1. Two different patterns of nuclear fluorescence were seen with BHLF1 expression, diffuse and punctate. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than B958 cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells, but BHLF1 expression was not detected in these cells, which harbor only latent EBV infection. (C) EBV-negative RHEK-1 epithelial cells did not hybridize to either EBER or BHLF1. (D) Hybridization with both the EBER and BHLF1 probes was seen in a band-like pattern in the upper spinous layer of oral hairy leukoplakia, consistent with the known localization of productive EBV replication in the oral epithelium. (E, F, G, and H) Nuclear EBER probe hybridization was always associated with nuclear cohybridization of the BHLF1 probe in the upper spinous layer of oral hairy leukoplakia. Nuclear chromatin margination was present, and the most intense EBER probe hybridization strongly colocalized with the BHLF1 probe in the punctate hybridization pattern. This phenomenon of EBER probe hybridization in the upper spinous layer of oral hairy leukoplakia does not represent latent EBV infection but instead is consistent with EBER probe cross-hybridization to EBER gene sequences present in single-stranded EBV DNA synthesized in the nuclei of these cells during productive EBV replication, as previously described for EBER in situ hybridization in oral hairy leukoplakia (28). In the cells with the strongest nuclear EBER hybridization, additional weaker EBER hybridization was often seen in the cytoplasm and likely represents EBER probe cross-hybridization to EBER gene sequences present in artifactually denatured double-stranded EBV DNA in maturing virions being prepared for release from the cells. Furthermore, the cells immediately below and immediately above the EBER-BHLF1 cohybridizing cells often showed nuclear hybridization with only the BHLF1 probe. This result is consistent with early gene expression both preceding and persisting after viral DNA synthesis in the differentiation-dependent cascade of replicative EBV gene expression in oral epithelium, as previously described in oral hairy leukoplakia (40, 52). (I, J, and K) Three tissue sections (panel I, normal tongue epithelium without EBV replication; panels J and K, tongue epithelium with oral hairy leukoplakia) each demonstrated a solitary EBER-expressing cell located in or immediately above the basal layer. The locations of the epithelial basement membrane and basal layer are illustrated by the white lines and circles. The tissue section in panel K represents a cut through the mucosal rete ridges (white circles) in a plane that is perpendicular to the plane represented by the tissue sections in panels I and J. In all three cases, the EBER probe localization was confirmed to be intranuclear in a single cell by computer-generated three-dimensional reconstruction of the cell with a sequential series of 0.6-μm-deep confocal microscopy images. This expression of EBER in the absence of BHLF1 indicates the presence of latent EBV infection in each of these three solitary cells, similar to that demonstrated in the Namalwa cell line (panel B). (L) A tissue section of tongue epithelium with oral hairy leukoplakia demonstrated a solitary EBER- and BHLF1-coexpressing cell in the basal or lower spinous epithelial layer. The location of the epithelial basement membrane is not evident in this photomicrograph, but the cell appears to be located at the top of a rete ridge and was distinctly distant from the EBV replication in the upper spinous epithelial layer. The punctate nuclear colocalization of BHLF1 with the more diffuse EBER and the absence of EBER in the cell cytoplasm together suggest that this cell represents EBV reactivation of early replicative gene expression in a previously latently infected cell, similar to that demonstrated in the B958 cell line (panel A).

FIG. 6.

EBV EBER/BHLF1-FISH of oral epithelial tissue. Control cells and oral surgical biopsy tissue sections from HIV-positive subjects were examined by in situ hybridization for latency-associated EBER transcription and replication-associated BHLF1 transcription. Tissues were imaged by fluorescence laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. (A) EBV-positive B958 lymphoblastoid cells express high levels of EBER in most cells, and nuclear saturation of fluorescence was consistently detected. Approximately 1 to 5% of B958 cells also express replicative genes, including the early replicative gene BHLF1. Two different patterns of nuclear fluorescence were seen with BHLF1 expression, diffuse and punctate. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than B958 cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells, but BHLF1 expression was not detected in these cells, which harbor only latent EBV infection. (C) EBV-negative RHEK-1 epithelial cells did not hybridize to either EBER or BHLF1. (D) Hybridization with both the EBER and BHLF1 probes was seen in a band-like pattern in the upper spinous layer of oral hairy leukoplakia, consistent with the known localization of productive EBV replication in the oral epithelium. (E, F, G, and H) Nuclear EBER probe hybridization was always associated with nuclear cohybridization of the BHLF1 probe in the upper spinous layer of oral hairy leukoplakia. Nuclear chromatin margination was present, and the most intense EBER probe hybridization strongly colocalized with the BHLF1 probe in the punctate hybridization pattern. This phenomenon of EBER probe hybridization in the upper spinous layer of oral hairy leukoplakia does not represent latent EBV infection but instead is consistent with EBER probe cross-hybridization to EBER gene sequences present in single-stranded EBV DNA synthesized in the nuclei of these cells during productive EBV replication, as previously described for EBER in situ hybridization in oral hairy leukoplakia (28). In the cells with the strongest nuclear EBER hybridization, additional weaker EBER hybridization was often seen in the cytoplasm and likely represents EBER probe cross-hybridization to EBER gene sequences present in artifactually denatured double-stranded EBV DNA in maturing virions being prepared for release from the cells. Furthermore, the cells immediately below and immediately above the EBER-BHLF1 cohybridizing cells often showed nuclear hybridization with only the BHLF1 probe. This result is consistent with early gene expression both preceding and persisting after viral DNA synthesis in the differentiation-dependent cascade of replicative EBV gene expression in oral epithelium, as previously described in oral hairy leukoplakia (40, 52). (I, J, and K) Three tissue sections (panel I, normal tongue epithelium without EBV replication; panels J and K, tongue epithelium with oral hairy leukoplakia) each demonstrated a solitary EBER-expressing cell located in or immediately above the basal layer. The locations of the epithelial basement membrane and basal layer are illustrated by the white lines and circles. The tissue section in panel K represents a cut through the mucosal rete ridges (white circles) in a plane that is perpendicular to the plane represented by the tissue sections in panels I and J. In all three cases, the EBER probe localization was confirmed to be intranuclear in a single cell by computer-generated three-dimensional reconstruction of the cell with a sequential series of 0.6-μm-deep confocal microscopy images. This expression of EBER in the absence of BHLF1 indicates the presence of latent EBV infection in each of these three solitary cells, similar to that demonstrated in the Namalwa cell line (panel B). (L) A tissue section of tongue epithelium with oral hairy leukoplakia demonstrated a solitary EBER- and BHLF1-coexpressing cell in the basal or lower spinous epithelial layer. The location of the epithelial basement membrane is not evident in this photomicrograph, but the cell appears to be located at the top of a rete ridge and was distinctly distant from the EBV replication in the upper spinous epithelial layer. The punctate nuclear colocalization of BHLF1 with the more diffuse EBER and the absence of EBER in the cell cytoplasm together suggest that this cell represents EBV reactivation of early replicative gene expression in a previously latently infected cell, similar to that demonstrated in the B958 cell line (panel A).

FIG. 6.

EBV EBER/BHLF1-FISH of oral epithelial tissue. Control cells and oral surgical biopsy tissue sections from HIV-positive subjects were examined by in situ hybridization for latency-associated EBER transcription and replication-associated BHLF1 transcription. Tissues were imaged by fluorescence laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. (A) EBV-positive B958 lymphoblastoid cells express high levels of EBER in most cells, and nuclear saturation of fluorescence was consistently detected. Approximately 1 to 5% of B958 cells also express replicative genes, including the early replicative gene BHLF1. Two different patterns of nuclear fluorescence were seen with BHLF1 expression, diffuse and punctate. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than B958 cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells, but BHLF1 expression was not detected in these cells, which harbor only latent EBV infection. (C) EBV-negative RHEK-1 epithelial cells did not hybridize to either EBER or BHLF1. (D) Hybridization with both the EBER and BHLF1 probes was seen in a band-like pattern in the upper spinous layer of oral hairy leukoplakia, consistent with the known localization of productive EBV replication in the oral epithelium. (E, F, G, and H) Nuclear EBER probe hybridization was always associated with nuclear cohybridization of the BHLF1 probe in the upper spinous layer of oral hairy leukoplakia. Nuclear chromatin margination was present, and the most intense EBER probe hybridization strongly colocalized with the BHLF1 probe in the punctate hybridization pattern. This phenomenon of EBER probe hybridization in the upper spinous layer of oral hairy leukoplakia does not represent latent EBV infection but instead is consistent with EBER probe cross-hybridization to EBER gene sequences present in single-stranded EBV DNA synthesized in the nuclei of these cells during productive EBV replication, as previously described for EBER in situ hybridization in oral hairy leukoplakia (28). In the cells with the strongest nuclear EBER hybridization, additional weaker EBER hybridization was often seen in the cytoplasm and likely represents EBER probe cross-hybridization to EBER gene sequences present in artifactually denatured double-stranded EBV DNA in maturing virions being prepared for release from the cells. Furthermore, the cells immediately below and immediately above the EBER-BHLF1 cohybridizing cells often showed nuclear hybridization with only the BHLF1 probe. This result is consistent with early gene expression both preceding and persisting after viral DNA synthesis in the differentiation-dependent cascade of replicative EBV gene expression in oral epithelium, as previously described in oral hairy leukoplakia (40, 52). (I, J, and K) Three tissue sections (panel I, normal tongue epithelium without EBV replication; panels J and K, tongue epithelium with oral hairy leukoplakia) each demonstrated a solitary EBER-expressing cell located in or immediately above the basal layer. The locations of the epithelial basement membrane and basal layer are illustrated by the white lines and circles. The tissue section in panel K represents a cut through the mucosal rete ridges (white circles) in a plane that is perpendicular to the plane represented by the tissue sections in panels I and J. In all three cases, the EBER probe localization was confirmed to be intranuclear in a single cell by computer-generated three-dimensional reconstruction of the cell with a sequential series of 0.6-μm-deep confocal microscopy images. This expression of EBER in the absence of BHLF1 indicates the presence of latent EBV infection in each of these three solitary cells, similar to that demonstrated in the Namalwa cell line (panel B). (L) A tissue section of tongue epithelium with oral hairy leukoplakia demonstrated a solitary EBER- and BHLF1-coexpressing cell in the basal or lower spinous epithelial layer. The location of the epithelial basement membrane is not evident in this photomicrograph, but the cell appears to be located at the top of a rete ridge and was distinctly distant from the EBV replication in the upper spinous epithelial layer. The punctate nuclear colocalization of BHLF1 with the more diffuse EBER and the absence of EBER in the cell cytoplasm together suggest that this cell represents EBV reactivation of early replicative gene expression in a previously latently infected cell, similar to that demonstrated in the B958 cell line (panel A).

FIG. 6.

EBV EBER/BHLF1-FISH of oral epithelial tissue. Control cells and oral surgical biopsy tissue sections from HIV-positive subjects were examined by in situ hybridization for latency-associated EBER transcription and replication-associated BHLF1 transcription. Tissues were imaged by fluorescence laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. (A) EBV-positive B958 lymphoblastoid cells express high levels of EBER in most cells, and nuclear saturation of fluorescence was consistently detected. Approximately 1 to 5% of B958 cells also express replicative genes, including the early replicative gene BHLF1. Two different patterns of nuclear fluorescence were seen with BHLF1 expression, diffuse and punctate. (B) EBV-positive Namalwa Burkitt's lymphoma cells express much lower levels of EBER (at least 100-fold lower than B958 cells). Variable levels of nuclear EBER expression were easily detected in most Namalwa cells, but BHLF1 expression was not detected in these cells, which harbor only latent EBV infection. (C) EBV-negative RHEK-1 epithelial cells did not hybridize to either EBER or BHLF1. (D) Hybridization with both the EBER and BHLF1 probes was seen in a band-like pattern in the upper spinous layer of oral hairy leukoplakia, consistent with the known localization of productive EBV replication in the oral epithelium. (E, F, G, and H) Nuclear EBER probe hybridization was always associated with nuclear cohybridization of the BHLF1 probe in the upper spinous layer of oral hairy leukoplakia. Nuclear chromatin margination was present, and the most intense EBER probe hybridization strongly colocalized with the BHLF1 probe in the punctate hybridization pattern. This phenomenon of EBER probe hybridization in the upper spinous layer of oral hairy leukoplakia does not represent latent EBV infection but instead is consistent with EBER probe cross-hybridization to EBER gene sequences present in single-stranded EBV DNA synthesized in the nuclei of these cells during productive EBV replication, as previously described for EBER in situ hybridization in oral hairy leukoplakia (28). In the cells with the strongest nuclear EBER hybridization, additional weaker EBER hybridization was often seen in the cytoplasm and likely represents EBER probe cross-hybridization to EBER gene sequences present in artifactually denatured double-stranded EBV DNA in maturing virions being prepared for release from the cells. Furthermore, the cells immediately below and immediately above the EBER-BHLF1 cohybridizing cells often showed nuclear hybridization with only the BHLF1 probe. This result is consistent with early gene expression both preceding and persisting after viral DNA synthesis in the differentiation-dependent cascade of replicative EBV gene expression in oral epithelium, as previously described in oral hairy leukoplakia (40, 52). (I, J, and K) Three tissue sections (panel I, normal tongue epithelium without EBV replication; panels J and K, tongue epithelium with oral hairy leukoplakia) each demonstrated a solitary EBER-expressing cell located in or immediately above the basal layer. The locations of the epithelial basement membrane and basal layer are illustrated by the white lines and circles. The tissue section in panel K represents a cut through the mucosal rete ridges (white circles) in a plane that is perpendicular to the plane represented by the tissue sections in panels I and J. In all three cases, the EBER probe localization was confirmed to be intranuclear in a single cell by computer-generated three-dimensional reconstruction of the cell with a sequential series of 0.6-μm-deep confocal microscopy images. This expression of EBER in the absence of BHLF1 indicates the presence of latent EBV infection in each of these three solitary cells, similar to that demonstrated in the Namalwa cell line (panel B). (L) A tissue section of tongue epithelium with oral hairy leukoplakia demonstrated a solitary EBER- and BHLF1-coexpressing cell in the basal or lower spinous epithelial layer. The location of the epithelial basement membrane is not evident in this photomicrograph, but the cell appears to be located at the top of a rete ridge and was distinctly distant from the EBV replication in the upper spinous epithelial layer. The punctate nuclear colocalization of BHLF1 with the more diffuse EBER and the absence of EBER in the cell cytoplasm together suggest that this cell represents EBV reactivation of early replicative gene expression in a previously latently infected cell, similar to that demonstrated in the B958 cell line (panel A).

FIG. 7.

CD207/Langerin immunostaining of LC in oral epithelial tissue. Oral surgical biopsy tissue sections of normal tongue epithelium were immunostained for CD207/Langerin. Tissues were imaged by fluorescent laser scanning confocal microscopy. Tissue section panels are oriented with the mucosal surface to the top. DIC, Nomarski differential interference contrast. LC were identified in the basal layer and the immediate suprabasal region of the lower spinous layer of the oral epithelium. The location of the epithelial basement membrane and basal layer is illustrated by the white lines. These results demonstrate that oral LC localize to the same lower epithelial layers as the solitary EBV-positive cells identified in Fig. 6, suggesting a possible LC identity for these EBV-positive cells.

FIG. 8.

Proposed model of EBV oral epithelial entry, persistence, and reactivation. EBV latently infects pre-LC in the blood. The pre-LC migrate from the blood, through the submucosa, and into the oral epithelium, transporting latent EBV infection. In the epithelium, the pre-LC differentiate into LC that reside in the lower epithelial layers and extend dendrites into the upper spinous layer. EBV may persist in LC as a latent infection or may reactivate in LC to productive replication. Infection of adjacent epithelial cells results in productive EBV replication in the upper spinous layer, sometimes causing the pathological changes of oral hairy leukoplakia and ultimately releasing infectious virions into the oral cavity.