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Tufts University School of Medicine, Boston, MA 02138
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous study, we described the preferential localization of latent EBV to memory B cells in the periphery based on characterizing the cells as sIg+ and sIgD- (22). However, at that time there was no specific marker for peripheral, human, memory B cells available to confirm this hypothesis. Recently, it has been shown that CD27 is such a marker (29, 30); therefore, we have fractionated peripheral B cells on the basis of CD27 to directly ask whether EBV is restricted to memory B cells. Identification of CD27 as a B cell memory marker also allowed the discovery of a new subset of memory cells within the IgD+ subset (30). This raised the possibility that the small numbers of infected cells we found in the IgD+ B cell population represented the presence of the virus within the memory subset of IgD+ cells. Previously, we had considered that the low frequency of infected cells in the IgD+ fraction probably represented either contamination with IgD- cells or, less likely, the presence of a small number of IgD+, latently infected cells. However, because naive IgD+ cells constitute 75–80% of the peripheral B cell pool (31), this would mean that numerically as many as 10–20% of the virus-infected cells could be naive. Therefore, we have performed experiments to distinguish these possibilities. We have used anti-CD27 Abs, magnetic activated cell sorting (MACS),3 and FACS techniques to obtain highly purified populations of naive (IgD+, CD27-), IgD+ memory (IgD+, CD27+), and IgD- memory (IgD-, CD27+) cells and then assayed quantitatively the frequency of virus-infected cells in each population. In addition, our model proposes that differentiation through a germinal center is essential for latently infected cells to enter the long-lived pool of persistently infected B cells. Therefore, we have separated the B cells on the basis of CD5 expression and again assayed quantitatively for the virus. CD5 expression defines the long-lived/self-renewing B1 subset of B cells (32, 33) that are important in antimicrobial defenses but are not believed to go through a germinal center. If our model is correct, then the virus should be excluded from the CD5+ subset in the peripheral blood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IB4 (gift of Elliot Kieff, Harvard Medical School, Boston, MA) is an EBV-positive lymphoblastoid cell line. It contains three or four copies of the EBV genome and was used as a positive control for all PCR. BJAB, an EBV-negative B cell lymphoma, or EBV-negative tonsillar mononuclear cells were used as negative controls for all experiments and as carrier cells when the cell number tested was <1 x 104 cells.
Heparinized peripheral blood (240–480 ml) was layered onto Ficoll-Hypaque Plus (Pharmacia, Piscataway, NJ) and centrifuged at 2000 rpm for 30 min at 25°C. Plasma was aspirated, and the resulting buffy coats were removed and washed two times with PBSA (1x PBS/0.5% BSA) at 1200 rpm for 15 min. PBMCs were then resuspended at a concentration of 2 x 107 cells/ml in the same buffer. Tonsils were obtained from patients undergoing routine tonsillectomies for obstructed breathing disorders at the Massachusetts General Hospital. Tonsils were minced in PBSA, and the resulting suspension was passed through silkscreen to remove any connective tissue. The cell suspension was diluted to 1 x 107 cells/ml, and the buffy coat was isolated by Ficoll-Hypaque (Pharmacia) centrifugation as described above.
Magnetic bead separations
PBMCs and tonsillar mononuclear cells were resuspended to 2
x 107 cells/ml in PBSA as 1-ml aliquots.
Biotinylated Ab was added to each tube and incubated on a rotator at
4°C for 30 min. The biotinylated Abs used were: 
IgD Ab (Southern
Biotechnology Associates, Birmingham, AL) for selection or depletion of
IgD+ cells and anti-CD19 (our laboratory) for
selection of B cells. All tubes were washed two times with PBSA and
resuspended to 180 µl in the same buffer. Streptavidin-coated
microbeads (20 µl; Miltenyi Biotec, Auburn, CA) were added to each
and incubated for 15 min at 4°C. Cells were again washed and
resuspended in 500 µl for separation using MACS columns (Miltenyi
Biotec) and kept at 4°C at all times. The type of MACS column used
depended on the amount of cells expected in the positive bound
fraction. For an expected number <3 x 107
total positive cells, the AS column (Miltenyi Biotec) was used, and for
expected numbers >3 x 107 but <2 x
108 total positive cells, the CS column was used.
The MACS column was prepared by rinsing with three column volumes of
PBSA and then inserted into a VarioMACS magnet. The flow rate of the
column was adjusted by attaching either a 21-, 23-, or 25-gauge needle
to the base of the stopcock. Cells were then loaded onto the column,
and the negative fraction was collected. The cells were washed through
by applying three column volumes of PBSA to the column while it was
still attached to the magnet. The retained population was then washed
by removing the column from the magnet and injecting one column volume
of PBSA from the bottom of the column using the side syringe supplied.
The needle was then replaced with a 21- or 23-gauge needle, the column
was reinserted into the magnet, and cells were allowed to flow through.
These cells were collected as the wash fraction and discarded. The
column was again rinsed with three column volumes of PBSA, as before,
to remove any remaining nonspecifically bound cells. The column was
then removed from the magnet, the needle was removed, and the column
was washed with five column volumes of PBSA to elute the retained
cells.
FACS separations
Either due to the lack of biotinylated Abs for the markers CD27, CD5, IgA, and IgG, or to obtain highly purified cells, populations selected for the expression of these markers were obtained by FACS separation. For CD27 separations, IgD+ and IgD- CD19+ B cells were first isolated by MACS from whole PBMC or tonsillar mononuclear cells as described above. Both populations were then stained with goat F(ab')2 anti-human IgD FITC (Southern Biotechnology Associates) and anti-CD27-PE (PharMingen, San Diego, CA). For CD5 separations, the CD19+ bulk B cell population was isolated by MACS as described above. The resulting cells were stained with either anti-CD20 FITC (Dako, Carpinteria, CA) (a pan B cell marker) or anti-human IgD FITC and anti-CD5 PE (Leu-1; Becton Dickinson, Mountain View, CA). For analysis of memory B cells expressing different isotypes, IgD- CD19+ B cells were isolated by MACS as described above and the cells were costained with anti-CD20-FITC (Dako) and PE-coupled anti-IgA or IgG (Southern Biotechnology Associates). Relevant populations were sorted either on a FACStarPlus (Becton Dickinson) or the MoFlo (Cytomation, Fort Collins, CO).
Flow cytometric analysis was used to assay the purity of all isolated
populations. All fractionated populations were analyzed using a Becton
Dickinson FACScan with Lysis II software. After separation, all
fractions were stained with an Ab of the appropriate isotype conjugated
to PE (Southern Biotechnology Associates) as well as FITC-coupled
CD20 (Dako). As negative controls, MOPC21 (IgG1 isotype control;
Sigma, St. Louis, MO), 1a2 (IgG2a isotype control; this laboratory),
and MOPC121 (IgG2b isotype control; Sigma) were used. FACS analysis
allowed for the determination of both the recovery and purity of the
isolated populations.
DNA PCR and limiting dilution DNA PCR
Limiting dilution DNA PCR analysis was performed on isolated populations as described previously (22). Isolated populations were serially diluted, and then replicates of each cell dilution were distributed to the wells of a V-bottom microtiter plate (Nunc, Naperville, IL) and centrifuged at 1500 rpm for 15 min at 4°C. The supernatant was aspirated and the cell pellets were resuspended in 10 µl of a lysis solution containing 0.45% Tween 20, 0.45% Igepal CA 630, 20 mM Tris pH 8.3, 50 mM KCl, 2.0 mM MgCl2, and 0.5 mg/ml Proteinase K (Sigma). The plate was sealed, incubated at 55°C for >2 h, and then centrifuged briefly to remove condensation. Five microliters of the cell lysate were used in DNA PCR. DNA PCR specific to the W-repeat region of the EBV genome was performed in a final volume of 50 µl. Amplimers and reaction conditions have been described previously (22). Briefly, each reaction contained 50 mM KCl, 20 mM HCl pH 8.3, 2.0 mM MgCl2, 0.2 mM dNTPs, 20 µM each primer, and 1 U AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). Reactions were conducted in a GeneAmp 9600 Thermocycler (Perkin-Elmer) at 95°C for 15 s and 66°C for 1 min for 30 cycles followed by a single 5-min 72°C extension step. PCR products were resolved on a 2% Nuseive Agarose (FMC, Chicago, IL), 1% Seakem LE Agarose (FMC) gel, and Southern blotted to Nytran Plus (Schleicher & Shuell, Keene, NH) as described by the manufacturer. Specific products were detected using random primed labeled, purified PCR product from Namalwa cells as previously described (17). Because the DNA PCR technique can detect a single copy of the viral genome, every sample with one or more infected cells will be detected. The fraction of negative samples was estimated for each cell dilution tested, and the frequency of virus-infected cells in the population was then estimated using Poisson statistics (for details of the quantitation, sensitivity, and confidence limits of the analysis see Ref. 34).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IgD+ and IgD- B
cell fractions were isolated from the peripheral blood using sequential
positive selection with biotinylated anti-IgD and anti-CD19 Abs
and the MACS magnetic bead system as described previously
(22). The B cell fractions were then stained with
FITC-coupled anti-IgD and PE-coupled anti-CD27. The
IgD+, CD27- (naive) cells,
IgD+, CD27+ (IgD memory),
and IgD-, CD27+ (memory)
cells were purified using the FACS. With this approach, we obtained
populations of cells that were always >95% pure and frequently >99%
pure (Fig. 1
, lower panels).
The purified cells were then subjected to limiting dilution DNA PCR
analysis (15, 34). For this assay, each sample is serially
diluted, and then replicates of each cell dilution are tested for the
presence of virus-infected cells by DNA PCR. By measuring the fraction
of samples negative at each cell dilution and applying Poisson
statistics, it is possible to calculate the frequency of virus-infected
cells in the population. The DNA PCR detects a single copy of the viral
genome (15). Therefore, the limiting dilution analysis
allows for the precise calculation of the absolute frequency of
virus-infected cells in a given population. The results of one such
analysis are shown in Fig. 1 (upper panels). EBV-infected
cells were readily detected in the IgD- memory
compartment (IgD-, CD27+).
However, the virus was barely detectable in both the highly purified
naive population (IgD+,
CD27-) and, more surprisingly, in the
IgD+ memory population
(IgD+, CD27+). We have
performed this experiment on three different donors, and a summary of
the results are presented in Table I for
the IgD+, CD27- population
and in Table II for the
IgD+, CD27+ population. In
two of the donors, no signals were observed at all in either
IgD+ population, and, in the third, only one
signal was detected. Therefore, the frequency of virus-infected cells
in these populations was so low as to be incalculable by our
techniques. For comparative purposes, we have presented the data as the
number of signals that we would expect to see, for the number of cells
tested, if the frequency in the IgD+ populations
was the same as in the IgD- memory population
(IgD-, CD27+). This
expected value is then compared with the actual observed number of
signals. By combining the results from all three experiments, it was
possible to conclude that <0.1% of the virus-infected cells in the
peripheral blood reside in the naive population and <0.4% reside in
the IgD+ memory population. We conclude that
latent or persistent virus is essentially absent from the
IgD+ memory and naive populations. Therefore, the
virus is tightly restricted to memory B cells that have switched away
from IgD expression.
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We have shown above that EBV is excluded from the
IgD+ memory subset. Recently, evidence has been
presented that it is even more narrowly restricted, being limited to
memory cells expressing the IgA isotype (35). To test this
claim, we have compared the frequency of virus-infected B cells in
total IgD, CD27+ memory B cells with highly
purified IgA+ and IgG+
memory B cells. These isotypes are expressed on widely differing
fractions of memory B cells. The IgG memory cells constitute about half
of the compartment, whereas the IgA memory cells constitute <10%
(31). If the virus was restricted to the IgA compartment,
we would expect to see a dramatic enrichment in the frequency of
virus-infected cells in the purified IgA+ subset
and a concomitant depletion in the IgG subset when compared with the
bulk of unfractionated memory cells. The result of this experiment is
shown in Fig. 2. The frequency of
virus-infected cells was essentially identical in all three populations
of cells, irrespective of surface isotype. We conclude that the virus
is not measurably enriched in IgA+ or
IgG+ memory B cells.
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We have hypothesized that EBV can only persist in vivo in the resting, recirculating, long-lived, memory B cell pool because there is only limited or no expression of viral proteins. The virus would achieve this quiescent state through the differentiation of latently infected lymphoblasts into resting memory cells via a germinal center.
CD5+ expression in the peripheral blood
identifies a subset of B cells that are important in antimicrobial
defenses (32). These cells constitute a long-lived,
self-renewing subset of B cells that do not pass through a germinal
center (33). Although CD5+ B cells
are readily infectable in tissue culture, our model would predict that
the virus will not be able to persist within this compartment in vivo.
To test this, we again used MACS to isolate CD19+
B cells and used the FACS to separate the CD5+
and CD5- populations. The result of one
experiment is presented in Fig. 3, and a
summary of results from three such experiments are presented in Table III. As predicted, the virus was excluded
from the CD5+ population and was tightly
restricted to the CD5- (B2) population.
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We have shown previously that EBV has no specificity for
IgD- cells in the tonsils, where infectious
virus is produced (22). We believe that this reflects the
promiscuous nature of EBV infection. This is known from in vitro
studies where the virus is able to latently infect essentially any
resting B cell that expresses the viral receptors. However, the studies
described above indicate that the only infected cells that survive to
persist in the peripheral blood are those that can exit the tonsil as
IgD- memory cells. To test for the lack of
cell-type specificity of latently infected cells in the tonsil, we have
performed the frequency analysis on a variety of tonsillar B cell
populations. We have tested cells fractionated on the basis of either
IgD and/or CD5 expression. The results of one such experiment using CD5
is shown in Fig. 4, and all of the
results obtained are summarized in Table IV. These studies provide a striking
contrast to the high degree of restriction seen in the peripheral
blood. The virus is found in all of the B cell subsets tested,
irrespective of surface IgD or CD5 expression. Furthermore, there is no
significant enrichment or depletion of virus-infected cells associated
with any of the subsets.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One striking observation of this study is that the highly restricted
nature of persistent infection in the peripheral blood exists despite
the fact that all subsets of B cells are latently infected at the site
of primary infection, the tonsil. We have shown previously that viral
replication is ongoing in most healthy tonsils (22), and,
consistent with the promiscuous nature of this virus, we see that every
subset of B cells in the tonsil is infected. A cursory glance at Table IV
demonstrates that subfractionation on the basis of CD5 and sIgD
expression, either alone or in combination, does not delineate any
obvious subset preference of the virus. In experiments not shown here,
we have also fractionated tonsillar B cells on the basis of IgD
expression and expression of CD10 and CD77, specific markers for
germinal center B cells (36). Again, we found no
significant or meaningful difference in the frequency of virus-infected
cells between any of the tonsillar subsets separated on the basis of
expression of these markers.
We also know that EBV has no subset preference for infection in vitro (3). The presence of the virus in all B cell types in the tonsil confirms that this lack of subset preference also occurs in vivo. Therefore, it would be very difficult to explain the high degree of selectivity of viral persistence in the periphery if the cells were simply derived through direct infection in the blood. Consequently, there must be highly restrictive mechanisms that only allow latently infected memory B cells to survive in the peripheral circulation. When EBV infects cells, including memory cells, in vitro, they become proliferating lymphoblasts (37), not resting cells. Yet we and others have shown that infected cells in the periphery are resting (17) and express little or no viral genetic information (18, 19, 20, 21). How does the virus achieve strict cell specificity and the resting state seen in the blood? One possibility is that only latently infected memory B cell blasts in the tonsil are able to leave the cell cycle and exit into the periphery as resting cells. We favor an alternate model that we have proposed previously, which suggests that B cell blasts activated by latent infection with EBV in lymph nodes can recapitulate the normal pathways of B cell germinal center differentiation (27, 28, 38) and thereby enter into the memory B cell pool. Memory cells produced through this pathway are CD27+, sIg+, sIgD-, and CD5- (28, 32). This is exactly the phenotype we observed for the latently infected cells in the peripheral blood. We believe that this differentiation process, which allows proliferating blasts to exit the lymph nodes as resting cells, provides a mechanism to down-regulate expression of the growth-promoting latent genes. The consequence of this is that the virus can establish a persistent infection in a cell type that is not pathogenic to the host and is not readily seen by CTL. Other infected subsets in the tonsil, like the B1 cell, that cannot undergo the differentiation process (32) will be destroyed by CTL before they can enter the peripheral circulation.
Our studies have also uncovered an interesting exception to the rule that EBV is associated with memory B cells in the periphery. This is the observation that the virus is not present in one particular group of memory cells, the IgD+ subset. The discovery of the IgD+ memory cell was made possible through the observation that CD27 expression was the first reliable marker for memory B cells and that lack of sIgD expression was not necessarily indicative of memory cell status (29, 30). To check that our isolated IgD+, CD27+ cells were bona fide memory cells, we have cloned and sequenced the expressed Ig genes on five separate occasions (data not shown). We confirmed the published studies that these cells had the mutations in the hypervariable regions of their Ig genes characteristic of Ag-selected memory cells (30). Nevertheless, it is apparent that these cells must be different from the other memory cells because they alone are incapable of sustaining a persistent infection with EBV. This means that the IgD+ memory cell is physiologically different from the rest of the memory pool and suggests that further investigation of the origin and significance of this subset would be worthwhile.
A recent report claimed that EBV in the peripheral blood was restricted to IgA-positive cells (35). However, in several studies, we have failed to repeat these observations. The rationale for the restriction was the high frequency of IgA-positive cells found in the mucosal epithelium where EBV is believed to initiate infection. However, this represents a misunderstanding. Although there is a high frequency of IgA-secreting plasma cells in the mucosal epithelium, the preponderance of memory cells at this site are IgM+ (39, 40). If EBV had a preference for a particular memory subset based on sIg isotype expression on mucosal memory cells, then it should be IgM. The reason for the discrepancy in the results is technical. In the paper of Ehlin-Henrikson et al. (35), the total IgM-bearing population of B cells was compared with the IgG- and IgA-bearing subsets. Because naive B cells are also IgM positive and constitute the bulk of peripheral B cells, the IgM+ fraction will be highly diluted by naive B cells that we have shown to be EBV negative. Similarly, selection for IgG+ cells frequently results in substantial contamination with monocytes that bear Ig on the surface Fc receptors. Because the Dynal system was used to purify the cells, it is not possible to confirm the absence of contaminating monocytes. Therefore, the IgM- and probably the IgG-positive populations used in that study contained large populations of contaminating uninfected cell populations that would lead to an apparently higher representation in the IgA+ subset. IgA+ memory cells constitute a very small fraction of the memory compartment (31); therefore, if EBV was restricted to this subset, then a striking enrichment should be seen in comparing the degree of infection in bulk memory cells vs the IgA+ subset and, conversely, a striking depletion should be seen on comparing the bulk to the IgG+ memory subset. Careful quantitation of the total number of genomes before and after fractionation would reveal whether a true enrichment in IgA+ cell was occurring. Such an enrichment was not seen in either our or the previously published studies (35).
In conclusion, we have defined the phenotype of virus-infected cells in the peripheral blood and shown that the virus is tightly restricted to a single subset, the isotype-switched memory B cell. This restriction does not hold in lymph nodes, leading to the conclusion that highly specific mechanisms must exist, allowing the selection of only latently infected memory cells into the long-lived memory pool.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Thorley-Lawson, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02138.
3 Abbreviations used in this paper: MACS, magnetic activated cell sorting; PBSA, 1x PBS/0.5% BSA.
Received for publication March 27, 2000. Accepted for publication June 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. Hochberg, J. M. Middeldorp, M. Catalina, J. L. Sullivan, K. Luzuriaga, and D. A. Thorley-Lawson Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo PNAS, January 6, 2004; 101(1): 239 - 244. [Abstract] [Full Text] [PDF]
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P. Lieby, A. Soley, A.-M. Knapp, M. Cerutti, J.-M. Freyssinet, J.-L. Pasquali, and T. Martin Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals Blood, October 1, 2003; 102(7): 2459 - 2465. [Abstract] [Full Text] [PDF]
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D. O. Willer and S. H. Speck Long-Term Latent Murine Gammaherpesvirus 68 Infection Is Preferentially Found within the Surface Immunoglobulin D-Negative Subset of Splenic B Cells In Vivo J. Virol., August 1, 2003; 77(15): 8310 - 8321. [Abstract] [Full Text] [PDF]
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I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman Maintenance of Long Term {gamma}-Herpesvirus B Cell Latency Is Dependent on CD40-Mediated Development of Memory B Cells J. Immunol., July 15, 2003; 171(2): 886 - 892. [Abstract] [Full Text] [PDF]
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E. Flano, I.-J. Kim, J. Moore, D. L. Woodland, and M. A. Blackman Differential {gamma}-Herpesvirus Distribution in Distinct Anatomical Locations and Cell Subsets During Persistent Infection in Mice J. Immunol., April 1, 2003; 170(7): 3828 - 3834. [Abstract] [Full Text] [PDF]
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 E. Flano, I.-J. Kim, D. L. Woodland, and M. A. Blackman {gamma}-Herpesvirus Latency Is Preferentially Maintained in Splenic Germinal Center and Memory B Cells J. Exp. Med., November 18, 2002; 196(10): 1363 - 1372. [Abstract] [Full Text] [PDF]
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C. Rose, M. Green, S. Webber, L. Kingsley, R. Day, S. Watkins, J. Reyes, and D. Rowe Detection of Epstein-Barr Virus Genomes in Peripheral Blood B Cells from Solid-Organ Transplant Recipients by Fluorescence In Situ Hybridization J. Clin. Microbiol., July 1, 2002; 40(7): 2533 - 2544. [Abstract] [Full Text] [PDF]
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S. Nikiforow, K. Bottomly, and G. Miller CD4+ T-Cell Effectors Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation J. Virol., April 15, 2001; 75(8): 3740 - 3752. [Abstract] [Full Text]
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G. J. Babcock and D. A. Thorley-Lawson Tonsillar memory B cells, latently infected with Epstein-Barr virus, express the restricted pattern of latent genes previously found only in Epstein-Barr virus-associated tumors PNAS, October 24, 2000; 97(22): 12250 - 12255. [Abstract] [Full Text] [PDF]
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