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CD19 Signaling Pathways Play a Major Role
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J Immunol 2002; 169:5607-5614; ;
doi: 10.4049/jimmunol.169.10.5607
http://www.jimmunol.org/content/169/10/5607
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Sonja M. Knoetig, Ted A. Torrey, Zohreh Naghashfar, Tom
McCarty and Herbert C. Morse III
The Journal of Immunology
CD19 Signaling Pathways Play a Major Role for Murine AIDS
Induction and Progression
Sonja M. Knoetig, Ted A. Torrey, Zohreh Naghashfar, Tom McCarty, and
Herbert C. Morse III1
M
ice infected with the LP-BM5 mixture of murine leukemia viruses (MuLV)2 develop a syndrome of lymphoproliferation and immunodeficiency, termed murine AIDS (MAIDS) (1). MAIDS is characterized by a rapid and
persistent proliferation of B and CD4⫹ T cells, hypergammaglobulinemia, phenotypic abnormalities of lymphocyte subsets, and increasingly severe defects of cellular and humoral immunity (2).
Later in the course of disease, malignant transformation of both T
and B cells may occur (1, 3, 4).
Induction of MAIDS requires expression of the MA and p12
portions (5) of the gag gene of replication-defective viruses designated BM5def (6, 7) or Du5H (8). MAIDS can be induced by
helper-free defective virus (9), but in disease induced by the LPBM5 virus mixture, nonpathogenic, replication-competent ecotropic, and mink cell focus-forming (MCF) viruses serve as helpers
for cell-to-cell transmission of the defective genome and are
known to accelerate development of disease (5, 7).
Following infection with helper-free virus, BM5def is expressed
most prominently in mature B cells (10). However, in mixed virus
inoculation, infection of B cells is required for efficient infection of
T cells and macrophages, because animals lacking functional mature B cells are resistant to MAIDS (11, 12). This conclusion was
based on studies in mice depleted of B cells from birth by adminLaboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
Received for publication May 8, 2002. Accepted for publication September 12, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Herbert C. Morse III, Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook I, 5640 Fishers Lane, Rockville, MD 20852.
E-mail address: [email protected]
2
Abbreviations used in this paper: MuLV, murine leukemia virus; BCR, B cell receptor; CR2, CD21/CD35; GC, germinal center; GFP, green fluorescent protein;
HPRT, hypoxanthine phosphoribosyltransferase; KO, knockout; MCF, mink cell focus-forming; MAIDS, murine AIDS; p.i., postinfection.
Copyright © 2002 by The American Association of Immunologists, Inc.
istration of a rabbit Ab to IgM or mice with a B cell deficiency due
to a targeted mutation of the membrane exon of the IgM H chain
gene. Consequently, these studies did not elucidate specific B cell
signaling pathways involved in MAIDS pathogenesis.
Signaling through the B cell receptor (BCR) directly regulates B
cell development, activation, and differentiation. Nevertheless, it is
important that B cells have appropriately established thresholds for
initiation of transmembrane signals to allow the generation of
physiologically relevant responses to foreign or self Ag. Potential
response regulators on the surface of B cells include CD19, which
forms a complex with CD21, CD81, and Leu13 (13). Ligation of
the CD19 complex initiates a cascade of biologic responses that
can modulate signal transduction through the B cell-Ag receptor
complex and other cell surface receptors. Recent studies with mice
that lack CD19 or CD21 as the result of gene knockouts (KO)
suggest that the CD19-CD21 complex serves as a general response
regulator that governs humoral immune responses. Decreased humoral immune responses in these mice correlate with a general
lack of secondary follicles and germinal center (GC) formation
within lymphoid tissues (14 –16). Phosphorylated CD19 interacts
with Vav, leading to subsequent phosphorylation of Vav by activated tyrosine kinases (17). Vav is a member of the large family of
guanine nucleotide exchange factors for the Rho family GTPases.
These proteins can stimulate signaling reactions via the c-Jun NH2terminal kinase and p38 mitogen-activated protein kinases (18).
Another function of Rho family GTPases is participation in phosphoinositol (4, 5) bisphosphate hydrolysis by stimulating phosphatidylinositol 4-phosphate 5-kinase that synthesizes phosphoinositol bisphosphate (19), thus generating more substrate for phospholipase C␥2. In agreement with these observations, Vav has
been shown to be important for BCR-induced proliferation, T helpdependent IgG class switching, and Ab responses to T cell-dependent Ag (20).
Another critical signal transduction element modulating BCR
responses is CD22 (21). Following BCR cross-linking, the Src
0022-1767/02/$02.00
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Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus
(BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions
between B cells and CD4ⴙ T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at
highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play
critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19
integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent
tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of
CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine
leukemia viruses. Infected CR2ⴚ/ⴚ mice developed MAIDS with a time course and severity indistinguishable from that of wildtype mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs
of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to
accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal
induction and progression of MAIDS. The Journal of Immunology, 2002, 169: 5607–5614.
5608
Materials and Methods
Mice
C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). B6.CD19⫺/⫺ (CD19⫺/⫺) mice were originally generated by
R. Rickert (University of Cologne, Cologne, Germany) (14). B6.Vav-1⫺/⫺
(Vav⫺/⫺) mice (27) were a gift from J. Rivera (National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of
Health, Bethesda, MD). B6.Cr2⫺/⫺ (hereafter referred to as CR2⫺/⫺) mice
(16) were obtained from M. Carroll (Harvard Medical School, Boston,
MA). The murine Cr2 locus encodes both mCR1 and mCR2 (complement
receptor 1 and 2); disruption of this locus renders mice deficient in both
mCR1 and mCR2 (CD35 and CD21). B6.CD22⫺/⫺ mice (26) were generated by T. Tedder (Duke University Medical Center, Durham, NC). Mice
were inoculated i.p. with 0.1 ml of LP-BM5 mixed virus pools at 6 –10 wk
of age.
Virus and virus assays
BM5eco and LP-BM5 virus stocks were prepared from the G6 clone of
chronically infected SC-1 cells as described previously (6). LP-BM5 virus
stocks contain a mixture of nonpathogenic ecotropic and MCF MuLV and
a disease-causing defective genome BM5def. At selected time points after
infection, mice were killed and bled; serum was stored at ⫺20°C until use.
Spleen weight, degree of lymphadenopathy, and histopathologic evaluations of selected tissues obtained at autopsy were used to stage the progression of MAIDS by criteria detailed previously (28 –30). The frequencies of spleen cells expressing infectious ecotropic or MCF MuLV were
determined by infectious center tests of mitomycin C-treated cells as described elsewhere (28, 31, 32).
Flow cytometry
All staining was done at 4°C. Cells were stained with FITC- or PE-conjugated Ab directed against Ig ␬ L chain, CD23, B220, Fc␥RII/III (CD16/
CD32), and CD11b (all from BD PharMingen, San Diego, CA). Propidium
iodide was added to allow gating on live cells. Flow cytometry was performed on a FACScan (BD Biosciences, San Jose, CA), and data were
analyzed with CellQuest software (BD Biosciences).
Spleen cell response to mitogens
Spleen cells (1 ⫻ 105) from control and experimental mice were plated
in triplicate into 96-well plates with medium containing 10% FCS,
L-glutamine, 2-ME, antibiotics, and a final concentration of 20 ␮g/ml
LPS and 2 ␮g/ml Con A or of 20 ng/ml PMA and 250 ng/ml ionomycin.
After 48 h, all wells were pulsed with 1 ␮ Ci [ 3 H]thymidine
(PerkinElmer, NEN, Boston, MA) and harvested 6 h later for assessment of thymidine incorporation by scintillation counting (Betaplate
system; PerkinElmer Wallac, Boston, MA).
Measurement of serum Ig levels
Serum IgE levels were measured by ELISA using Ab and standards purchased from BD PharMingen according to their recommendations. Briefly,
96-well microtiter plates (Corning, Corning, NY) were coated with 2
␮g/ml of the 02111D anti-IgE Ab overnight. After blocking the plates with
BSA and incubation with the samples, biotinylated anti-mouse IgE Ab
(02122D) was added, followed by incubation with streptavidin-HRP.
Plates were developed with ABTS peroxidase substrate (Kirkegaard &
Perry Laboratories, Gaithersburg, MD).
For ELISA determinations of serum IgG1, IgG3, and IgM, affinitypurified goat anti-mouse IgG1, IgG3, or IgM Ab (Southern Biotechnology
Associates, Birmingham, AL) were diluted to 5 ␮g/ml to coat 96-well
microtiter plates overnight at 4°C. The plates were then blocked with BSA,
and sera were plated and allowed to incubate for 2 h at 37°C. The plates
were washed, and HRP-labeled goat anti-mouse IgG1, IgG3, or IgM
(Southern Biotechnology Associates) was then added. After 2 h at 37°C,
plates were developed with ABTS peroxidase substrate.
RNA purification, reverse transcription, Southern blotting, and
real-time PCR
Mouse spleen samples were stored at ⫺70°C in TRIzol Reagent (Invitrogen, Carlsbad, CA) until further processing. RNA was extracted according
to the manufacturer’s directions. RNA (1 ␮g) was reverse transcribed using
MuLV reverse transcriptase (Invitrogen) according to the recommendations of the manufacturer. The cDNA solution was diluted to 200 ␮l, and
10 ␮l were used for specific amplification by PCR. The primers and probe
used for BM5def were 5⬘-CCTTTTCCTTTATCGACACT-3⬘ (sense), 5⬘ACCAGGGGGGGAATACCTCG-3⬘ (antisense), and 5⬘-CTCTGCC
AAAGGGACCAGTT-3⬘ (probe). The primers and probe for hypoxanthine
phosphoribosyltransferase (HPRT) were 5⬘-GTTGGATACAGGCCA
AGACTTTGT-3⬘ (sense), 5⬘-GATTCAAACTTGCGCTCATCTTAGGC-3⬘
(antisense), and 5⬘-GTTGTTGGATTGCCCTTGAC-3⬘ (probe). Amplifications were done for 22 cycles (BM5def) and 24 cycles (HPRT). The PCR
products were separated on 1.5% agarose gels, blotted onto a nylon membrane
(Hybond-N⫹; Amersham Pharmacia Biotech, Buckinghamshire, U.K.), and
hybridized with the fluorescein-labeled probe using the Gene Images 3⬘-oligolabeling and ECF signal amplification system (Amersham Pharmacia
Biotech).
For real-time PCR, 1 ␮l of diluted cDNA was amplified using Sybr
Green PCR Master Mix (PE Applied Biosystems, Foster City, CA) and
0.33 ␮M each of sense and antisense primers. The primers used for
BM5def were 5⬘-CCTCCTAAGTCCCGGGTTCTC-3⬘ (sense) and 5⬘-CG
GCCGCCTCTTCTTAACT-3⬘ (antisense); the primers for HPRT were 5⬘GGGAGGCCATCACATTGTG-3⬘ (sense) and 5⬘-TCCAGCAGGTCAG
CAAAGAAC-3⬘ (antisense). All PCR were performed on an ABI PRISM
7700 sequence detector system (PE Applied Biosystems). Serial logarithmic dilutions of a pool of cDNA obtained from three B6 mice 12 wk
postinfection (p.i.) with LP-BM5 were prepared in triplicate to generate
standard curves. The fluorescence signals of each well were collected every
7 s, and threshold cycles were set by determining the point at which the
fluorescence intensity increased in a linear manner. The ratio between the
BM5def and HPRT content in the samples was defined as the normalization factor. Relative BM5def mRNA quantities were determined by dividing the interpolation-derived values from the standard curve by the normalization factor.
Virus-binding assays
Virus binding was detected as previously described (33, 34) with modifications. To reduce virus interference with non-B cells, spleen cells were
treated with ACK lysis buffer (Biofluids, Rockville, MD) to lyse RBC and
subsequently depleted of CD43⫹ and CD4⫹ cells using anti-CD43 and
anti-CD4 paramagnetic beads (Miltenyi Biotech, Auburn, CA) and column
purification according to the manufacturer’s protocol (VarioMACS; Miltenyi Biotech). Cells (1 ⫻ 106) were pelleted and resuspended in 100 ␮l of
BM5eco virus supernatant plus 8 ␮g/ml polybrene. Cells were incubated at
37°C for 40 min with gentle agitation to maintain cell suspension. Following incubation with virus, cells were washed twice with 2 ml of ice-cold
PBS-5% FBS. Cells were then incubated for 30 min on ice with PE-conjugated anti-B220 (BD PharMingen) and biotinylated mAb 83A25, which
recognizes ecotropic viral envelope gp70. Cells were then washed and
incubated with FITC-conjugated avidin (Zymed Laboratories, San Francisco, CA) for 30 min at 4°C. After another wash, cells were analyzed by
flow cytometry. The human lymphoblastoid cell line WIL2-NS (American
Type Culture Collection, Rockville, MD), which lacks ecotropic virus receptor was assayed for nonspecific viral binding using the same methods as
described without B220 staining.
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homology protein 1 tyrosine phosphatase associates with tyrosinephosphorylated CD22, which induces Src homology protein 1
phosphatase activity (22–24). This results in suppression of mitogen-activated protein kinase activation and counterregulation of
CD19 effects (25). B cells from CD22-deficient mice have the
phenotype of chronically activated cells and are hyperresponsive
to BCR ligation, exhibiting increased Ca2⫹ flux and proliferation
(26). This indicates that the normal role of CD22 is to negatively
regulate receptor signaling, although some indications of positive
regulation have also been reported (26).
In the present study, we examined the role of BCR response
regulators in MAIDS by infection of CD21-, CD19-, Vav-1-, and
CD22-deficient mice. The results show that CD21 is not required
for MAIDS induction and that LP-BM5 infection can induce GC
formation and isotype switching to IgG and even IgE in CD21deficient animals. Second, we provide evidence that the CD19-Vav
signaling pathway is required for LP-BM5 replication and subsequent development of lymphadenopathy and immunodeficiency.
Finally, we show that CD22 deficiency accelerates disease progression, suggesting a negative regulatory role for this molecule in
MAIDS.
BCR RESPONSE REGULATORS CONTROL MAIDS
The Journal of Immunology
5609
In vitro infection and integration
Spleen cells (106/ml) were cocultured with ecotropic MuLV-green fluorescent protein (GFP)-producing NIH 3T3 cells (35) in the presence of 2
␮g/ml polybrene in RPMI 1640 supplemented with 10% FCS and 50 mM
2-ME. For B cell stimulation, LPS (Sigma-Aldrich, St. Louis, MO) at a
concentration of 20 ␮g/ml, anti-IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) at a concentration of 15 ␮g/ml, or PMA (20 ng/ml;
Sigma-Aldrich) and ionomycin (250 ng/ml; Sigma-Aldrich) were added.
Cells were harvested after 48 or 72 h, after plating, and infection was
analyzed by flow cytometry using determination of green fluorescence. To
exclude contaminating producer cells from the analysis, cells were stained
with B220-PE Ab as a B cell marker.
In vivo BrdU labeling
BrdU (Sigma-Aldrich) was added to the drinking water of wild-type and
CD19- and Vav-1-deficient mice at a concentration of 1 mg/ml 6 days
before harvesting spleens. The incorporation of BrdU was determined by
flow cytometry using a BrdU Flow kit (BD PharMingen). Splenocytes were
labeled with PE-conjugated anti-B220 to detect B cells, washed, fixed, and
stained for incorporated BrdU using FITC-conjugated anti-BrdU Ab according to the manufacturer’s instruction.
MAIDS-associated lymphoproliferative disease and histologic
changes in wild-type and CR2-, CD22-, CD19- and Vav-1deficient mice
At 4, 8, and 12 wk p.i., mice were screened for MAIDS-associated
splenomegaly, lymphadenopathy, and histopathologic changes by
criteria previously described. Spleens and lymph nodes of infected
B6 and CR2-deficient mice were found severalfold enlarged compared with those of uninfected mice (Table I). In contrast, the
CD19-deficient mice displayed only slight splenomegaly or
lymphadenopathy after infection with LP-BM5 (Table I). The his-
Table I. Development of MAIDS in wild-type, CR2-, CD19-, Vav-, and
CD22-KO micea
Tissue Weight (mg)
Genotypeb
B6
CR2⫺/⫺
CD19⫺/⫺
Vav⫺/⫺
CD22⫺/⫺
Weeks After
Infection
Spleenc
Lymph Nodesc
Histopathologyd
0
4
8
12
0
4
8
12
0
4
8
12
0
4
8
12
0
4
8
12
80
155 (80)
453 (80)
1150 (103)
83
223 (80)
593 (83)
933 (90)
85
87 (80)
120 (90)
150 (107)
70
88 (67)
110 (70)
145 (70)
80
309 (83)
406 (85)
1010 (90)
⬍10
32
207
630
⬍10
52
243
540
⬍10
15
38
47
⬍10
15
18
20
⬍10
67
153
763
N
R-1
2
3
N
1
2
3
N
N
N-1
N-1
N
N-R
N-1
1
N
1
2
3
a
Data are for three mice per data point and are representative of two experiments
involving three mice compared at each time point.
b
Mice of the indicated genotype were infected with LP-BM5 virus mixture and
killed at the indicated time points.
c
Spleens and cervical lymph nodes were weighed and material was saved for
histopathology. Numbers in parentheses indicate spleen weights of age-matched
controls.
d
Histopathologic criteria for staging progression of MAIDS have been presented
elsewhere (28). Briefly, N indicates no change from normal; R indicates a reactive
prodromal stage; and stages 1, 2, and 3 indicate changes characteristic of MAIDS of
increasing severity.
Phenotypic changes in spleen cells from wild-type and CR2-,
CD19-, Vav-1-, and CD22-deficient mice
FACS analysis of spleen cells from B6 and CR2⫺/⫺ mice obtained
4 and 12 wk p.i. revealed that these mice had comparable phenotypic changes typical for MAIDS at these time points (data not
shown). At 4 wk p.i., spleen cells obtained from infected CD22⫺/⫺
mice showed FACS profiles indicative of a more advanced disease
state than that revealed by studies of B6 spleen cells (Fig. 1A).
Blast populations, indicated by increased forward angle scatter,
were seen along with reduced expression of Ig ␬ L chain as signs
of B cell activation. Surface labeling of CD23 was reduced in
infected CD22⫺/⫺ mice. Expression of IgG FcR was greatly increased, and the size of the CD11b⫹ population was expanded.
Furthermore, an expanded subpopulation of CD4⫹Thy-1⫺ cells
characteristic of MAIDS could be detected (37). At 12 wk p.i.,
spleen cells from infected B6, CR2⫺/⫺, and CD22⫺/⫺ mice
showed identical profiles for all markers tested (Fig. 1B and data
not shown). In contrast to the marked changes in B6, CR2⫺/⫺, and
CD22⫺/⫺ mice induced by LP-BM5 infection, the phenotype of
spleen cells from infected CD19⫺/⫺ and Vav-1⫺/⫺ mice was very
similar to that of uninfected mice at 4 and 12 wk p.i., with no
evidence of B cell activation or differentiation (Fig. 1B and data
not shown). To evaluate further B cell activation markers, we
screened B6 and CD19⫺/⫺ B cells for CD80 and CD86 as well as
for MHC class II expression at 8 wk p.i. As expected, B cells from
B6 mice carried increased levels of these molecules, whereas B
cells from CD19⫺/⫺ mice showed no significant changes in expression (data not shown).
Immunodeficiency in CR2- and CD22-deficient mice but not in
CD19- and Vav-1-KO animals
Immunodeficiency of increasing severity is a second prominent
feature of MAIDS (38, 39). At 4, 8, and 12 wk p.i., spleen cells
were prepared from wild-type and KO mice and stimulated for
48 h with LPS, Con A, and PMA/ionomycin. As soon as 4 wk p.i.,
cells from infected B6, CR2⫺/⫺, and CD22⫺/⫺ mice showed decreased proliferative response to mitogenic stimulation compared
with cells from uninfected control mice (Fig. 2A). It was notable
that cells from infected CD22-deficient mice were significantly
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Results
topathological picture in splenic sections ranged from no lesions to
appearance of follicular hyperplasia and development of a few
small active GC. To determine whether MAIDS induction is impaired because of the reduced number of B cells in CD19-deficient
mice (14) or as a result of the disrupted signaling pathway initiated
by CD19 ligation, we infected Vav-1-deficient mice with the disease-inducing virus mixture. Signaling through CD19 induces formation of a CD19-Vav-phosphatidylinositol 3 kinase complex that
activates the Vav-mitogen-activated protein kinase pathway. Vav1-deficient mice have normal numbers of conventional B cells
(36). When infected with the MAIDS-inducing virus mixture,
Vav-1-deficient mice showed somewhat increased spleen and
lymph node sizes with spleen weights doubling at 12 wk p.i. This
was accompanied by mild immunopathologic changes characterized by the appearance of proliferating immunoblasts and plasma
cells in splenic follicles (Table I).
CD22 is another critical cell surface molecule capable of modulating signals through the BCR as well as BCR-independent signals (26). Notably, infected CD22-deficient mice displayed a rapid
onset of splenomegaly and lymphadenopathy; at 4 wk p.i., spleen
and lymph node weights were ⬃2-fold higher than those of infected B6 mice (Table I). However, this hyperreactivity was not
observed at later times, indicating an acceleration in disease progression rather than an increased susceptibility.
5610
BCR RESPONSE REGULATORS CONTROL MAIDS
less responsive to all mitogens tested at this early time point ( p ⬍
0.001; Fig. 2A). In contrast, mitogenic responses of infected
Vav-1- and CD19-deficient mice were not affected. At 12 wk p.i.,
spleen cells from infected B6 and CR2- and CD22-deficient mice
were almost totally nonresponsive to both B and T cell mitogens
(Fig. 2B). In contrast, cells from infected CD19- and Vav-1-deficient mice showed no or moderate proliferative defects ( p ⬍ 0.05;
Fig. 2B). It should be noted that the intrinsic ability of CD19⫺/⫺
spleen cells to proliferate in response to LPS was reduced in all
experiments (two experiments per time point); the same was true
for the ability of Vav-1-deficient spleen cells to respond to Con A.
LP-BM5 infection induces polyclonal Ig production, including
switch to IgE in CR2- and CD22-deficient mice
Elevated levels of polyclonal Ab in serum are a well-known feature of MAIDS; however, the absence of CR2, CD19, or Vav
results in severely impaired humoral responses to T cell-dependent
Ag (14, 16, 20). Therefore, we measured serum Ab levels by
ELISA in groups of control and BM5-infected B6 and KO mice at
8 wk p.i. (Fig. 3). Increased levels of IgE, IgM, IgG1, and IgG3
were detected in LP-BM5-infected B6 and CR2- and CD22-deficient mice. When comparing Ig levels in infected CD19⫺/⫺ and
Vav-1⫺/⫺ mice vs uninfected controls, only Vav⫺/⫺ mice showed
slightly increased IgE and IgM levels. Thus, the polyclonal B cell
response that leads to accumulation of serum Ig does not occur in
CD19-deficient mice and only marginally in Vav-1-deficient mice.
Furthermore, these results suggest that MAIDS infection can induce class switching in the absence of CR2.
Suppression of viral replication in CD19- and Vav-1-deficient mice
To examine whether the mild disease course in CD19- and Vav1-deficient mice is due to impaired virus replication in these mice,
we used RT-PCR followed by Southern blotting of amplified transcripts to detect levels of BM5def mRNA in spleen samples at 12
wk p.i. Of interest, BM5def transcripts in spleen cells from CD19deficient mice were below the limits of detection, and Vav-1⫺/⫺
mice showed clearly reduced levels compared with infected B6
mice (Fig. 4A). Real-time PCR confirmed these results; no
BM5def transcripts were detected in spleen cells from CD19⫺/⫺
mice, and BM5def levels in Vav-1-deficient animals ranged from
30 to 64% of those found in infected wild-type mice 12 wk p.i.
(data not shown).
The frequencies of spleen cells producing ecotropic and MCF
viruses also differed among the strains (Table II). Cells from B6
mice expressed both classes of MuLV, whereas cells from
CD19⫺/⫺ mice did not harbor any ecotropic MuLV, and Vav-1deficient mice expressed ecotropic MuLv at much lower levels
(Table II). Infectious MCF viruses were also not detected in the
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FIGURE 1. Phenotype of spleen cells from mice infected with LP-BM5 viruses for 4 (A) or 12 (B) wk. Histograms and dot plots show expression of
the indicated molecules and are gated on propidium iodide-negative cells. FSC, Forward angle scatter. MFI, Mean fluorescence intensity.
The Journal of Immunology
spleens of CD19-deficient mice. However, after passage of infected Mus dunni cells, low to moderate amounts of MCF could be
noticed in blind-passaged cultures. Levels of MCF-producing cells
were ⬃10-fold lower in Vav-1-KO mice compared with B6 mice.
In summary, replication of all LP-BM5 virus types was clearly
suppressed in CD19- and Vav-1-deficient mice.
FIGURE 4. Viral load and virus binding in wild-type and KO mice. A,
Semiquantitative RT-PCR analyses of BM5def and HPRT transcripts in
spleens of B6 and CD19- and Vav-1-deficient mice either left uninfected
(⫺) or infected (⫹) for 12 wk. The length of the amplified products for
BM5def and HPRT were 245 and 163 bp, respectively. The electrophoresed amplified products were transferred to a nylon membrane and detected by ECL as described in Materials and Methods. B, Virus binding to
wild-type and CD19⫺/⫺ B cells. Spleen cells were assayed for ecotropic
virus binding using mAb 83A25 as described in Materials and Methods.
The human lymphoblastoid cell line WIL2-NS was used to test for specificity of the assay. Histogram overlays are gated on propidium iodidenegative, B220⫹ cells and show cells incubated with ecotropic virus (filled)
over noninfected control cells (unfilled).
cells. B cell-enriched spleen cells obtained from wild-type as well
as CD19 KO mice were incubated with ecotropic virus for 40 min
and subsequently labeled with mAb 83A25, which recognizes ecotropic viral envelope gp70. A human lymphoblastoid cell line that
lacks receptors for ecotropic virus was used as a control for specificity of the assay. As shown in Fig. 4B, virus binding was equivalent in both wild-type and CD19⫺/⫺ B cell cultures as opposed to
minimal binding of ecotropic virus to the human cell line. Thus,
absence of CD19 does not influence virus binding to B cells.
Virus binding is not affected by CD19 deficiency
Viral integration is not impaired in CD19- and Vav-1-deficient
B cells
To further investigate the nature of viral inhibition in CD19-deficient B cells, we examined the capacity of virus to bind to these
Another critical step in viral replication is integration of newly
synthesized viral DNA into the host genome. To test whether
Table II. Viral recovery in wild-type, CD19-, and Vav-KO micea
Virus Recovery
MCF
Virus
Genotype Inoculatedb
B6
CD19⫺/⫺
Vav⫺/⫺
FIGURE 3. Serum Ig levels in control mice (E) and mice infected with
LP-BM5 8 wk p.i. (F). Sera were assayed for the indicated Ig by isotypespecific ELISA. Each circle represents a different animal, and data shown
are the mean of triplicate values within a typical experiment.
⫺
⫹
⫺
⫹
⫺
⫹
Ecotropic PFUc
(log10/107 cells)
FFU (log10/107 cells)
Passaged
⬍1
3.5
⬍1
⬍1
⬍1
ⱕ1
⬍1.3
3.1
⬍1
⬍1
⬍1
2.1
⫺
⫹
⫺
⫹
⫺
⫹
c
a
Infectious center assays in tissue culture were performed by cocultivation of
mitomycin C-treated spleen cells with SC-1 cells for assays of ecotropic MuLV by the
XC plaque test (32) and with Mus dunni cells for assays of mink-infectious viruses by
a fluorescent Ab focus-forming test (31).
b
Mice of the indicated genotype were infected (⫹) or not (⫺) with LP-BM5 virus
mixture and killed 12 wk p.i.
c
Titers of ecotropic virus are expressed as log10 PFU/107 cells and titers of mink
infectious virus as log10 focus forming unit (FFU)/107 cells. Numbers indicate the
mean for assays of spleen cells from three mice per group.
d
In MCF virus assays, cultures were subcultured at 9 –12 days; ⫺, negative, no
foci detected on passage; ⫹, positive, MCF foci detected on passage.
Downloaded from http://www.jimmunol.org/ by guest on July 12, 2017
FIGURE 2. Proliferative responses of spleen cells to stimulation with
mitogens or PMA plus ionomycin. Cells (1 ⫻ 105/well) from spleens of
uninfected mice (䡺) or mice infected for 4 and 12 wk (f) were cultured
for 48 h in triplicate in 96-well plates with Con A (2 ␮g/ml), LPS (20
␮g/ml), or PMA (20 ng/ml) plus inonomycin (250 ng/ml). Cultures were
pulsed with [3H]thymidine for the last 6 h of culture. Data are average
values ⫾ 1 SEM for three mice per time point. The p values were derived
by Student’s t test. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; and ⴱⴱⴱ, p ⬍ 0.001.
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BCR RESPONSE REGULATORS CONTROL MAIDS
complexes enter the nucleus only after the nuclear envelope is
transiently disassembled during mitosis (40). Therefore, impaired
in vivo B cell turnover might be a limiting factor for LP-BM5
replication in CD19- and Vav-1-deficient mice. To analyze in vivo
B cell proliferation in B6 and CD19- and Vav-1-KO animals,
BrdU was added to the drinking water. We then surface labeled the
splenic B cell pool with PE-anti-B220 and counterstained with
FITC-anti-BrdU to detect cells that had divided during the 6-day in
vivo labeling. On average, 12.9% of wild-type B cells, 15.6% of
CD19⫺/⫺ B cells, and 11.3% of B cells from Vav-1-deficient mice
were positive for BrdU (Fig. 6). Thus, in vivo B cell proliferation
is not impaired in CD19- or Vav-1-deficient animals.
Discussion
CD19 or Vav-1 deficiency interferes with this process, we cocultured spleen cells from wild-type and CD19- and Vav-1-deficient
animals with an ecotropic MuLV-GFP-producing cell line (35).
Viral integration was assessed by flow cytometry. Wild-type and
CD19- and Vav-1-deficient spleen cells were infectable in culture
and expressed GFP after stimulation with anti-IgM, LPS, and
PMA/ionomycin (Fig. 5 and data not shown). Although reduced
GFP expression could be noted only in Vav-1-deficient B cells
after anti-IgM treatment (Fig. 5B), this was due to an impaired
proliferative response to anti-IgM as determined by [3H]thymidine
uptake (data not shown), which had been shown previously (20).
These findings indicate that the reduced viral load in CD19- and
Vav-1-deficient animals is not due to defective integration.
In vivo proliferation
Integration was reduced in Vav-1-deficient B cells in vitro, most
likely due to impaired proliferation after BCR engagement. This is
in accordance with previous studies that suggested that retroviral
integration might depend on cell division because viral integration
FIGURE 6. In vivo B cell proliferation in wild-type and CD19- and
Vav-1-deficient mice. BrdU was added to the drinking water 6 days before
sacrificing mice (n ⫽ 3 per group). Spleens were harvested, and cells were
surface labeled with PE-anti-B220, fixed, permeabilized, and stained with
FITC-labeled anti-BrdU Ab. Numbers indicate percentage of positive
cells ⫾ SD.
Downloaded from http://www.jimmunol.org/ by guest on July 12, 2017
FIGURE 5. In vitro infection and integration. Spleen cells from B6 and
CD19- and Vav-1-deficient animals were cocultured with ecotropic
MuLV-GFP-producing NIH 3T3 cells as described in Materials and Methods. Cells were harvested 48 h after plating, and infection was analyzed
flow cytometrically by determination of green fluorescence in B220⫹ B
cells. Histogram overlays show B cells cocultured with virus producer cells
(filled) over noninfected control cells (unfilled). For B cell stimulation,
LPS (20 ␮g/ml; A) or anti-IgM (15 ␮g/ml; B) was added to the cultures.
Although the presence of B cells and B cell-T cell interactions
have been shown to be critical for development of MAIDS (11, 12,
41, 42), little is known about functional features of B cells that
would favor or inhibit MAIDS progression. Possible candidates for
affecting MAIDS development are the B cell surface molecules
CD19 and associated CR2. The role of these receptors in B cell
activation and differentiation has been demonstrated in vitro as
well as in vivo (14, 16, 43, 44). For this reason, we looked at
responses of CD19- and CR2-deficient mice to LP-BM5 infection.
CR2⫺/⫺ and wild-type mice were found to have equivalent levels
of lymphadenopathy and splenomegaly. FACS and histopathologic studies revealed that mice of both genotypes had comparably
advanced disease at all time points tested. Surprisingly, LP-BM5infected CR2⫺/⫺ mice generated GC reactions and Ig of all isotypes tested, with the magnitude of their responses being equivalent to that of B6 mice. This could be attributable to a strong and
persistent antigenic stimulus provided by LP-BM5, because it has
been shown that CR2⫺/⫺ B cells respond to abundant Ag/adjuvant
with GC that are quantitatively and qualitatively comparable to
those of wild-type mice (45). It should be noted that the CR2⫺/⫺
mice used in this study were found to express low levels of a
hypomorphic CD21/35 molecule (46); however, it has not been
established whether expression of this molecule is functionally
relevant.
In CD19⫺/⫺ mice, all of the defining features of MAIDS were
nearly absent due to impaired replication of LP-BM5 MuLV. It is
unlikely that reduced B cell numbers in these mice were the cause
for restricted viral load, because Vav-1-deficient mice, which have
normal B cell numbers, showed reduced viral load and mild disease course as well. Furthermore, impaired viral replication and
limited MAIDS progression are probably not due to the lack of
marginal zone B cells, which are virtually absent in CD19-deficient mice. In general, histological studies do not provide evidence
for an involvement of marginal zone cells in MAIDS-related pathology, because progressively expanding GC B cells are the dominating
cell type in splenic sections from LP-BM5-infected mice (47).
The mechanisms that cumulatively result in resistance of CD19and Vav-1-deficient mice to infection with LP-BM5 and MAIDS
development appear complex. In vitro infections revealed that virus binding was not affected by the absence of CD19, nor was
retroviral integration affected in either CD19- or Vav-1-deficient B
cells. These results do not point to an intrinsic B cell defect in these
animals, particularly because in vivo B cell turnover was not impaired by the lack of CD19 or Vav-1. However, in vitro infection
might represent an optimal system where B cells are adequately
stimulated and in close proximity to virus-producing cells. In vivo,
initial infection of a number of B cells might occur but be abortive
due to unresponsive CD19⫺/⫺ and Vav-1⫺/⫺ target cells. The
demonstration of dramatically decreased tyrosine phosphorylation
of Src-family kinases and multiple downstream effector molecules
The Journal of Immunology
vation and subsequent B cell changes (42), Vav-1 deficiency might
limit MAIDS on the B cell as well as on the T cell side.
Loss of CD22 altered early LP-BM5 responses, because spleen
cells from CD22-deficient mice demonstrated more advanced
signs of MAIDS soon after infection than spleen cells from wildtype animals. Differences in signs of MAIDS were greatest at 4 wk
p.i., although viral load was not increased at that time point (data
not shown). Therefore, the exaggerated response to LP-BM5 infection in CD22-deficient mice is likely to result directly from
alterations in the activation state or the signaling capacity of their
B cells rather than being due to an increased susceptibility to virus
infection. Later in the course of disease, the reduced number of
circulating B cells in CD22-deficient animals (26) might limit target and effector cells, which could explain why enhanced disease
is not seen in CD22⫺/⫺ mice at 8 and 12 wk p.i.
This is the first report showing the crucial role of the CD19-Vav
signaling pathway in retroviral replication; however, it has been
shown before that blocking accessory molecules CD54 and CD11a
was effective in suppressing LP-BM5 replication (69). Both studies indicate that blocking the immune response suppresses active
retroviral replication and point to a strategy for interfering with
retrovirus-associated disease.
Acknowledgments
We thank J. Hartley for helpful suggestions, D. Bodine for MuLV-GFP
producer cells, T. Fredrickson for histopathologic evaluations, and R. Rickert, M. Carroll, J. Rivera, and T. Tedder for mice.
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in CD19-deficient B cells following BCR ligation suggests that
CD19 coordinates the initiation of multiple signaling pathways
(48 –51), the sum of which seems to be critical to retroviral replication and MAIDS pathogenesis. Furthermore, CD19 has recently been shown to potentiate the signaling initiated through
MHC class II cross-linking as well (52). Class II-mediated signals
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BCR RESPONSE REGULATORS CONTROL MAIDS