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Journal of Virology, April 2001, p. 3581-3589, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3581-3589.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of the CD154-Positive and
CD40-Positive Cellular Subsets Required for Pathogenesis in
Retrovirus-Induced Murine Immunodeficiency
Kathy A.
Green,
Randolph J.
Noelle,
Brigit G.
Durell, and
William R.
Green*
Department of Microbiology and Immunology,
Dartmouth Medical School and Norris Cotton Cancer Center, Lebanon,
New Hampshire 03756
Received 7 September 2000/Accepted 12 January 2001
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ABSTRACT |
Genetically susceptible C57BL/6 (B6) mice that are infected with
the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell
lymphomas, and an immunodeficiency state bearing many similarities to
the pathologies seen in AIDS. Because of these similarities, this
syndrome has been called murine AIDS (MAIDS). We have previously shown
that CD154 (CD40 ligand)-CD40 molecular interactions are required both
for the initiation and progression of MAIDS. Thus, in vivo anti-CD154
monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in
LP-BM5-infected wild-type mice when either a short course of anti-CD154
MAb treatment was started on the day of infection or a course was
initiated 3 to 4 weeks after LP-BM5 administration, after disease was
established. Here, we further characterize this required CD154-CD40
interaction by a series of adoptive transfer experiments designed to
elucidate which cellular subsets must express CD154 or CD40 for LP-BM5
to induce MAIDS. Specifically with regard to CD154 expression,
MAIDS-insusceptible B6 nude mice reconstituted with highly purified
CD4+ T cells from wild-type, but not from CD154 knockout,
B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast,
nude B6 recipients that received CD8+ T cells from
wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6
CD40 knockout mice, which are also relatively resistant to
LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease
after reconstitution with highly purified wild-type B cells but not
after receiving purified wild-type dendritic cells (DC) or a combined
CD40+ population composed of DC and macrophages obtained
from B6 SCID mouse donors. Based on these and other experiments, we
thus conclude that the cellular basis for the requirement for
CD154-CD40 interactions for MAIDS induction and progression can be
accounted for by CD154 expression on CD4+ T cells and CD40
expression on B cells.
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INTRODUCTION |
The LP-BM5 murine leukemia
retrovirus (MuLV) isolate causes an immunodeficiency syndrome in
genetically susceptible mice such as the highly susceptible C57BL/6
(B6) strain. This MuLV mixture includes ecotropic, recombinant mink
cell cytopathic focus-inducing, and replication-negative or -defective
viruses, with the defective genome serving as the proximal agent
causing the syndrome (3, 7, 18, 22, 36). Many of the
described disease features of this LP-BM5-induced mouse
immunodeficiency syndrome are similar to those seen in human
immunodeficiency virus (HIV)-infected individuals, hence the
designation murine AIDS (MAIDS). Noted disease similarities are
activation-related parameters such as hypergammaglobulinemia (hyper-Ig), splenomegaly, and lymphadenopathy; severely dampened T- and
B-cell responses; increased susceptibility to infection, disease
progression, and death upon exposure to environmental pathogens
that normally cause limited infections; and the development of terminal
B-cell lymphomas (5, 6, 26, 27, 29, 34, 37, 40, 53).
It has been suggested that CD4+ T cells and B cells are
required for LP-BM5 MAIDS induction (6, 53, 47). In these
experiments, in vivo depletion of CD4+ T cells before
LP-BM5 infection rendered genetically susceptible mice resistant to the
development of MAIDS (53). Similarly, after in vivo
depletion of B cells from birth in neonatal mice by the administration
of rabbit antibody to immunoglobulin M (IgM), followed by infection
with LP-BM5 virus, MAIDS failed to develop (6). These
studies, however, did not exclude the required involvement of other
cellular subsets in MAIDS pathogenesis and did not experimentally address the possible interaction of these subsets or the molecular basis for interaction.
We have recently examined the effect of CD154-CD40 interactions in
MAIDS initiation and progression (15, 16). CD154 is transiently expressed on activated T cells, especially murine CD4+ T cells after stimulation through the T-cell receptor
for antigen (2, 39), and once expressed serves as the
ligand for CD40, a 50-kDa membrane signaling protein responsible for
driving B-cell activation and differentiation to antibody secretion
(51). In view of the fact that CD154-CD40 interactions had
been shown to be important in a wide array of immunological responses
(1, 23, 44), we treated LP-BM5-infected B6 mice in vivo
with blocking anti-CD154 monoclonal antibody (MAb), either during the
first week of infection (15) or chronically, starting at 3 to 4 weeks after LP-BM5 infection (16). Both time courses
of administered anti-CD154 MAb were very effective in inhibiting
MAIDS-associated splenomegaly, hyper-Ig, and immunodeficiency. The data
from these two experimental approaches strongly suggested that
CD154-CD40 interactions are necessary for both the initiation and the
progression of LP-BM5-induced MAIDS.
In the study presented here, we further characterize the CD154-CD40
interaction in MAIDS by a series of in vivo adoptive transfer experiments designed to elucidate which cellular subsets in a susceptible B6 mouse must express CD154 or CD40 for LP-BM5
virus-induced pathogenesis. Progression to MAIDS was evaluated in
reconstituted, LP-BM5-infected nude CD154 knockout (k.o.) and CD40 k.o.
mice, all on the B6 genetic background, by the following standard
readouts of MAIDS-associated symptoms, which we (15, 16)
and others (3, 5, 6, 7, 18, 21, 36) have previously
established: (i) spleen size, with enlargement indicating
MAIDS-associated B- and T-cell lymphoproliferation; (ii) serum IgG2a
levels, with increases due to MAIDS polyclonal B-cell activation; (iii)
splenic B-cell responses to lipopolysaccharide (LPS) stimulation, with decreases indicative of B-cell-associated MAIDS-induced
immunodeficiency; and (iv) T-cell responses to either concanavalin A
(ConA) mitogen stimulation or allogeneic major histocompatibility
complex stimulation (as determined by cytotoxic T-lymphocyte [CTL]
generation), with decreases indicative of MAIDS-induced T-cell immunodeficiency.
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MATERIALS AND METHODS |
Mice.
Seven-week-old male B6 mice were purchased from the
National Institutes of Health (Bethesda, Md.), housed in the Dartmouth Medical School Animal facility, and used when 8 to 10 weeks of age as
control mice or spleen cell donors. Breeding pairs of CD154 k.o. mice
(fully backcrossed to B6) were obtained from the Jackson Laboratory
(Bar Harbor, Maine); the mice were originally derived by Immunex
Corporation (Seattle, Wash.) as previously described (45).
B6-backcrossed CD40 k.o. breeding pairs, derived as reported elsewhere
(24), for initial experiments were obtained from Anthony Hayward's laboratory (University of Colorado Health Science Center, Denver); mice for later experiments were obtained from the Jackson Laboratory. Six-week-old B6 nude mice were purchased from the Jackson
Laboratory. Equal numbers of nude and k.o. mice, either three or four
in a given experiment, were reconstituted with various cell
preparations (see below) before infection with LP-BM5 virus. B6 SCID
mice, purchased from the Jackson Laboratory were used at 6 weeks of age
as spleen cell donors in a reconstitution experiment.
Cell lines.
LB27.4, a murine B-cell hybridoma
(H-2b,d), and P815, a murine
mastocytoma (H-2d), were cultured at
37°C with 5% CO2 in RPMI 1640 with 5% fetal calf serum
(FCS), L-glutamine, 2-mercaptoethanol and antibiotics (Gibco BRL, Life Technologies, Grand Island, N.Y.).
LP-BM5 virus inoculations.
LP-BM5 was prepared in our
laboratory as previously described (26). G6 cells, cloned
from SC-1 cells infected with the LP-BM5 virus mixture and originally
provided by Janet Hartley and Herbert Morse, were cocultured with
uninfected SC-1 cells. Mice were infected intraperitoneally with 0.25 ml of a virus stock which was quantitated by an XC plaque assay
(46) to contain approximately 5 × 105
ecotropic PFU/ml and which postinoculation caused MAIDS-related splenomegaly, as shown by at least a doubling of spleen weight as early
as 4 weeks after inoculation into B6 mice.
ELISA determinations of serum Ig.
For measuring hyper-Ig,
affinity-purified goat anti-mouse IgG2a antibody (Southern
Biotechnology Associates, Birmingham, Ala.), was diluted to 7 µg/ml
in phosphate-buffered saline (PBS) to coat 96-well enzyme-linked
immunosorbent assay (ELISA)-grade plates (Becton Dickinson, Oxford,
Calif.) overnight at room temperature. The plates were then washed
three times with PBS and blocked for 1 h with 5% bovine serum
albumin-PBS (Sigma, St. Louis, Mo.) at 37°C. Sera obtained at the
termination of all LP-BM5 infection experiments were then plated and
allowed to incubate for 2 h at 37°C. The plates were washed
three times with PBS, and alkaline phosphatase-labeled goat anti-mouse
Ig (Southern Biotechnology Associates) was added. After a 2-h, 37°C
incubation, p-nitrophenyl phosphate (Sigma) provided a
colorimetric change which was then quantitated at 405 nm by an ELISA
reader (Dynatech Laboratories, South Hampton, United Kingdom).
Spleen cell responses to mitogens.
Spleen cells (4 × 105) from control and experimental mice were plated in
triplicate into 96-well plates with medium containing 5% FCS,
L-glutamine, antibiotics, and a final concentration of 2 µg/ml for ConA or 10 µg/ml for LPS. After 72 h, all wells were pulsed with 1 µCi of [3H]thymidine (Dupont NEN, Boston,
Mass.) and harvested (Packard, Meriden Conn.) 6 h later for assessment
of thymidine incorporation by scintillation counting (Packard).
Generation of primary allogeneic CTL (allo-CTL) and
51Cr release assays.
Responder splenocytes
(107) from experimental or control mice were mixed with
irradiated (8,000 rad) LB27.4 tumor cells at a responder-to-stimulator
ratio of 35:1 in RPMI 1640 containing 5% FCS, antibiotics, and
L-glutamine. These cultures were maintained at 37°C in a
5% CO2 incubator for 6 days. P815 target cells were resuspended in 100 µl of FCS and labeled with 100 µl of sodium chromate (2 mCi/ml; Dupont NEN). After washing, 5 × 103 51Cr-labeled P815 target cells were plated
in duplicate with various numbers of effector cells to achieve
effector-to-target ratios of 100:1, 20:1, and 4:1. The plates were
centrifuged briefly and incubated at 37°C in 5% CO2 for
4 h. Aliquots of cell-free supernatant (100 µl) were collected
for counting on a gamma counter (Wallac, LKB Gaithersburg, Md). Percent
specific lysis was defined as [(a 
b)/c] × 100,
where a is experimental cpm released, b is
spontaneous cpm released, and c is freeze-thaw releasable
(about 80% of total) cpm. The values for spontaneous target cell
release were 15% or lower, with the duplicate experimental percent
specific lysis values ranging ± 10% of the mean value.
Splenocyte subpopulation preparation.
Splenocyte suspensions
were labeled with antibody-coupled paramagnetic beads (MACS; Miltenyi
Biotec, Auburn, Calif.) and subjected to column purification according
to the manufacturer's protocol. The following purified cellular
subsets were used in reconstitution experiments.
(i) CD4+ T-cell preparations.
Spleen cell
suspensions were incubated with anti-CD8a beads, positively selecting
for CD8+ T cells. The flowthrough cellular preparation was
then labeled with anti-CD4 (L3T4) beads, and positive selection yielded
cell preparations which were 
90% CD3+ CD4+,
7% B220+, and 1% CD8+ as detected by flow
cytometric analysis.
(ii) CD8+ T-cell preparations.
Spleen cell
suspensions were incubated with anti-CD4 (L3T4) beads, and the
flowthrough of this positive selection was then incubated with
anti-CD8a beads, with positive selection yielding cell preparations
which were 89% CD8a+, 9% B220+, and 1%
CD4+.
(iii) B-cell preparations.
Spleen cell suspensions were
incubated with a cocktail of anti-CD43 (Ly48, leukosialin) and
anti-CD11c magnetic beads. The flowthrough of this positive selection
was then positively selected with anti-CD19 magnetic beads and yielded
cell preparations which were 98% CD19+.
(iv) DC preparations.
Spleen cell suspensions were obtained
from B6 mice which had received injections of 10 µg per day of
purified Flt3 ligand subcutaneously for 9 days. These cell suspensions
were incubated with mouse Ig as specified by the manufacturer (Miltenyi
Biotech) to block Fc receptor-mediated magnetic labeling of macrophages and subsequently with anti-CDIIc paramagnetic beads. After column purification, positively selected fractions were evaluated by direct
immunofluorescence for cell surface markers as described for DC subsets
in Flt3 ligand-treated mice (43) and were 71% CDIIc+ I-Ab+, 18% CDIIc+
I-Ab
, and 68% CD40+. Based on the reported
number of DC present in a collagenase-treated spleen from a
non-Flt3-treated mouse (0.5% of the viable lymphocytes) (8), we reconstituted each experimental CD40 k.o. mouse
with >10 DC splenic equivalents.
SCID spleen cell preparation.
A cell suspension prepared
from SCID spleens contained no detectable CD4-, CD8-, or
CD19-expressing cells and was 19% CD40+ CDIIc+
and 27% CD40+ CD1lb+ by flow cytometric analysis.
All of the above cell preparations were injected intravenously via the
tail vein, and those recipient mice whose transfers we rated
technically as suboptimal were not used in these experiments.
PCR amplification.
DNA was extracted from splenic tissues of
reconstituted and nonreconstituted CD40 k.o. mice and wild-type (w.t.)
B6 mice as specified by the manufacturer (Qiagen Inc., Valencia,
Calif.). PCR amplification was performed with primers specific for the CD40 gene product (CD40upG, 5'-dGGCAGTAAGACGATGTGACAACAGAG-3'; CD40Holo, 5'-dGAGATGAGAAGGAAGAATGGGAAAAC-3') according
to standard procedures. The predicted 2,100-bp band was detected for
all CD40 k.o. mice, and a 900-bp band indicative of w.t. CD40 was
detected only from amplified DNA samples obtained from control w.t. B6 mice.
Flow cytometry.
Spleen cells were incubated with fluorescein
isothiocyanate (FITC)- or phycoerythrin-conjugated antibodies, and
direct immunofluorescence by linear amplification (FACScan) was used to
detect the expression of murine CD4 (L3T4), CD8a (Ly2), CD3e, CD40,
Thyl.2 (CD90.2), B220, CD19, CD11b, CD11c, I-Ab, NK1.1
(NKR-PIC Ly-55), CD43 (Ly48, leukosialin), and CD5 (Ly-1) (Pharmingen,
San Diego, Calif.). The appropriate FITC- or phycoerythrin-conjugated Ig isotype of irrelevant specificity were used to control for each antibody.
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RESULTS AND DISCUSSION |
Determination of which cellular subset(s) must express CD154 for
MAIDS pathogenesis.
We challenged B6 CD154 k.o. mice with LP-BM5
virus and found that, as with anti-CD154 MAb-treated LP-BM5-infected B6
mice from our previous studies (15, 16), such k.o. mice
are resistant to developing MAIDS. As shown in Fig.
1, LP-BM5-infected CD154 k.o. mice did
not exhibit any indications of LP-BM5-induced MAIDS. LP-BM5-infected
and uninfected B6 CD154 k.o. mice were essentially the same with
respect to spleen weights, terminal IgG2a values, and spleen cell
mitogenic responses to LPS and ConA. Positive control LPBM5-infected B6
mice, in this same experiment and consistent with our previous reports
(15, 16), compared to uninfected B6 (or CD154 k.o.) mice,
had approximately three- to fivefold-larger spleens,
severalfold-increased IgG2a values, completely or partially inhibited
LPS mitogen responses, and totally inhibited ConA responses. Thus,
taken together with our anti-CD154 MAb blocking studies, these results
confirmed that functional CD154 expression was required for
LP-BM5-induced MAIDS pathogenesis.
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FIG. 1.
B6 CD154 k.o. mice do not develop MAIDS after LP-BM5
infection. B6 w.t. or CD154 k.o. mice infected with LP-BM5 virus were
sacrificed 8 weeks later. LP-BM5-infected w.t. B6, but not CD154 k.o.,
mice exhibited all of the positive readouts denoting LP-BM5-induced
MAIDS which were statistically different from those responses seen for
uninfected mice; splenomegaly, hyper-IgG2a, and diminished or
nonexistent spleen cell response to LPS or ConA stimulation. Each bar
represents a value for an individual mouse. P values were
derived by the Student t test and are represented as
follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS (nonsignificant),
P > 0.05. This experiment is representative of three
other experiments.
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To determine which cellular subset(s) that expresses CD154 is required
for induction of MAIDS, various in vivo reconstitution
experiments were
undertaken. The first approach involved reconstituting
B6 CD154 k.o.
mice with purified CD4
+ T cells (see Materials and Methods)
from w.t. B6 donors, since
CD154 expression has been found most
prominently on the CD4
+ T subpopulation. In three different
experiments, approximately
70% of the CD4
+
T-cell-reconstituted CD154 k.o. mice developed some clear signs
of
MAIDS after LP-BM5 challenge (data not
shown).
Because the results from this experimental approach were only
suggestive that CD4
+ T cells were the basis for the CD154
requirement in MAIDS pathogenesis,
we alternatively reconstituted
T-cell-deficient B6 nude mice (
41),
in which there was no
possibility of dilution of w.t. donor, with
resident k.o. recipient,
CD4
+ T cells. Nude mice, in particular B6 nude mice, have
previously
been shown to be resistant to MAIDS induction (
37,
47). CD4
+ T cells positively selected from B6 w.t.
or CD154 k.o. spleen
cell suspensions were transferred 2 days before
inoculation with
LP-BM5 virus. In four different experiments, only the
CD154
+ CD4
+ T-cell-reconstituted,
LP-BM5-infected nude mice exhibited MAIDS,
and this was consistent
across all disease readouts examined.
This result was in sharp contrast
to that for the LP-BM5-infected
mice which had received
CD154
CD4
+ T cells. For example, as shown by
the representative experiment
in Fig.
2,
comparision of the mean responses for the uninfected
and infected
CD154
+ CD4
+ T-cell-reconstituted nude mice
showed that LP-BM5 infection led
to spleen weights that were
approximately 7-fold higher and serum
IgG2a levels around 5,000-fold
higher, and the LPS mitogenic response
was almost completely inhibited.
These MAIDS-related changes in
the LP-BM5-infected, CD154
+
CD4
+ T-cell-reconstituted mice were also significantly
different from
results for both the infected nude mice that were not
reconstituted
and those that received CD154
CD4
+ T cells prior to infection (Fig.
2). In addition, the
ConA-dependent
mitogenic response, another index of LP-BM5-induced
immunodeficiency,
also demonstrated the critical expression of CD154 by
the CD4
+ T-cell population for MAIDS pathogenesis. Thus,
after infection,
the ConA response for the CD154
+
CD4
+ T-cell-reconstituted nude mice was statistically
significantly
lower (
P = 0.0146) than the response for the
CD154
CD4
+ T-cell-reconstituted mice (data
not shown). Of note, we have
reported in Fig.
2 and throughout the
subsequent experiments all
readout responses that we have routinely
used to define MAIDS
pathology (Fig.
1) except those that directly
measure the responsiveness
of the specifically transferred cell subset.
These responses,
although showing the same trend as the other disease
parameters,
were somewhat more variable, possibly because of slightly
different
effective cellular reconstitutions on a mouse-to-mouse basis.
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FIG. 2.
LP-BM5-infected B6 nude mice, which are normally MAIDS
resistant, become MAIDS susceptible after reconstitution with
CD154+ CD4+ T cells but not CD154
CD4+ T cells. B6 nude mice were intravenously reconstituted
with 2 × 107 purified B6 w.t. CD154+ or
CD154 CD4+ T cells and 2 days later infected
with LP-BM5 virus. At 8 weeks after LP-BM5 infection, only the
CD154+ CD4+ T-cell-reconstituted mice had
developed all of three positive disease readouts indicating MAIDS
susceptibility (splenomegaly, hyper-IgG2a, and diminished spleen cell
response to LPS), which were all statistically different from those
obtained for control, reconstituted, uninfected mice and
CD154 CD4+ T-cell-reconstituted,
LP-BM5-infected nude mice. Each bar represents a mean value ± standard deviation. P values were derived as detailed in the
legend to Fig. 1. There were three mice for the reconstituted,
uninfected group and four mice for the reconstituted, LP-BM5-infected
group. This experiment is representative of three other experiments.
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In contrast to CD154
+ CD4
+ T-cell-reconstituted
nude mice, in uninfected and infected CD154
CD4
+ T-cell-reconstituted mice, the mean spleen sizes and
LPS responses
(Fig.
2) and ConA responses (data not shown) were
essentially
the same, indicating that MAIDS was not induced. The
10-fold elevation
of the serum IgG2a level detected for the infected
mice was minimal
compared to the 5,000-fold elevation seen in the
CD154
+ CD4
+ T-cell-reconstituted and
LP-BM5-infected nude mice, and approximately
10-fold increases in IgG2a
levels were also found for the nonreconstituted,
infected nude mice.
This pattern of slight IgG2a elevation, compared
to the level for the
CD154
+ CD4
+ T-cell transfer and infected group,
was also observed in the
three repeat experiments conducted and may
represent the reported
LP-BM5 virus-dependent but MAIDS-independent
increases in multiple
Ig isotypes that have been observed by two other
laboratories
in different k.o. mouse models (
35,
54; see
below). Similarly,
there was a slight (statistically
nonsignificant) increase in
spleen weight for infected
CD154
CD4
+ T-cell-reconstituted nude mice,
but a similar (and somewhat greater)
increase was observed for
nontransferred infected nude mice. FITC
anti-CD4 labeling of spleen
cells obtained on the day of sacrifice
from CD154
+ and CD
154
donor CD4
+ T-cell-reconstituted nude
mice, followed by flow cytometric analysis
(see Materials and Methods),
ruled out the possibility that the
MAIDS-insusceptible
CD154
CD4
+ T-cell-transferred mice were not
successfully reconstituted with
CD4
+ T cells (data not
shown). From these data, we can conclude that
CD4
+ T cells
are required and must express CD154 for LP-BM5-induced
MAIDS
pathogenesis.
To test the possibility that cell subsets other than CD4
+ T
cells might also provide the CD154 expression requisite for MAIDS
pathogenesis, we examined CD8
+ T cells, a portion of which
have been reported to express CD154
in response to antigenic
stimulation (
9,
19). We reconstituted
B6 nude mice with
w.t. CD8
+ T cells (see Materials and Methods). In two
experiments, no CD8
+ T-cell-reconstituted, LP-BM5-infected
nude mice presented with
any positive readouts indicating
LP-BM5-induced MAIDS. As shown
by the representative experiment in Fig.
3, LP-BM5-infected, w.t.
CD8
+
T-cell-reconstituted nude mice exhibited mean responses for spleen
size, serum IgG2a levels, and LPS stimulation that were not
significantly
different from the mean responses for control,
uninfected, CD8
+ T-cell-reconstituted nude mice. Spleen
cells from the CD8
+ T-cell-reconstituted, LP-BM5-infected
nude mice were also able
to mount an allo-CTL response (see Materials
and Methods) that
was not significantly different from that for
uninfected CD8
+ T-cell-transferred nudes (
P = 0.1237 [data not shown]). Compared
to the LP-BM5-infected, w.t.
CD8
+ T-cell-reconstituted mice represented in Fig.
3,
LP-BM5-infected,
CD154
+ CD4
+ but not
CD154
CD4
+ T-cell-reconstituted nude mice,
which were also included in the
experiment, exhibited the clear signs
of MAIDS as shown in Fig.
2 (data not shown). Indeed, the responses of
the CD154
CD4
+ and w.t. CD154
+
CD8
+ T-cell-reconstituted and LP-BM5-infected mice were
similar. These
data strongly suggest that w.t. CD8
+ T cells
are not capable of providing the CD154 expression required
for
LP-BM5-induced MAIDS pathogenesis.
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FIG. 3.
CD154+ CD8+ T-cell-reconstituted
B6 nude mice remain MAIDS insusceptible after LP-BM5 infection. B6 nude
mice were reconstituted with 107 purified
CD154+ CD8+ T cells and 2 days later were
infected with LP-BM5 virus. At 8 weeks postinfection, all recipient
mice were sacrificed and the indicated disease parameters indicative of
MAIDS were determined. Each bar represents a mean value ± standard deviation. This is representative of one other experiment.
P values shown were derived by the Student t test
(NS [not significant], P > 0.05).
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Thus, CD4
+ T cells appear to be uniquely able to provide
the CD154 expression required for induction and progression of MAIDS.
This finding is consistent with a variety of studies indicating
defects
in priming and/or expansion of CD4
+ T cells in CD154 k.o.
mice (
17,
30,
33,
49,
52). Whether
this requirement for
CD154 in these previous studies and the present
study reflects a
signaling defect normally mediated by CD154 to
directly activate the
CD4
+ T cell per se, as was originally suggested in some
early studies
(
17,
52), or a failure to activate and
mature CD40
+ B cells or professional antigen-presenting
cells to function
and/or traffic appropriately (
33) was
unclear. However, because
the bulk of the literature now generally
favors an effect mediated
by the lack of signaling through CD40
(
30), we focused next
on identification of the possible
CD40
+ cell subsets required for
MAIDS.
Determination of which cellular subset(s) must express CD40 for
MAIDS pathogenesis.
We and others (54) have found
that, like CD154 k.o. mice, B6 CD40 k.o. mice are resistant to
LP-BM5-induced MAIDS, although the degree of resistance is somewhat
less dramatic than for CD154 k.o. mice. These findings provided
confirmation of our conclusion that CD154-CD40 interactions are
required, based on the results from our studies showing an interference
with MAIDS initiation and progression by treatment with blocking
anti-CD154 MAb (15, 16). CD40, originally identified as a
B-cell molecule, has also been reported to be expressed prominently by
DC and macrophages (48), as well as by other cell types.
To determine which cellular subsets must express CD40 for
LP-BM5-induced MAIDS pathogenesis, we performed parallel
reconstitutions of normally MAIDS-resistant B6 CD40 k.o. mice with
three different B6 background-derived sources of CD40+
cells: (i) highly purified CD40+ B cells; (ii) purified
CD40+ DC; and (iii) B6 SCID spleen cells, representing a
combined population of CD40+ DC and macrophages. We found
that only CD40+ B-cell-reconstituted CD40 k.o. mice became
MAIDS susceptible after LP-BM5 infection. Thus, compared to the minimal
to moderate effects seen for LP-BM5 infection of nonreconstituted CD40
k.o. mice, CD40+ B-cell-reconstituted, LP-BM5-infected CD40
k.o. mice exhibited MAIDS by statistically significant differences for
mean spleen size, serum IgG2a levels, allo-CTL responsiveness (Fig. 4),
and LPS responsiveness (P = 0.0272, data not shown). Of
note, the observed serum hyper-Ig levels were 2 fold higher than that
seen in LP-BM5-infected B6 control mice (data not shown). These
responses for CD40+ B-cell-reconstituted, LP-BM5-infected
mice were also statistically different from those obtained for
CD40+ DC-reconstituted, LP-BM5-infected CD40 k.o. mice (see
also below). A similar pattern of results was found in another
experiment in which CD40 k.o. mice were again reconstituted with 7 × 107 CD40+ B cells, representing
approximately 1.1 splenic equivalents, before they were infected with
LP-BM5. In two earlier experiments in which CD40 k.o. mice were
reconstituted with either 2 × 107 or 5 × 107 CD40+ B cells, LPBM5-induced MAIDS also
occurred, but there was more mouse-to-mouse variability.
To address the possible ability of alternative CD40-expressing cellular
subsets to provide the CD40 expression required for
MAIDS pathogenesis
in the absence of CD40-expressing B cells,
we reconstituted CD40 k.o.
mice in parallel with 5 × 10
6 (> 10 splenic
equivalents) purified CD40
+ DC. No MAIDS induction was
observed in such CD40
+ DC-reconstituted CD40 k.o. mice 11 weeks after LP-BM5 infection
compared to nontransferred,
LP-BM5-infected CD40 k.o. controls
(Fig.
4). In addition to the allo-CTL data,
there was no evidence
for immunosuppression based on LPS responsiveness
(
P = 0.1066
[data not shown]). A repeat experiment in
which more (10
7) DC were used for reconstitution produced
the same result: all
LP-BM5-infected, DC-reconstituted CD40 k.o. mice
had spleen weights,
IgG2a levels, and LPS and allo-CTL responses
similar to those
seen in LP-BM5-infected, nonreconstituted CD40 k.o.
mice. Of note,
CD40
+ CD11c
+ donor DC were
detected in about half of the mice by flow cytometric
analysis on the
day of sacrifice in the spleens of reconstituted
mice (mean delta
percent positive of 1 to 2%). These results were
confirmed by the
presence of the expected 900-bp band indicative
of w.t. CD40 following
PCR amplification of DNA also extracted
at sacrifice from the
reconstituted recipients (see Materials
and Methods). While it seems
likely that more dramatic evidence
controlling for the presence of the
donor DC populations would
have been obtained if recipients could have
been analyzed before
their sacrifice at 11 weeks after transfer and
infection, and
considering that because no disease occurred, there was
no expansion
of the transferred cells, we take these results to be
reasonable
evidence of the success of the reconstitution (see also
Materials
and Methods regarding the transfers).
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 4.
MAIDS-resistant LP-BM5-infected B6 CD40 k.o. mice become
MAIDS susceptible after reconstitution with CD40+ B cells
but not after reconstitution with CD40+ DC. CD40 k.o. mice
were intravenously reconstituted with 7 × 107 highly
purified B6 CD40+ B cells or 5 × 106
CD40+ purified DC obtained from Flt3-treated B6 mice. Two
days later they were infected with LP-BM5 virus, and at 11 weeks
postinfection, the recipients were sacrificed for the indicated disease
parameters indicating MAIDS susceptibility: splenomegaly, hyper-IgG2a,
and diminished allo-CTL responses. Serum IgG2a levels for
LP-BM5-infected nonreconstituted CD40 k.o. mice are higher than those
for noninfected k.o. mice, apparently in accordance with the published
observation that LP-BM5 causes hyper-IgG2a due to a virus-dependent but
non-MAIDS-related isotype class switching in CD40 k.o. mice
(54). Each bar represents the mean response ± standard deviation. P values were derived as detailed in the
legend to Fig. 1. This experiment is representative of one other. A
less consistent but similar trend of responses was seen in two other
experiments in which 2 × 107 and 5 × 107 purified CD40+ B cells were transferred
(see text). E:T, effector:target.
|
|
As an alternative approach to assessing non-B-cell sources of
CD40
+ cells for their possible role in MAIDS, CD40 k.o.
mice were alternatively
reconstituted with 5 × 10
6 B6 SCID (
4) spleen cells as a source
of both CD40
+ DC and macrophages. Of note, SCID mice have
previously been shown
by Simard et al. to be resistant to induction of
MAIDS, albeit
in experiments using the alternative Du5H (Du5H/Mo-LTR)
rescued
chimeric defective virus (
47). Such transferred
SCID mouse spleen
cell subsets, which have been reported as competent
antigen-presenting
cells (
10), did not render CD40 k.o.
recipients MAIDS susceptible.
Thus, there were no positive readouts
indicating LP-BM5-induced
MAIDS at 11 weeks after LP-BM5 infection
(Fig.
5).
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 5.
B6 CD40 k.o. mice do not develop LP-BM5-induced MAIDS
after reconstitution with a cell preparation enriched for
CD40+ DC and macrophages. CD40 k.o. mice were intravenously
reconstituted with 5 × 106 spleen cells obtained form
B6 SCID mouse donors; they were infected with LP-BM5 2 days later and
assessed for the indicated MAIDS disease parameters at 11 weeks
postinfection. Each bar represents the mean ± standard deviation.
Serum IgG2a levels for LP-BM5-infected nonreconstituted CD40 k.o. mice
are higher than those for uninfected k.o. mice, apparently in
accordance with the published observation that LP-BM5 causes hyper-IgG1
and -IgE, (54) due to a virus-dependent but
non-MAIDS-related isotype class switching in CD40 k.o. mice.
P values were derived by the Student t test (NS
[nonsignificant], P > (0.05). E:T,
effector:target.
|
|
In conclusion, collectively these data strongly suggest that with
respect strictly to the CD154
+ and CD40
+
cellular requirements for disease, CD4
+ T cells expressing
CD154 and B-cells expressing CD40 are necessary
and sufficient for
LP-BM5 MuLV-induced MAIDS pathogenesis. Further,
in the absence of
CD40
+ B cells, macrophages, and/or DC, despite their CD40
positivity,
are unable to provide the functional CD40 expression for
LP-BM5-induced
MAIDS. However, we cannot exclude a role for macrophages
or DC
in MAIDS pathogenesis in a manner that does not involve their
display of CD40. Of note, flow cytometric analyses indicated that
the
highly purified B-cell preparations, which allowed for LP-BM5
induction
of MAIDS in reconstituted CD40 k.o. mice, were negative
not only for
CD11c- or CD11b-expressing DC or macrophages but
also for
CD5
+ CD19
+ double-positive B cells. Thus, it
would appear from our data
that this latter B-la subset of B cells is
not essential as the
cellular basis of the CD40 requirement in MAIDS.
This finding
is relevant to the ongoing debate as to the importance of
B-la
B cells in MAIDS pathogenesis (
20,
50). Our data
clearly imply
that if B-la B cells have any role in MAIDS pathogenesis,
then
at least this putative function does not depend on the expression
of
CD40.
The results from our previous studies and herein, in which we have
defined CD154
+ CD4
+ T cells and
CD40
+ B cells as the cellular subsets which must express
these two
paired interacting molecules for LP-BM5-induced MAIDS
initiation
and progression, thus provide the basis for further
experimentation
in which we will explore the molecular signaling
cascades emanating
from CD40 (and possibly CD154) that appear central
to MAIDS pathogenesis.
However, other receptor-ligand pairs may also be
critical to disease
induction. Along these lines, De Leval et al.
recently reported
that MAIDS pathogenesis in LP-BM5-infected B6 mice
could be partially
blocked by in vivo treatment with soluble CTLA4Ig
(
12) or more
completely by the transgenic overexpression
of soluble CTLA4 (
11).
Whether the critical manifestation
of CD40 signaling in MAIDS
is indeed the upregulation on B cells of
CD80 and/or CD86 and
a subsequent interaction with T cells expressing
CD28 and CTLA4
will await future studies. Similarly, evidence
suggesting that
the receptor ligands CD11a/CD18 (LFA-1) and CD54
(ICAM-1) are
required for MAIDS development has been presented
(
31). Whether
these molecules aid in the interaction of
CD4
+ T cells with B cells prior to delivery of signals from
CD154
to CD40 or there is a role for LFA-1-ICAM-1 interactions
subsequent
to CD40 signaling is
unclear.
Our results on the requirement for interactions between
CD40
+ B cells and CD154
+ CD4
+ T
cells in the initiation and progression of MAIDS are interesting
in the
context of other retrovirus-induced immunodeficiencies,
specifically
human AIDS. Particularly relevant to the present
study, several recent
reports have defined CD154-CD40 interactions
that may be important to
HIV infectivity and/or pathogenesis.
In a study by Poulin et al.
(
42), human B lymphocytes activated
in vitro with murine
cell surface-presented CD154 in the presence
of interleukin-4 became
infectable by HIV type 1 (HIV-1). When
such infected B cells were
cocultured with an HIV-1-susceptible
CD4
+ T-cell line, B-T
cell fusion and syncytium formation led to direct
infection of the T
cells (
42). In a follow-up report, it was
further found
that culturing freshly isolated B lymphocytes with
soluble oligomeric
CD154 plus interleukin-4 upregulated B-cell
CD4 and CXCR4 receptor
expression, with subsequent greatly increased
susceptibility to
infection by T-cell-tropic and dualtropic strains
of HIV but not by
macrophagetropic strains. The authors speculated
that CD40
activation-dependent conversion of B cells to HIV-1
susceptibility may
make the B lymphocyte a potential viral reservoir
in AIDS patients
(
32). Similarly, Gras et al. (
14) have
provided
data suggesting that activation through CD40 augments HIV
replication
in B cells. Furthermore, Muller et al. (
38)
have reported that
the polyclonal hyper-Ig characteristic of
HIV-infected patients
may relate to the observed increases in the
percentage of CD4
+ T cells expressing CD154 and in the
density of cell surface CD40
expressed by B
cells.
Because evidence has been provided that in MAIDS B cells are the
primary cell type for expression of the disease-causing defective
retrovirus (
21,
25), further study of the effects of
CD154-CD40
activation and downstream molecular signaling interactions
in
LPBM5-infected B cells may provide additional insights into the
role
of B cells in MAIDS and potentially the pathogenesis of other
retrovirus-induced immunodeficiencies, including AIDS. Such new
information, particularly if B cells do constitute a retroviral
reservoir, may be useful in improving existing, or developing
new,
strategies involving the use of antiviral drug therapies
or other
approaches to eliminate residual
retrovirus.
| |
ACKNOWLEDGMENTS |
We thank Herbert C. Morse III, Jim Gorham, Hillary White, Bob
Rich, On Ho, Sue Eszterhas, Loren Erickson, and Burkhard Becher for
helpful discussions, and we thank Barbara Peterson-Cremer and Darshan
Sappal for technical assistance and discussions in helping to complete
the experiments described in this report.
This work was supported by Public Health Service grant CA50157. The DMS
irradiation facilities and the flow cytometers were the generous gift
of the Fannie Rippel Foundation and are partially supported by National
Institutes of Health Core grant CA23108 for the Norris Cotton Cancer Center.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Microbiology, Dartmouth Medical School and Norris Cotton
Cancer Center, Borwell Building, 1 Medical Center Dr., Lebanon, NH
03756. Phone:(603) 650-8607. Fax: (603) 650-6223. E-mail:
William.R.Green{at}Dartmouth.edu.
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Journal of Virology, April 2001, p. 3581-3589, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3581-3589.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
