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*
Department of Patholoy, University of Massachusetts Medical School, Worcester, MA 01655; and
Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice and
by using SCID mice reconstituted with CD40-/- B cells,
that the lack of CD40 signaling in B cells results in a 50% decrease
in TI IgG secreted in response to PyV. SCID mice reconstituted with
CD40L-/- B cells also responded to PyV infection with
diminished IgG secretion compared with that of SCID mice reconstituted
with wild-type B cells. This finding suggests that B cells may provide
the CD40L for CD40 signaling in the absence of T cell help during acute
virus infection. Our studies demonstrate that, although about half of
the TI IgG responses to PyV are independent of CD40-CD40L interactions,
these interactions occur in T cell-deficient mice and enhance antiviral
TI Ab responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently we reported that polyomavirus (PyV) infection elicits protective TI IgM and IgG responses in T cell-deficient mice (9, 10). In contrast to infection with PyV, immunization with the viral capsid protein 1 (VP1) or with virus-like particles assembled from VP1 into highly organized, repetitive structures almost identical with those of the intact virions does not lead to TI IgG synthesis (11). These findings suggest that signals generated by live virus infection, such as the secretion of cytokines and the induction of membrane determinants on certain cell types, may be essential for generating efficient isotype-switched humoral responses in the absence of T cells.
The studies reported here were initiated to test whether CD40-CD40L
interaction plays any role in the antiviral TI IgG responses to PyV.
Although it was long believed that CD40L was expressed only on
activated CD4+ Th cells, CD40L recently has been
detected on the surface of CD8+ T cells, NK
cells, mast cells, basophils, eosinophils, dendritic cells, and human B
cells (12, 13, 14, 15, 16, 17, 18, 19, 20). Therefore, CD40L expressed on cells other
than T cells may provide costimulatory signals that have a positive
effect on TI Ab responses to viruses. In this report, we show by
blocking CD40-CD40L interactions in vivo with anti-CD40L Ab
treatment in TCR ßx-/- mice and by using
SCID mice reconstituted with CD40-/- B cells
that the lack of CD40 signaling in B cells results in a decrease in the
TI IgG secreted in response to PyV. Our data also suggest that B cells
themselves may provide functional CD40L for this interaction, because
SCID mice reconstituted with CD40L-/- B cells
also responded to PyV infection with diminished IgG secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and C57BL/CD40L-/- mice
were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/SCID
and C57BL/TCR ßx-/- mice were bred and
housed under specific pathogen-free conditions in the Department of
Animal Medicine of the University of Massachusetts Medical School.
C57BL/CD40-/- mice were kindly provided by Dr.
Hitoshi Kikutani (Osaka University, Osaka, Japan). Age-matched groups
of 6- to 19-wk-old mice were used for the experiments. The mice were
infected i.p. with 2 x 107 PFU PyV strain
A2, which was originally a gift from Dr. Michel Fluck (Michigan State
University, East Lansing, MI).
In vivo CD40L blocking
The mAb MR1 (21) (produced as ascites and kindly provided by Dr. Tom Markees, University of Massachusetts Medical School, Worchester, MA) was used to block CD40L in vivo. Mice were injected i.p. with 0.25 mg of MR1 on days -1, 2, 6, and 9 and were infected with PyV on day 0.
Adoptive transfers
Adoptive transfer of B cell-containing splenocyte populations of wild-type and mutant (CD40-/- and CD40L-/-) mice into C57BL/SCID mice was performed as described previously (9). Briefly, spleen cell suspensions were obtained by homogenizing spleens between frosted glass microscope slides, and the erythrocytes were lysed by treatment with 0.83% ammonium chloride. After in vitro T cell depletion (see below), the cells were pooled, and the number of viable cells was counted. Aliquots of the T cell-depleted splenocytes were stained with anti-B220-peridinin chlorophyl protein and anti-CD19-FITC or with anti-CD4-FITC and anti-CD8-PE (PharMingen, San Diego, CA) and were analyzed by FACS to determine the number of B cells and to test the quality of T cell depletion (see next paragraph). Spleens of C57BL/6 and CD40L-/- mice yielded comparable numbers of cells after T cell depletion (108/spleen and 1.3 x 108/spleen, respectively), and in the C57BL/6 and CD40L-/- adoptive transfer experiments, cells obtained from one spleen (on average) were injected i.v. into each SCID mouse. Spleens of CD40-/- mice yielded four to five times fewer T cell-depleted cells than C57BL/6 mice did; therefore, the same number of pooled T cell-depleted CD40-/- or C57BL/6 spleen cells were given to each SCID mouse (in the experiment shown, 1.12 x 107 cells/per mouse). The T cell-depleted C57BL/6 spleen cell population contained 60% B220+/CD19+ B cells, determined by FACS analysis, and the T cell-depleted CD40L-/- splenocytes and CD40-/- spleen cells contained 67.5% and 66%, respectively.
In vitro T cell depletions
Spleen cell suspensions were incubated with rat anti-mouse Thy1.2 Ab (PharMingen) for 45 min on ice, washed with RPMI 1640 containing 10% FCS, and then incubated with a 1:10 dilution of rabbit complement (Pel-Freez Clinical Systems, Brown Deer, WI) at 37°C for 45 min. The cell suspensions were washed repeatedly with media, subjected to one more round of Thy1.2 depletion, counted with trypan blue to obtain the viable cell count, suspended in PBS, and used in adoptive transfer experiments. The efficiency of T cell depletion was tested by FACS analysis employing anti-CD4-FITC and anti-CD8-PE Abs (PharMingen), and the samples were found to be 99% free of CD4+ and CD8+ T cells.
ELISA assays
Virus Ag-specific ELISA assays were done using purified VP1 PyV capsid Ag (50 ng/well) that was produced by recombinant baculovirus expression vectors in Sf9 insect cells and purified as described (22). The serum samples were tested in duplicates using biotinylated goat anti-mouse IgM or IgG and streptavidin-HRP (Vector Laboratories, Burlingame, CA) to detect IgM or IgG, respectively. A Southern Biotechnology Associates (Birmingham, AL) isotyping kit was used to measure IgG isotypes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab responses to PyV infection were tested in mice that had a
targeted mutation in the gene encoding CD40
(CD40-/- mice). Early IgM responses specific
for the PyV major capsid Ag VP1 were observed in
CD40-/- mice and were not significantly
different from those seen in C57BL/6 and TCR
ßx-/- mice (Fig. 1
A). On day 14 postinfection,
PyV-specific IgG was detectable in serum samples obtained from
CD40-/- mice (Fig. 1, B and
C). Comparison of the magnitude of this IgG response
indicated that the absence of CD40 signaling led to a 10-fold decrease
in virus-specific IgG titers, and this virus-specific IgG titer was
approximately the same in magnitude as that detected in TCR
ßx-/- mice (Fig. 1C). Thus,
about 10% of the antiviral IgG synthesis is independent of CD40
signaling in PyV-infected mice.
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mice decreases antiviral TI IgG
responsesMice lacking T cells respond to PyV infection with the generation of protective IgM and IgG Abs (10). Assuming that Th cells are the only cell type expressing significant levels of CD40L, the TI Ab responses should be completely independent of CD40 signaling. However, recent findings that CD40L can be expressed on cells other than T cells suggest the possibility that CD40L expressed on non-T cells might enhance TI Ab responses by interacting with CD40 on B cells.
The first approach to test whether CD40L signaling has an influence on
the TI IgG responses to PyV was to block in vivo CD40-CD40L
interactions in TCR ßx-/- mice using the
CD40L-specific mAb MR1. This Ab was administered to TCR
ßx-/- mice following the protocol and
dosage previously shown to successfully inhibit CD40-CD40L interactions
in immunocompetent C57BL/6 mice (see Materials and Methods
and Ref. 23). Serum samples taken on day 14 after PyV
infection from T cell-deficient anti-CD40L Ab-treated mice
consistently had 2-fold lower PyV-specific IgG levels than TCR
ßx-/- mice that did not receive treatment
to block CD40L (Fig. 2, A and
B). Treatment of C57BL/6 mice with the anti-CD40L Ab
resulted in the expected decrease in VP1-specific IgG, indicating the
efficient blocking of CD40L achieved by the in vivo administration of
the MR1 Ab in this experiment (Fig. 2A).
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-/-
mice treated with anti-CD40L Ab, similarly to the TCR ßx mice
that received no such treatment, showed no symptoms of PyV-induced
disease. DNA samples prepared from kidneys of PyV-infected TCR
ßx-/- mice treated or untreated with
anti-CD40L Abs were analyzed by Southern blot to test whether the
diminished IgG responses in the former group were associated with
differences of viral clearance. In these experiments no viral DNA was
detected in the kidney DNA samples of either group of mice, whereas DNA
from SCID mice kidneys had a strong PyV-specific band (data not shown).
Thus, the TI Ab responses generated in the absence of CD40-CD40L
interactions were sufficient for protection and viral clearance.
TI IgG responses to PyV are predominantly IgG2a and IgG2b with a
variable amount of IgG3 also synthesized, whereas the IgG1 response is
strictly TD (11). It is of interest that anti-CD40L Ab
treatment of TCR ßx-/- mice did not lead
to significant changes of this isotype composition.
CD40-/- B cells respond to PyV infection with diminished TI IgG synthesis in vivo
The other approach to test whether CD40 signaling in B cells
facilitates virus-specific TI IgG responses was to ask how a
CD40-/- B cell population will respond in the
absence of T cells to PyV in vivo, compared with the response of
wild-type B cells. To address this question, we performed adoptive
transfer experiments. Spleen cell suspensions prepared from
CD40-/- or C57BL/6 mice, respectively, were
depleted of T cells in vitro by treatment with anti-Thy1 Ab and
complement. These splenocyte populations devoid of T cells but
containing B cells were then transferred into SCID mice, and the
reconstituted mice were infected with PyV on the day after the cell
transfer. Virus-specific serum IgG levels were lower in mice that
received CD40-/- Thy-depleted spleen cells than
in mice reconstituted with wild-type cells at all three time points
tested: on day 14 (data not shown), day 21 (Fig. 3A), and day 37 (Fig. 3B) postinfection. These results confirmed the findings
obtained in the in vivo CD40L blocking experiments, suggesting that
CD40 signaling in B cells may enhance TI IgG responses to PyV. In
addition, these experiments raised an important question: in the
absence of T cells, which cell types provide CD40L for stimulating CD40
on B cells?
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We next tested whether B lymphocytes could stimulate each other by
expressing CD40L in the absence of T cell help. Spleen cells from
CD40L-/- mice were prepared and subjected to T
cell depletion in vitro. The resulting cell suspension was transferred
into B6/SCID mice, generating T cell-deficient mice with intact CD40L
gene in all cell types except in B cells. These mice, as well as SCID
mice reconstituted with T cell-depleted splenocytes from wild-type
(CD40L+/+) mice, were infected with PyV, and
their virus-specific Ab responses were measured. Remarkably, SCID mice
reconstituted with CD40L-/- B cells had
significantly lower VP1-specific IgG levels in their sera on day 21
(Fig. 4) as well as on day 56
postinfection (data not shown) than did SCID mice that had received
wild-type B cells. This difference in IgG production was not due to a
generalized defect of CD40L-/- B cell function
or to a lack of survival of CD40L-/- B cells
during adoptive transfer, because serum levels of virus-specific IgM
tested on day 5 were the same or higher in SCID mice with
CD40L-/- B cells than they were in the control
group of SCID mice reconstituted with wild-type B cells (Fig. 4A). The presence of functional Th cells in the
reconstituted mice was also excluded because their serum samples lacked
detectable VP1-specific IgG1 (day 21 serum samples; tested in 1:100
dilutions), and this isotype is strictly TD (6, 11).
Importantly, our results showing an 
50% drop in antiviral IgG
responses in SCID mice reconstituted with
CD40L-/- or CD40-/- B
cells, respectively, compared with the ones with wild-type B cells
suggested that B cells may provide functional CD40L to each other, thus
enhancing the antiviral TI IgG responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immune system evolved to provide protection against invading pathogens, and it reacts differently to infectious viruses than to inert Ags. Inert proteins are TD Ags, and the generation of isotype-switched Abs in response to these Ags requires T cell help (24). Accordingly, immunization with the PyV VP1 capsid protein or with its repetitively structured form assembled into virus-like particles does not induce VP1-specific IgG secretion in the absence of T cell help. In contrast, infection with PyV elicits protective TI IgG responses in T cell-deficient mice (11). Because infection with live viruses activates several cell types and induces numerous cytokines produced by cells other than T cells, these signals acting on B cells may enable them to secrete isotype-switched TI Abs.
The virus-induced signals and pathways involved in TI IgG synthesis in response to viruses are unknown. It is possible that in the absence of T cells, the "usual" T cell-derived helper signals are supplied by non-T cells. Alternatively, in the course of TI IgG responses, a different set of signals may activate distinct TI pathways of B cell activation, differentiation, isotype switching, and Ab secretion. Interaction of CD40 on B cells with CD40L on activated T cells is an essential component of T cell help, and it is required for several steps of TD Ab responses to noninfectious protein Ags (e.g., OVA or SRBC) (25). CD40-/- or CD40L-/- mice immunized with these Ags lack germinal center formation and isotype switching and do not establish B cell memory (2, 3, 4, 5). In this study, first we demonstrated that PyV infection leads to readily detectable virus-specific IgG synthesis in CD40-/- mice, although the antiviral IgG titer in the serum of CD40-/- mice is 10-fold lower than that in wild-type C57BL/6 mice. This finding is consistent with observations that other viruses, such as LCMV (6, 7) and vaccinia virus (7), induce virus-specific IgG responses in CD40L-/- mice that are 1–10% of the responses measured in immunocompetent wild-type mice. Thus, IgG responses to live, infectious viruses have a CD40- independent component.
Next, we questioned whether TI IgG responses to PyV previously observed
in TCR ßx-/- mice are completely
independent of CD40 signaling. Assuming that CD40L is expressed only on
Th cells, TI responses would be necessarily CD40-independent. However,
if cells other than T cells express CD40L in virus-infected mice, these
cells then may stimulate TI IgG responses. Our experiments suggest that
indeed this is the case in that TI IgG responses are reduced in the
absence of CD40 signaling by 50%. This means that although a
fraction (half) of the TI IgG responses are independent of CD40, the
induction of TI IgG responses is enhanced by CD40-CD40L
interactions.
Which cell types provide CD40L for these interactions? Importantly, the finding that B cells with a disrupted CD40L gene also produced decreased amounts of antiviral IgG when transferred into SCID mice may indicate indirectly that CD40L expression is induced on murine B cells in vivo by the virus infection. This result also suggests that B cells are able to provide "help" for each other when they encounter infectious Ags in the absence of Th cells. Alternatively, the lower IgG response to PyV in mice reconstituted with CD40L-/- B cells could indicate that B cells evolved in an environment entirely free of CD40L have altered effector functions. We feel that this interpretation of the data is less likely. The synthesis of same or slightly elevated levels of virus-specific IgM in mice carrying CD40L-/- B cells in comparison with mice that received normal B cells indicates that CD40L-/- B cells are able to respond to Ags with Ab secretion. Moreover, humans with the hyper IgM syndrome who carry a mutation in the gene coding for CD40L and therefore do not express functional CD40L have B cells that are fully functional in vitro (26).
Previous reports described the induction of CD40L in human peripheral B
cells in vitro by ionomycin and phorbol ester stimulation and on human
B cell lymphomas (20, 27). Moreover, most recently a study
demonstrated CD40L expression on human tonsillar B cells ex vivo and in
situ (28). In the mouse, hyperreactive B cells from
autoimmune male BXSB mice have been shown to express CD40L
(29). The induction of CD40L expression on murine B cells
in vitro by anti-IgM or anti-CD38 Ab treatment has also been
reported (30). Our data suggest that B cells may express
functionally active CD40L, which could enhance antiviral TI responses
in mice. It is an important task for future investigations to define
what are the signals inducing CD40L expression on B cells under these
circumstances. Thus far we have been unable to demonstrate the
induction of CD40L on splenic B cells of PyV-infected TCR
ßx-/- mice directly by FACS staining ex
vivo. A possible reason for this result is that only a very small
fraction of the B cells in the spleen may be activated by the
virus.
The CD40-CD40L interaction provides essential costimulatory second
signals for the activation of T and B cell responses to "inert" or
noninfectious Ags (27). However, some infectious agents
(such as LCMV, vaccinia virus, and B. burgdorferi) have the
ability to elicit isotype-switched Ab responses even in the absence of
CD40 signaling (6, 7, 8). IgG responses to PyV are 10-fold
lower in CD40-/- mice than in their wild-type
C57BL controls, and TI IgG titers (which amount to 10% of the IgG
responses seen in normal immunocompetent mice) have an additional
2-fold reduction in the absence of CD40 signaling. Remarkably, these
greatly diminished Ab responses are still sufficient to ensure
protection from PyV infection, in that TCR
ßx-/- mice treated with Abs against CD40L
survive the acute phase of PyV infection without visible signs of
PyV-induced disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to
Dr. Eva Szomolanyi-Tsuda, Department of Pathology, University
of Massachusetts Medical School, 55 Lake Avenue North,
Worcester, MA 01655.
3 Abbreviations used in this paper: CD40L, CD40
ligand; TD, T cell-dependent; TI, T cell-independent; LCMV, lymphocytic
choriomeningitis virus; PyV, polyomavirus; VP1, viral capsid protein
1.
Received for publication November 24, 1999. Accepted for publication March 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes. J. Exp. Med. 181:1239.This article has been cited by other articles:
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; n = 4), and C57BL/6 (
;
n = 3) mice, determined by ELISA assays. Mean OD +
SE shown; the OD values obtained in the assay with uninfected mouse
serum were below 0.13. B, VP1-specific serum IgG levels
on day 14 of CD40-/- (•; n = 3),
TCR ßx
; n = 3) mice. Mean + SE shown;
the OD values obtained with uninfected mouse serum in this assay were
below 0.07. The differences between the two groups are statistically
significant (p = 0.018 (1:200 dilution), 0.004
(1:400), and 0.005 (1:800)).