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*
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, 38105; and Departments of
Microbiology and Immunology, and
Pediatrics, University of Tennessee, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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HV68) results in acute replication in respiratory epithelial cells
and persistent, latent infection of B cells and macrophages. HV68
elicits virus-specific Ab, and also nonspecifically activates B cells
to Ab production through a CD4+ T cell-dependent process.
The current analysis characterizes virus-specific and nonspecific Ab
production at the single cell level and investigates the requirements
and nature of the nonspecific response. Virus-specific Ab-forming cell
(AFC) numbers were dwarfed by the increase in total AFC in all sites
examined, indicating substantial nonspecific Ab production. Clear
increases and decreases in specific and total AFC numbers occurred in
the lymph nodes draining the respiratory tract and the spleen, but AFC
numbers in the bone marrow (BM) increased to a plateau and remained
constant. The longevity of the BM response was reflected in a sustained
increase in virus-specific and total serum Ab levels. Generally, the
IgG2a and IgG2b isotypes predominated. Analysis of cytokine-deficient
mice, CD40 ligand-deficient mice, and radiation BM chimeras lacking MHC
class II expression specifically on B cells indicated that nonspecific
Ab production is independent of IL-6 or IFN-, and dependent on
cognate CD4+ T cell help. Several observations were
consistent with polyclonal B cell activation by HV68, including the
induction of durable serum levels of IgG reactive with mammalian dsDNA
and murine type II collagen. Our findings indicate new directions for
studies of this valuable model of -herpesvirus
pathogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HV68, also called
MHV-68)5, a virus
isolated originally from small rodents (1), is a member of
the -herpesvirus subfamily, which includes EBV, the Kaposi’s
sarcoma-associated human herpesvirus 8 (HHV-8), and herpesvirus
saimiri. The HV68 sequence is more similar to HHV-8 (2, 3), though the pathogenesis of HV68 infection in the
laboratory mouse has a number of biological and immunological features
in common with human EBV infection. Little, if anything, is known about
the acute phase of HHV-8 infection. The initial site of pathology in
mice infected with HV68 by the intranasal (i.n.) route is the lower
respiratory tract where alveolar epithelial cells and mononuclear cells
support virus replication (4). Hematogenous spread of the
virus results in low level, transient replication in distal sites,
particularly the thymus and adrenal glands (4, 5).
Life-long latent infection of B lymphocytes (6) and
macrophages (7) is established concurrently. Resolution of
the pulmonary phase begins as infectious virus is cleared from the lung
within 10–12 days of challenge (4). Prominent features of
the host response are a transient, CD4+ T
cell-dependent splenomegaly peaking after approximately 2 wk (4, 8, 9), and an infectious mononucleosis-like condition
characterized by an increased frequency of activated
CD8+ T cells in the peripheral blood
(10, 11, 12).
Control of acute HV68 infection in the lung can be mediated by
CD8+ cytotoxic T cells (8), and also
CD4+ effector T cells that function via secretion
of IFN- (13). A slight delay in the clearance of virus
from the lungs of Ig-deficient µMT mice (14) indicates
that virus-specific Ab makes only a minor contribution to this process.
However, virus-specific Ig may be important in controlling the
long-term, persistent phase of infection. This is suggested by
experiments in which adult thymectomized mice were depleted of
CD4+ and CD8+ T cells 1 mo
after HV68 infection. The mice retained high circulating levels of
virus-specific IgG after depletion, and recrudescent pulmonary
infection was not detected (15). In contrast, elimination
of the CD4+ and/or CD8+ T
cell subsets in persistently infected µMT mice resulted in the
reemergence of infectious virus in the lungs and increased numbers of
latently infected cells (13, 16). A likely explanation is
that Ab neutralization of free virus, and perhaps also Ab-dependent
cellular cytotoxicity (17), supplement CTL-mediated
surveillance of latently infected cells.
Analysis of the Ab response to HV68 infection has been limited to
measurements of circulating Ig. Serum levels of virus-specific IgG and
neutralizing activity increase sharply from days 10–20 postinfection,
and then continue to rise more gradually over several months (18, 19). In addition, HV68 infection induces a dramatic and
sustained CD4+ T cell-dependent increase in total
serum IgG, indicating widespread, nonspecific B cell activation
(18, 19). The infection of B cells in vitro with HV68
has a direct activating effect, resulting in proliferation and
differentiation to IgM-producing cells. However, significant Ig class
switching does not occur, even in the presence of
CD4+ T cells, indicating the importance of in
vivo interactions that are not readily reproduced in cell culture
(19). The current study was undertaken to characterize the
virus-specific and nonspecific Ab response to HV68 at the single
cell level. This defines for the first time the kinetics and isotype
profile of the Ab response in different anatomical compartments during
the acute and persistent phases of a -herpesvirus infection. Some
insights were also gained into the requirements and nature of the
nonspecific component of the Ab response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clone G2.4 of HV68 was originally obtained from Dr. A.
A. Nash (Edinburgh, U.K.), and virus stocks were grown in owl monkey
kidney cells and titrated on NIH-3T3 cells (5). Infectious
HV68 administered to mice was free of contamination with LPS
(concentration <0.005 EU/ml) as determined by the Limulus
amebocyte lysate assay (BioWhittaker, Walkersville, MD). Sendai virus
administered i.n. (20) was used as a control agent in some
experiments. Purified HV68 for use in the immunoassays was prepared
by differential centrifugation and sucrose banding of tissue
culture grown virus. Influenza virus A/HKx31 (H3N2) for use as a
control Ag was purified from stocks grown in the allantoic cavity of
embryonated hen’s eggs. Protein concentrations were determined by the
method of Bradford (21).
Mice
Wild-type (+/+) C57BL/6J (B6) and (B6 x
129)F2 mice, and mice deficient (-/-) for IL-6
(B6, 129-Il6) (22), IFN-
(C57BL/6-Ifng) (23), or CD40 ligand (CD40L)
(B6, 129-Cd40l) (24) were purchased from The
Jackson Laboratory (Bar Harbor, ME). MHC class II (H-2
I-Ab)-deficient B6C2D mice
(C2D-/-) (25) and B cell-deficient
C57BL/6-Igh-6 mice (µMT) (26) were bred at
St. Jude Children’s Research Hospital.
Radiation chimeras deficient in the expression of MHC class II
molecules specifically on the B cell compartment were generated by i.v.
injection of mixtures of C2D-/- and µMT bone
marrow (BM) cells into 8- to 10-wk-old B6 mice that had been lethally
irradiated (950 rad) 1 day previously. Control chimeras with +/+ B
cells were made using a mixture of B6 and µMT donor BM. Recipient
mice were given Sulfatrim in their drinking water and held for at least
10 wk to allow full reconstitution before virus challenge. Chimeric
mice were analyzed by flow cytometry (27) at the time of
sampling to determine the prevalence and characteristics of various
lymphocyte subsets. The staining reagents were FITC-conjugated mAbs to
CD4 (RM4-4), I-Ab (AF6-120.1), or CD44 (IM7),
PE-conjugated mAbs to CD4 (RM4-4), CD8
(53-6.7), or CD19 (1D3), and
biotinylated mAbs to CD62L (MEL-14), all of which were purchased from
PharMingen (San Diego, CA).
Mice were housed under specific pathogen-free conditions until HV68
infection, and thereafter in BL3-level containment. Females were used
in all studies and, with the exception of the BM chimeras, were
infected at 8–12 wk of age.
Infection and sampling
Mice were anesthetized with Avertin (2,2,2-tribromoethanol)
given i.p., and then infected i.n. with 1 x
104 PFU of HV68 (in 30 µl PBS). Anesthetized
mice were exsanguinated via the retroorbital plexus before tissue
sampling. Cervical lymph nodes (CLN, a pool of the superficial CLN and
facial lymph nodes), the right posterior mediastinal lymph node (MLN),
and spleen were collected and gently disrupted to generate single-cell
suspensions in IMDM (Life Technologies, Grand Island, NY) supplemented
with L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin
(100 IU/ml), streptomycin (100 µg/ml), gentamicin (10 µg/ml), and
15% FCS (complete medium). BM cell suspensions were obtained by
flushing both femurs and tibiae. RBCs were removed from the spleen and
BM preparations by ammonium chloride lysis.
The enzyme-linked immunosorbent spot-forming (ELISPOT) assay for Ab-forming cells
The ELISPOT assay (28) was adapted to enumerate
HV68-specific Ab-forming cells (AFC). Purified HV68 was
detergent-disrupted, diluted in PBS, and plated at 1 µg/well in
nitrocellulose-bottomed 96-well Multiscreen HA filtration plates
(Millipore, Bedford, MA). After overnight incubation at 4°C, wells
were washed with PBS and blocked with complete medium. Serial (5x)
dilutions of single cell suspensions were prepared in complete medium,
and 100-µl volumes were added to the plates beginning at 5 x
105 cells/well. Plates were incubated for 3–4 h
at 37°C in a humid atmosphere containing 5%
CO2 and then washed thoroughly. Alkaline
phosphatase (ALP)-conjugated isotype-specific goat anti-mouse Abs
(Southern Biotechnology Associates, Birmingham, AL) diluted 1/500 in
PBS plus 5% BSA were added, and the plates were incubated overnight at
4°C. After extensive washing of both sides of the nitrocellulose
filters, spots were developed at room temperature with 1 mg/ml of
5-bromo-4-chloro-3-indolyl phosphate (Sigma, St. Louis, MO) in
diethanolamine buffer. Plates were washed and dried after optimal spot
development. Blue spots representing individual AFC were counted using
an Olympus SZH Stereozoom microscope. Negative control plates were
coated with purified A/HKx31 (H3N2) influenza A virus. Total numbers of
AFC were determined as described above except that plates were coated
with goat anti-mouse 
light chain Abs (Southern Biotechnology
Associates) diluted in PBS to give 0.5 µg/well. Plates were similarly
coated with goat anti-mouse 
light chain Abs (Southern
Biotechnology Associates) to enumerate AFC secreting light
chain Abs.
ELISA for virus-specific Ab
Nunc ImmunoMaxiSorp plates (Fisher Scientific, Pittsburgh, PA)
were coated with purified, detergent-disrupted HV68 diluted in PBS
and plated at 0.25 µg/well. After overnight incubation at 4°C,
plates were washed with PBS-Tween (0.05%), blocked with PBS/BSA (3%),
and washed again. Serum dilutions (3x) starting at 1/100 (1/300 for
IgM determinations) were prepared in PBS-Tween (0.05%)-BSA (0.5%) and
added. The plates were incubated overnight at 4°C, then washed
extensively, and bound Abs were detected with ALP-conjugated
isotype-specific goat anti-mouse Abs (Southern Biotechnology
Associates) diluted optimally in PBS-BSA (1%). After 
3 h incubation
at room temperature, plates were washed thoroughly, and color was
developed with p-nitrophenyl phosphate (Sigma) in
diethanolamine buffer. Absorbance at 405 nm was read using a model 3550
microplate reader (Bio-Rad, Hercules, CA). The virus-specific serum Ab
titer is expressed as the reciprocal of the highest dilution giving an
absorbance value more than twice that for simultaneously titrated
samples from naive mice.
Absorption with purified HV68 was used to remove virus-specific Abs
from serum (29). Briefly, pooled sera from infected mice
were diluted 1/50 or 1/100 in HBSS and incubated for 2.5 h at
4°C with 25 or 50 µg of purified virus. Viral particles were
pelleted by centrifugation, and Igs remaining in the supernatant were
measured by ELISA.
ELISA for total serum Ig
Total serum levels of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3
were determined by a sandwich ELISA employing the washing, blocking,
and incubation steps described above. Plates were coated with 5 µg/ml
goat anti-mouse Abs with specificity for IgM, IgA, IgG1, IgG2a, or
IgG3 (Southern Biotechnology Associates), or with anti-mouse IgG2b
mAb, clone R9-91 (PharMingen). Bound Ig was detected using ALP-goat
anti-mouse IgM, IgA, IgG1, IgG2a, or IgG3 (Southern Biotechnology
Associates), or ALP-anti-mouse IgG2b mAb, clone R12-3 (PharMingen)
followed by p-nitrophenyl phosphate substrate.
Concentrations were calculated from curves constructed using purified
mouse Ig standards (Southern Biotechnology Associates). Serum levels of
IgG and IgG were determined using goat anti-mouse or
goat anti-mouse (Southern Biotechnology Associates) as capture
Abs, ALP-goat anti-mouse IgG (Southern Biotechnology Associates) to
detect specific binding, and mouse IgG2a (clone G155-178) or IgG2a
(clone HOPC-1) mAb (PharMingen) as standards.
ELISA for anti-collagen Ab
Serum Abs to native murine type II collagen (CII) were measured by ELISA (30). Briefly, sera were incubated in collagen-coated plates, and bound Abs were detected using peroxidase-conjugated goat anti-mouse IgG. The level of anti-CII IgG is reported as the absorbance of a 1/200 serum dilution. An inhibition assay was used to confirm the specificity of binding in the anti-CII ELISA. Briefly, serum was incubated with a range of concentrations of murine CII for 4 h at 4°C before being added to collagen-coated plates, and the ELISA was repeated as described above.
ELISA for anti-dsDNA Ab
Serum Abs binding to mammalian dsDNA were measured by ELISA (31). Briefly, a range of serum dilutions starting at 1/50 were incubated in plates coated with native calf thymus DNA, and bound Abs were detected using alkaline phosphatase-conjugated goat anti-mouse IgG. Titers are expressed as the reciprocal serum dilution that gave 50% of maximum binding determined by reference monoclonal IgM and IgG anti-DNA Abs.
Isoelectric focusing
Isoelectric focusing (IEF) of serum proteins (32) was performed in polyacrylamide Ampholine PAGplates, pH range 3.5–9.5 (Pharmacia Biotech, Piscataway, NJ) on a Multiphor II flatbed electrophoresis system (Pharmacia Biotech) set at 10°C. Electrode strips saturated with 1 M NaOH (catholyte) or 1 M H3PO4 (anolyte) were applied to the gel, and 20-µl aliquots of diluted sera were loaded on application strips positioned near the anode. Proteins focused for 90 min at 1500 V were transferred by capillary action to a methanol-treated and distilled water-moistened polyvinylidene difluoride (PVDF) Immobilon-P transfer membrane (Millipore). The membrane was carefully removed from the gel and sequentially treated with blocking buffer, biotinylated anti-mouse IgG2ab mAb, clone 5.7 (PharMingen), and HRP-conjugated streptavidin (Pierce, Rockford, IL). The blot was developed with the ECL Western blotting analysis system (Amersham, Arlington Heights, IL) and exposed to Biomax film (Sigma) to record the IgG2a spectrotype pattern.
Statistics
Statistical comparisons of mean values were performed using the nonparametric Mann-Whitney U test for unpaired samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B cell ELISPOT assay was applied to analyze both the
virus-specific and nonspecific AFC response in B6 mice infected i.n.
with a sublethal HV68 dose. At various times after infection, AFC
responses were measured in the CLN and MLN, which receive lymphatic
drainage from the upper and lower respiratory tract, respectively
(33), and in the spleen and BM. The CLN, MLN, and spleen
participate in the early CD4+ and
CD8+ T cell responses to HV68 infection
(18, 34, 35) and are, in addition, sites where latently
infected B cell populations are first established (5). The
BM is recognized as a major site for the localization of long-lived AFC
(36) and is considered to maintain high circulating levels
of virus-specific Ab long after infectious virus has been cleared
(37, 38, 39).
AFC responses in the CLN, MLN, spleen, and BM. The
kinetics of the HV68-specific and total AFC responses in the CLN,
MLN, spleen, and BM are shown in Figs. 1
and 2, respectively. The virus-specific
response in the MLN slightly preceded responses in the CLN and spleen.
In these sites, an early peak of virus-specific IgM AFC gave way to
increased numbers of cells producing IgG isotypes, particularly IgG2a
and IgG2b. Peak numbers of virus-specific AFC were attained in the CLN
and MLN subsequent to 2 wk after infection, a somewhat delayed response
compared with that associated with other respiratory viruses
(38). Generally, the numbers of virus-specific IgG AFC in
the CLN, MLN, and spleen progressively decreased from 2 to 3 wk after
virus infection. Surprisingly, essentially no virus-specific IgA AFC
were generated in the CLN, a site where IgA generally contributes
substantially to the response following respiratory infection with
negative strand RNA viruses (20, 38).
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and 2),
measured as the number of lymphocytes producing Ig containing the
light chain. Such lymphocytes represent 90–95% of AFC in normal
mice (40). The virus-specific assays applied in this study
used purified, disrupted virions as target Ags. Responses to
HV68-encoded proteins that are not incorporated into the virion were
therefore not detected. Nevertheless, the striking difference between
total and specific AFC numbers (for instance, 250-fold and 49-fold
in the CLN and MLN, respectively, on day 10) indicates that the vast
majority of AFC induced by HV68 infection are not virus-specific.
Total AFC numbers were maximal in the CLN and MLN from days 8–12
postinfection and preceded the peak of the HV68-specific response by
approximately 5 days, suggesting that there may be subversion of the
humoral response to the virus by extensive, nonspecific B cell
activation. Interestingly, the isotype profiles of the virus-specific
and total Ig responses were remarkably similar, and there was no
increase in total IgA AFC numbers. Total AFC numbers in the CLN, MLN,
and spleen decreased gradually after 2–3 wk.
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HV68-specific AFC response in the BM was quite
distinct from that in the CLN, MLN, and spleen, where responses clearly
peaked and then declined. Virus-specific AFC appeared in the BM between
2 and 3 wk after infection, and numbers thereafter remained roughly
constant, with IgG2a and IgG2b predominating. In addition, a
significant increase in total IgG2a AFC in the BM
(p < 0.05 for day 15 and all subsequent
sampling times compared with day 3) followed the same kinetics as the
much smaller virus-specific AFC response in this location. Apparently,
the nonspecific B cell activation associated with HV68 infection
generates a long-sustained population of BM AFC secreting
non-virus-specific Ab.
Serum Ab responses. Serum levels of virus-specific Ab
(Figs. 3, A and B)
closely reflected the single cell analysis of the response. Specific
IgM levels peaked early and subsided, whereas IgG subclass Ab titers
increased rapidly from 1–3 wk after infection to a relatively stable
plateau. The kinetic analysis of the AFC response (Fig. 1) indicates
that the elevated virus-specific serum Ab levels were maintained in the
long-term by AFC in the BM, an observation consistent with studies of
other virus systems (37, 38, 39). No virus-specific serum IgA
was detected during the acute response, reflecting the absence of
significant numbers of specific IgA AFC in the respiratory lymph nodes
and spleen. Infection with HV68 induced increases in the total serum
levels of a number of Ab isotypes (Fig. 3, C and
D), but most striking was a greater than 10-fold increase in
the IgG2a concentration by day 10 after infection. Indeed, the increase
in serum IgG2a primarily accounts for the previously reported
HV68-associated increase in serum IgG levels (18). The
proportion of serum IgG2a that was HV68 specific was evaluated by
absorbing sera from infected mice with purified virus. Treatment of
pooled sera collected on day 10 after infection removed 70% of the
virus-specific IgG2a detected by ELISA. However, the total serum IgG2a
concentration was unaffected, indicating that most of the Ig produced
in the course of infection is not specific for the virus. Substantially
elevated serum IgG2a levels were maintained for the duration of
sampling, apparently due in large part to the non-virus-specific AFC
population that was established in the BM (Fig. 2).
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The production of non-virus-specific Ab in vivo following HV68
infection is dependent on CD4+ T cells
(19) and may be driven by cytokines, perhaps acting in
concert with virus infection of B cells. Previous studies of other
virus systems have identified IL-6 and IFN- as factors contributing
to the magnitude of the nonspecific Ab response (41, 42),
and high levels of these cytokines are generated in vivo by HV68
(43). IL-6 is well recognized as a factor acting on B
cells to induce Ig secretion (44), and the production of
IL-6 by B cells infected in vitro with HV68 (43)
suggests a role for the cytokine as a virus-induced autocrine growth
factor. IFN- induces Ig production by activated B cells
(45) and is also the major cytokine promoting class
switching to IgG2a (46). These functions suggest a
significant role for IFN- in the vigorous, IgG2a-biased, nonspecific
Ab response associated with HV68 infection.
Total AFC frequencies were determined in the respiratory lymph nodes of
IL-6-/-, IFN--/-,
and appropriate +/+ control mice at the peak of the response, and serum
IgG1, IgG2a, and IgG2b concentrations at this time were compared with
preinfection levels. Neither the isotype profile nor the magnitude of
the AFC response (Fig. 4,
A–C) were obviously modified in the
IL-6-/- mice. Total serum IgG2a and IgG2b
levels, but not IgG1 levels, were significantly increased
(p < 0.05) in IL-6-/-
and IL-6+/+ mice (data not shown). Apparently,
IL-6 is not required to drive the nonspecific Ig response. The absence
of IFN- had a much more profound effect, resulting in significantly
increased frequencies (p < 0.05) of IgA, IgG1,
and IgG2b AFC in both the CLN and MLN (Fig. 4, D and
E) and total AFC in the MLN (Fig. 4F). Serum IgG2a and IgG2b
levels were significantly increased (p < 0.05)
in both IFN--/- and
IFN-+/+ mice, with the rise in IgG2b being
particularly marked in the IFN--/- mice. In
addition, the serum IgG1 level was significantly increased
(p < 0.05) in the
IFN--/- mice, but not in the
IFN-+/+ mice (data not shown). The increase in
the nonspecific B cell response in the
IFN--/- mice could be thought to reflect
that this cytokine is inhibitory, though it is also the case that the
lytic phase of infection is controlled less rapidly in the absence of
IFN- (13, 34), so the magnitude of the antigenic
stimulus will be greater.
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The CD4+ T cell dependence of
non-virus-specific Ab production following HV68 infection in vivo
(19) indicates a requirement for T cell-derived factors
and/or cognate CD4+ T cell help to drive B cell
activation. A key molecular interaction in T-B collaboration is the
binding between CD40L, which is expressed on activated
CD4+ T cells, and CD40 on B cells
(47). The contribution of CD40-CD40L signaling to
nonspecific Ab production induced by HV68 was assessed in
experiments comparing CD40L-/- and
CD40L+/+ mice. Determination of total AFC
frequencies at the peak of the response in the respiratory lymph nodes
demonstrated absent or relatively weak responses in
CD40L-/- mice, in contrast to vigorous
responses in CD40L+/+ mice (Fig. 5, A and B). In a
second experiment using a lower virus dose (600 PFU) and sampling the
CLN and MLN on days 7, 15, and 35 postinfection, a strong, nonspecific
AFC response developed in +/+ mice, whereas total AFC numbers did not
increase in CD40L-/- mice (data not shown).
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HV68 infection is dependent on CD40-CD40L
interactions, this could be attributed to greatly reduced activation of
CD4+ T cells in CD40L-/-
mice (12, 48) and not necessarily to the contribution of
CD40-CD40L binding for cognate T-B help. Radiation BM chimeras were
therefore generated to provide a system in which complete
CD4+ T cell activation can occur, but specific,
contact-dependent CD4+ T cell help to drive B
cell responses is prevented by the absence of MHC class II expression
on B cells. Normally, cognate CD4+ T cell help
for thymus-dependent humoral responses is initiated by engagement of
the TCR with the Ag-MHC class II complex on the B cell. Chimeric mice
with MHC class II-deficient B cells were constructed by reconstituting
irradiated recipient B6 mice with donor BM from
C2D-/- (MHC class II-deficient) mice and µMT
(B cell-deficient) mice. BM elements from µMT mice will generate the
MHC class II-expressing APC required to stimulate
CD4+ T helper cells. Control +/+ chimeric mice
were produced as above, but with B6 BM substituted for
C2D-/- BM. The two sets of chimeras were
infected with HV68, and total AFC numbers were determined at the
peak of the response in the respiratory lymph nodes. Total AFC
frequencies remained at low levels in mice with MHC class
II-/- B cells, whereas a vigorous response
developed in the control animals (Fig. 5, C and
D). The cellularity of the lymph nodes at the time of
sampling was similar in the two sets of chimeras, as were the
proportions of B cells (CD19+) and
CD4+ and CD8+ T cells
demonstrated by flow cytometry (data not shown). As expected, all B
cells in the recipients of C2D-/- + µMT BM
were MHC class II-/-. The proportions of
activated (CD44high,
CD62Llow) CD4+ T cells were
also similar in the two groups (data not shown), an observation
consistent with recent evidence that the initiation of a
CD4+ T cell response does not require B cells
expressing class II molecules (49). Thus, even in the face
of apparently complete CD4+ T cell activation,
HV68-induced nonspecific B cell activation and Ab production
remained dependent on cognate help, perhaps as a prerequisite for the
participation of activated B cells in normal germinal center processes.
An implication is that the activated T cells are recognizing some
antigenic change on substantial numbers of B cells that are not
specific for viral components. Nonspecific Ab production reflects polyclonal B cell activation
Vigorous, non-virus-specific Ab production following HV68
infection is strongly suggestive of polyclonal B cell activation, a
feature of infection with a number of herpesviruses (41, 50, 51). The clonality of the nonspecific response to HV68 was
evaluated using isoelectric focusing to generate spectrotype profiles
of serum IgG2a Abs, since this isotype dominated the nonspecific
response. Spectrotyping of sera collected on day 10 after infection
established that the IgG2a increase elicited by HV68 represented a
very diverse molecular population (Fig. 6), indicating polyclonal B cell
activation. Indeed, virus infection amplified at least as many bands as
could be identified by spectrotyping IgG2a in sera pooled from 10
uninfected mice. Absorption of the day 10 sera with purified virus
removed 70% of the virus-specific IgG2a detected by ELISA, but had no
effect on the spectrotype, indicating that the bands reflect
nonspecific Ab production.
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HV68 generated an 10-fold increase in
total serum IgG2a levels within 2–3 wk, the ratio of - to
-containing IgG molecules remained constant during this period (data
not shown), a result consistent with extensive, nonselective B cell
activation.
Autoantibody induction by HV68
There is evidence that polyclonal B cell activation results in the
production of Abs directed against self components and may lead to the
development of systemic autoimmune disease (52). Sera
collected at different times following HV68 infection were thus
tested for IgG Abs reactive with mammalian dsDNA and murine CII.
Comparisons were made with sera from mice infected with Sendai virus, a
negative strand RNA virus that activates B cells nonspecifically
(29, 53). Infection with Sendai virus, like HV68,
induced a substantial and sustained increase in total serum IgG2a and
IgG2b concentrations, although the maximum IgG2a levels were 1.5x
higher following exposure to HV68 (data not shown). Significant
levels of anti-dsDNA and anti-CII Abs were present in more than
50% of mice from day 21 after HV68 infection and remained high in
some mice for at least 12 wk (Fig. 7,
A and B). The specificity of the anti-CII Abs
was demonstrated in an inhibition assay in which the preincubation of
serum with CII completely abolished high binding in the anti-CII
ELISA. No significant anti-dsDNA or anti-CII binding was
detected in sera from mice infected with Sendai virus. Total serum Ig
levels were relatively constant in HV68-infected mice from day 21
after challenge (Fig. 3D), indicating that the greater
reactivity to dsDNA and mCII in only a proportion of the mice cannot be
attributed to a general increase in assay background. This is also
indicated by the absence of any correlation between high levels of
binding on plates coated with distinct Ags (dsDNA, CII, or the
conventional Ag OVA). The induction by HV68 of IgG Abs reactive with
two distinct self Ags further supports the polyclonal nature of B cell
activation caused by the virus, and raises the possibility that
-herpesvirus infections may trigger autoimmune disease.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HV68 infection takes into account both the generation of
virus-specific AFC, and the nonspecific B cell activation that is a
feature of many virus infections (29) and may be
particularly relevant to the pathogenesis of the B lymphotropic
-herpesviruses. The specific response to HV68 had features in
common with the response to other respiratory viruses, namely, the
kinetics of Ab production in different anatomical compartments
(38), and the general predominance of the IgG2a and IgG2b
isotypes (38, 56). There was no indication that the
response was modified by potentially continuous antigenic stimulation
that may result from reactivation of HV68 from sites of latency in
the lung (14, 16) and lymphoid tissue (5).
The HV68-specific AFC response in the respiratory lymph nodes was
somewhat delayed and of a smaller magnitude compared with responses in
the same locations following infection with influenza or Sendai virus
(20, 38). Although this may reflect differences in the Ag
load, a significant factor may be the B cell tropism of HV68 and the
resultant loss of B cells by lytic infection (4),
apoptosis (19), or CD8+ T
cell-mediated clearance (8). The peak HV68-specific AFC
response in the CLN, MLN, and spleen was slightly preceded by a
dramatic increase in nonspecific AFC numbers, suggesting that
virus-driven, nonspecific B cell activation may also contribute to a
dampening of the specific humoral response. However, numerous viruses
including influenza and Sendai have the capacity to activate B cells
nonspecifically (29, 57), and factors such as the
mechanism and extent of this process may well determine the effect, if
any, on virus-specific Ab production.
A surprising feature of the Ab response to HV68, in marked contrast
to the response to other respiratory viruses (20, 28, 38),
was the almost complete absence of HV68-specific IgA AFC in the CLN
and MLN. The CLN in particular is recognized as a site biased toward
the production of IgA (58), and virus-specific IgA AFC
characteristically constitute a significant proportion of the response
in the CLN following i.n. immunizations with live or inactivated virus
(20). The processes by which IgA responses are generated
in the respiratory tract are not well understood, but there is evidence
(59) that B cells in close association with the
respiratory epithelium (60) are particularly important.
Conceivably, lytic infection of these cells at an early stage of
HV68 infection may subvert the induction of specific IgA AFC. The
response to HV68 in the CLN follows approximately the same kinetics
as that in the MLN and is well developed before any response is
apparent in nonrespiratory lymph nodes such as the axillary and
brachial (data not shown). This indicates that HV68 accesses the CLN
from the respiratory tract. The possibility that the virus replicates
in epithelial cells in the nasopharynx or other regions of the upper
respiratory tract that would be expected to drain to the CLN
(60) has not been analyzed. A possible scenario is that
HV68 spreads to the CLN via B cells that are directly infected in
the upper respiratory tract. There is recent evidence (61)
that EBV enters the human host by B cell infection alone
(62).
The HV68-specific response in the CLN, MLN, and spleen was
accompanied by a dramatic increase in the number of non-virus-specific
AFC (Figs. 1 and 2). Both the virus-specific and nonspecific responses
displayed similar kinetics and isotype profiles, consistent with
speculation (19) that the nonspecific response (like the
specific response) requires the participation of activated B cells in
normal germinal center processes. Surprisingly, HV68 infection
induced a substantial and long-sustained increase in nonspecific AFC
numbers in the BM. These cells presumably maintain the elevated levels
of circulating, non-virus-specific Ig. To date, the BM as a long-term
reservoir of AFC has been described only in the context of specific
responses to viral infection (37, 38, 39) or immunizations
with inert, thymus-dependent Ags (63, 64, 65). The observation
that HV68 infection generates increased numbers of
non-virus-specific AFC in the BM is further evidence that these cells
originate in germinal center reactions, since BM AFC appear to be
derived from cells that emerge from germinal centers (65, 66). It will be of interest to determine whether long-lasting,
nonspecific AFC populations in the BM also result from infection with
the many other viruses that are able to nonspecifically activate B
cells (29, 57). These viruses represent a broad range of
taxonomic groups, and the process of nonspecific B cell activation is
therefore unlikely to have a uniform mechanistic basis.
Experiments were conducted to define more precisely the requirements
for the CD4+ T cell-dependent production of
non-virus-specific Ab following HV68 infection (19).
Analysis of cytokine-deficient mice (Fig. 4) demonstrated that the
nonspecific Ab response is not dependent on IL-6 or IFN-. However,
nonspecific Ab production induced by HV68 was substantially
diminished in CD40L-/- mice and in chimeric
mice lacking MHC class II expression specifically on B cells (Fig. 5).
Engagement of the TCR with MHC class II-peptide on B cells cannot take
place in the chimeric mice, with consequent disruption of a series of
downstream events that normally lead to the participation of B cells in
germinal center reactions and the production of thymus-dependent Ab
(67). Perhaps pivotal in this process is up-regulation of
the transiently expressed CD40L on helper T cells that results from
signaling through the TCR (47). Our results indicate a
requirement for cognate CD4+ T cell help to drive
the nonspecific B cell activation associated with HV68 infection,
with CD40-CD40L interaction as an essential signaling component.
Signaling to B cells through CD40 is a prerequisite for normal germinal
center processes (68), and it follows that the production
of non-HV68-specific Ab may depend on the germinal center for B cell
proliferation and differentiation. A key question concerns the nature
of the CD4+ T cell help that drives the process,
which may well be the normal, Ag-specific CD4+ T
cell response. Conceivably, a virus-encoded molecule may mediate a
non-Ag-specific interaction between MHC class II on the surface of
infected B cells and the appropriate TCR on CD4+
T cells (69).
Our observations indicate that the production of nonspecific Abs that
is such a prominent feature of HV68 infection is a consequence of
generalized polyclonal B cell activation. In this regard, HV68 is
similar to a number of other herpesviruses including the human
pathogens EBV (50, 70) and CMV (51). Both EBV
and CMV also induce Abs that bind to host Ags (71, 72), a
consequence that may relate to the activity of these viruses as
polyclonal B cell activators (52). Herpesvirus-induced
polyclonal B cell activation and autoantibody production have been most
thoroughly studied in mice infected with murine CMV (MCMV). Although
some antigenic cross-reactivity between host and MCMV proteins has been
described (73, 74), the production of multiple
autoantibodies of different specificities during MCMV infection
(75) suggests that generalized polyclonal B cell
activation (41, 76) is largely responsible. Autoantibody
production in HV68-infected mice may also derive from nonselective,
polyclonal B cell activation. However, isotype-switched Abs reactive
with mammalian dsDNA and murine CII were generated in only a proportion
of mice infected with HV68 (Fig. 7, A and B),
suggesting the involvement of some stochastic processes. This feature
may indicate a role for Ag-driven selection in the production of some
autoantibodies, particularly anti-DNA. There is evidence
(77) that Abs specific for host dsDNA, and
non-cross-reactive with viral dsDNA, may be selectively induced by the
binding of virus-encoded proteins to cellular DNA. Serum levels of
anti-dsDNA and anti-CII Abs remained high in some
HV68-infected mice for the duration of sampling, raising the
possibility of autoantibody-mediated pathology, though a role for the
virus in the induction of autoimmune disease in the laboratory mouse
has not otherwise been investigated. It will be of interest to
determine whether the potentially long-lived (36),
non-virus-specific AFC in the BM generated by HV68 infection include
autoantibody-secreting cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark Y. Sangster, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail address:
3 Current address: Center for Vaccine Biology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642.
4 Current address: Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105.
5 Abbreviations used in this paper: HV68, murine gammaherpesvirus 68; AFC, Ab-forming cell(s); ALP, alkaline phosphatase; B6, C57BL/6J; BM, bone marrow; CII, type II collagen; CD40L, CD40 ligand; CLN, cervical lymph nodes; ELISPOT, enzyme-linked immunosorbent spot-forming (assay); HHV-8, human herpesvirus 8; i.n., intranasal(ly); MCMV, murine cytomegalovirus; MLN, mediastinal lymph node.
Received for publication September 23, 1999. Accepted for publication December 7, 1999.
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B6 and B6 + µMT 
