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-Transgenic Mice1
Departments of
*
Immunology and
Neurology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, MN 55905;
Department of Pathology, University of Chicago, Chicago, IL 60637; and
Shanghai International Cancer Institute and Eastern Hospital of Heptaobiliary Surgery, Shanghai, China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter. The 4-1BBL-transgenic mice
show progressive splenomegaly and selective depletion of
B220+ B cells accompanied with low levels of circulating
IgG and defective humoral responses to Ag challenge. In addition,
splenocytes from the transgenic mice fail to provide stimulation for
allogeneic T cells in both lymphoproliferative and CTL responses in
vitro, whereas their T cells remain functionally normal. Our results
reveal unexpected functions of 4-1BBL in the regulation of humoral
immune responses and Ag presentation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and costimulation of T cell
proliferation and cytokine production in vitro (1, 2, 3, 4, 5, 6).
Triggering of 4-1BB on T cells in vivo using agonistic mAb or
4-1BBL-transfected tumor cells induces preferential expansion of
CD8+ CTLs that recognize and reject tumors and
allogeneic transplants (1, 7, 8), inhibits T
cell-dependent Ab production (9), and prevents
superantigen-induced T cell death (10).
Expression of 4-1BBL has been detected on activated T cells, B cells,
dendritic cells (DCs), macrophages, mouse lymphomas, and human
carcinoma lines of epithelial origin (1, 2, 11). Two
recent studies using 4-1BBL-knockout
(4-1BBL-/-) mice indicate that lack of 4-1BBL
induces a moderate decrease of CTL responses to influenza virus
challenge (12) and to lymphocytic choriomeningitis virus
peptide immunization (13) but does not alter CTLs to
lymphocytic choriomeningitis virus and allogeneic skin graft (12, 14). In addition to interaction with 4-1BB, several lines of
evidence indicate that 4-1BBL transmits a reverse signal. Cross-linking
of 4-1BBL on activated T cells by 4-1BB-Ig, a fusion protein of the
extracellular domain of 4-1BB and the Ig C region, inhibits
proliferation of activated T cells and induces programmed cell death
(15). Ligation of 4-1BBL also activates monocytes to
produce IL-6, IL-8, and TNF- and inhibits IL-10 (16).
An increase in apoptosis of activated monocytes was also seen after
4-1BBL ligation (17). Binding 4-1BBL on B cells in the
presence of anti-µ-chain Abs increases proliferation of mouse
spleen B cells (18). These results suggest that, in
addition to providing a costimulatory signal to 4-1BB, 4-1BBL may have
broader functions on regulation of immune responses.
Although these in vitro studies suggest roles of 4-1BBL in regulation
of proliferation, growth, and apoptosis of different types of cells, it
is not clear whether these observations virtually happen in vivo. In
this report, we describe that transgenic (Tg) mice expressing 4-1BBL
under the control of MHC class II I-E
promoter, which allows the transgene exclusively expressed on APCs,
display unexpected phenotypes that suggest a predominant function of
4-1BBL on regulating B cell survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate 4-1BBL-Tg mice, a 980-bp cDNA fragment containing
the complete open reading frame of mouse 4-1BBL was amplified by PCR
and inserted into the EcoRI site of the pDOI-5 vector
(19). The XbaI-EagI fragment excised
from the resulting plasmid, which included the promoter of MHC class II
E gene, the 4-1BBL open reading frame, and the
intron and polyadenylation site of the rabbit 
-globin gene, was
microinjected into the fertilized eggs of C57BL/6 mice as described
previously (20). The Tg founders were identified by
detecting the presence of a 1-kb PCR fragment in DNAs isolated from
mouse tails using a pair of primers. The 5' primer (GAG AAC TAT CCT TCT
TGT GAC TCC TAG TTG) corresponds to the sequence of 4-1BBL,
and the 3' primer (GCG ATA ATG GCC TGC TTC TCG CCG AAA CGT) corresponds
to the sequence of the vector (Fig. 1
).
Expression of the transgene was determined by reverse
transcriptase-coupled PCR amplifying a 540-bp cDNA fragment from
splenocytes using two specific primers, Tgp and Tg540 (Fig. 1). Tgp
(TTA ATT CTG CCT CAG TCT GCG) is a 5' primer containing the
sequence of the E promoter transcription
initiation site +1 to +12 (underlined) (21), and the Tg540
(GTT GTA TTG CAC AAC GAT GCT) is a 3' primer corresponding to the
sequence of murine 4-1BBL (Fig. 1). The RT-PCR was performed for 4
cycles at 94°C for 45 s, 38°C for 1.5 min, and 72°C for 1
min followed by 35 cycles at 94°C for 45 s, 58°C for 45
s, and 72°C for 1 min.
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Normal BALB/c mice and C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). For immunization, mice were injected i.p. with keyhole limpet hemocyanin (KLH) (Biosearch Technologies, Novato, CA) at 100 µg/mouse in PBS on day 0. On day 21, the mice were challenged with the same dose of KLH (boost). Sera were collected from the mice, 10 days after primary immunization, or 7 days after secondary challenge.
Cell culture and reagents
FITC- and PE-labeled Abs against mouse B220, CD2, Mac1, and NK1.1 were purchased from BD PharMingen (San Diego, CA). Anti-mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO). Biotin-conjugated anti-mouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-anti-rat IgG was purchased from BioSource International (Camarillo, CA). EL4 (H-2b) thymoma and P815 (H-2d) mastocytoma cells were cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 25 mM HEPES, 0.3 mg/ml L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 55 µM 2-ME.
Immunohistochemistry
Spleens and lymph nodes (LNs) were harvested, embedded in the Tissue-Tek OCT compound (Miles, Elkhart, IN), and frozen at -70°C. Frozen sections (8 µm thick) were fixed in cold acetone. Endogenous peroxidase was quenched with 0.2% H2O2 in methanol. After washing in PBS, the sections were stained first by incubating with FITC-conjugated anti-B220 for B cells and biotinylated anti-mouse CD90.2 (Thy1.2) for T cells or biotinylated anti-CD11c (BD PharMingen) for DCs at 1/50–1/100 dilutions. HRP-conjugated rabbit anti-FITC (DAKO, Glostrup, Denmark) and alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, CA) were added 1 h later. Color development for an alkaline phosphatase and HRP was performed with an alkaline phosphatase reaction kit (Vector Laboratories) and 3,3'-diaminobenzidine (Sigma-Aldrich).
ELISA
Sera from the Tg mice and non-Tg littermates (LtM) were collected by tail-bleeding method and were diluted with PBS as indicated. To measure total IgG levels in sera, 100 µl of 2 µg/ml anti-mouse IgG (Sigma-Aldrich) was precoated to 96-well flat-bottom plates overnight at 4°C. The plates were blocked with 100 µl of PBS-10% FBS for 30 min followed by the addition of 90 µl of diluted sera. After incubating for 2 h, the plates were washed and further incubated with 100 µl of 2 µg/ml biotin-labeled anti-mouse IgG (The Jackson Laboratory, Bar Harbor, ME) for 30 min. A total of 50 µl of HRP-conjugated streptavidin (1/3000, 100 µl/well) was then added to the plates for another 30 min. Color was developed with 50 µl of tetramethylbenzidine substrate (Moss, Pasadena, MD) followed by 50 µl of stop solution (0.5 M H2SO4). Absorbance (OD) of samples was read by the TECAN Spectra Image (TECAN, Salzburg, Austria) microplate reader at 450 nm. Results were determined in triplicate and expressed as the mean ± SD.
Flow cytometric analysis
A total of 1 x 106 spleen cells of the Tg mice and LtM were incubated for 30–60 min with Abs conjugated with FITC or PE in 50 µl staining buffer (PBS, 3% FBS, 0.02% sodium azide). Cells were subjected to another 30 min of incubation with FITC-labeled secondary Abs (anti-rat IgG) if necessary. Fluorescence was analyzed on a FACScan flow cytometer with CellQuest software (BD Biosciences, Mountain View, CA).
T cell assays
For allogeneic MLRs, 3 x 105/well responder splenocytes were cocultured with irradiated (4000 rad) allogeneic stimulator cells at the ratio of 1:1, 3:1, and 10:1 for 3 days in 96-well plates. A total of 1 µCi/well of [3H]TdR was added 16 h before harvesting. For induction of CTLs to allogeneic Ags, 10–15 x 106 responder splenocytes were cocultured with equal numbers of irradiated allogeneic stimulator splenocytes in T-25 flasks in 15 ml RPMI medium for 7 days. Cells were then harvested, and their CTL activities were determined in standard 51Cr-release assays against allogeneic target cells and syngeneic target cells at indicated E:T ratios as described previously (22). Incorporated 3H in MLR assays and released 51Cr in CTL assays were detected by 1450 MicroBeta liquid scintillation counter (Wallac PerkinElmer, Turku, Finland).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We constructed a plasmid by insertion of the entire coding region
of 4-1BBL cDNA into pDOI-5 vector (Fig. 1
A). The pDOI-5
contains an MHC class II E promoter that
allows specific expression of the 4-1BBL transgene in MHC class
II+ cells (19). After screening by
dot blot and PCR using specific probes and primers, respectively, four
transgene-positive founders were identified and bred. Two lineages,
line 9 and line 16 (4-1BBL-Tg+), were studied in
detail.
To distinguish the expression of 4-1BBL transgene from that of
endogenous 4-1BBL, we designed an upstream primer in which part of the
sequences corresponded to the I-E promoter
(bases +1 to +12) and a downstream primer, which corresponded to the
sequences of mouse 4-1BBL (bases 520–540). Therefore, this pair of
primers is specific for RNA of the transgene. In a RT-PCR analysis,
this pair of primers was able to specifically detect a 0.5-kb
transcript of spliced Tg 4-1BBL cDNA in spleen cells of both lines 9
and 16 but was unable to do so in splenocytes of LtM (Fig. 1B). However, FACS analysis of freshly isolated
B220+ B cells, Mac1+
macrophages, as well as bone marrow-derived DCs by 4-1BB-Ig fusion
proteins or anti-4-1BBL mAb did not detect the
expression of 4-1BBL in both lines of Tg mice (data not shown). Our
results indicate that 4-1BBL is not constitutively expressed on the
surface of B cells and macrophages from Tg mice, although specific
transcript of Tg 4-1BBL could be detected.
Phenotypes of 4-1BBL-Tg+ mice
The 4-1BBL-Tg+ mice did not appear to have significant differences from their LtM in gross appearance, size, body weight, activities, and development up to 18 mo of age. However, the reproductive potential of both lines 9 and 16 started to decrease at the age of 2–3 mo and was completely lost when they passed 5 mo of age. LtM of both lines remained normal for reproduction.
The most striking observation in gross anatomy of major
organs of the Tg mice is splenomegaly. At the age of 1–2
mo, the spleens of most Tg mice are larger than those of LtM. The
splenomegaly of the Tg mice progressed by age. In 5- to 12-mo-old
Tg mice, size of spleens could be five or more times larger than those
of LtM. However, thymus and LNs of the Tg mice developed normally. Fig. 2 shows two representative spleens of Tg
mice vs LtM of lines 9 and 16 at the ages of 11 and 12 mo,
respectively. However, cell numbers of Tg spleens were not consistently
higher than those of LtM, although we did observe that in <25% of Tg
mice, the total cell counts of spleen cells could increase up to 3-fold
compared with those of their LtM. A large part of the spleens of Tg
mice are connective tissue. When Tg spleens were ground to prepare
splenocytes, their leftover tissue residues were consistently larger
than those of LtM.
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We next examined cellular composition of spleens from Tg mice of
different ages by FACS analysis. The splenocytes of Tg mice at 2–3 mo
of age displayed similar profiles of B220+ B
cells, CD3+ T cells, and
Mac1+ macrophages to those of LtM. Interestingly,
when splenocytes of aged Tg mice (4–12 mo) were subjected for
analysis, B220+ B cells were virtually
undetectable, whereas the number of Mac1+ cells
was triplicated in comparison with that of the splenocytes of LtM. This
was observed in lines 9 and 16 as well as all other Tg lineages. The
number of CD3+ T cells remained unchanged.
FACS-staining profiles of a representative experiment using 2- and
12-mo-old Tg mice as well as their littermates are summarized in Fig. 3. In addition, the numbers of
Gr-1+ granulocytes, NK1.1+
NK cells, and CD11c+ cells in Tg mice were also
lifted slightly. Such an increase could be due to the proportion
augmentation of each subset of cells in the absence of B cells. Our
results suggest a selective elimination of B220+
B cells in 4-1BBL-Tg+ mice.
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). We also noticed that
architectural structures of the Tg spleens had been damaged (Fig. 4).
CD11c+ cells in both spleens and LNs of
4-1BBL-Tg+ mice showed no reduction compared with
the LtM (data not shown). Consistent with this finding, basal levels of
total serum IgG of aged Tg mice were drastically reduced. Fig. 5 shows IgG levels of some representative
mice of line 16. A 4-1BBL-Tg+ mouse had normal
levels of IgG at the age of 3 mo (Fig. 5A). FACS analysis
revealed that B220+ cells in an 85-day-old
4-1BBL-Tg+ mouse had been diminished to one-fifth
of the normal mice (data not shown). Mice of the same line at the ages
of 4 (Fig. 5B) and 12 mo (Fig, 5C) had
drastically reduced levels of IgG. Immunization (Fig. 5D)
and even boost (Fig. 5E) of the 5-mo-old
4-1BBL-Tg+ mice by KLH failed to increase Ab
production. We conclude that the loss of potential to produce Abs to
Ags by Tg mice is due to the depletion of B cells in these
mice.
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As a preliminary step to analyze T cell responses in
4-1BBL-Tg+ mice, we first determined the
capability of spleen cells from 4-1BBL-Tg+ mice
(H-2b) as stimulators or responders to allogeneic
Ags. To test the stimulator function, T cells were purified from BALB/c
mice (H-2d) and were cocultured with irradiated
spleen cells of 4-1BBL-Tg+ mice. T cell responses
were examined in MLRs and in alloreactive CTL assays. A representative
experiment is summarized in Fig. 6A. Spleen cells from a
12-mo-old 4-1BBL-Tg+ mouse stimulated neither
proliferative responses nor alloreactive CTLs against the allogeneic
target EL4 cell line compared with those from a LtM. In contrast, the
splenocytes from a 1.5-mo-old 4-1BBL-Tg+ mouse
stimulated T cell responses similar to those from a LtM in both MLRs
and CTL assays. To examine responder functions of
4-1BBL-Tg+ T cells, T cells from the Tg mice were
cocultured with allogeneic BALB/c spleen cells as the stimulator.
Normal CTL responses (Fig. 6B) and proliferative responses
(data not shown) to allogeneic Ags were observed. Our results suggest
that T cell response to allogeneic Ags in Tg mice is normal, whereas
the stimulatory abilities of splenocytes are impaired.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although our experiments reveal an unexpected function of 4-1BBL on B
cells, the mechanisms leading to this phenotype in our Tg mice are not
yet known. Tg mice up to 3 mo of age produce Ab to KLH in levels
comparable to those from normal littermates, suggesting there is no
general defect in B cell function. There are several possible
explanations for progressive deletion of B cells from our Tg mice. The
4-1BBL on B cells could potentially deliver a signal leading to death.
It has been shown that immobilized 4-1BB proteins stimulate expression
of Fas in resting human B cells in vitro (23). Therefore,
it is possible that increased interaction between Tg B cells and other
4-1BB+ cells leads to B cell apoptosis in vivo.
Human monocytes isolated from some individuals constitutively express
4-1BB (24). A recent study by Kienzle and von Kempis
(24) shows that immobilized 4-1BB proteins can activate
human monocytes that in turn induce apoptosis of B cells through an
unknown mechanism. Our observation that macrophages are increased
significantly in aged Tg mice (Fig. 3) suggests this possibility.
Spleen cells from 4-1BBL-Tg+ mice are unable to
stimulate allogeneic T cell responses, including both proliferative
responses mediated by CD4+ T cells (Fig. 6 and
data not shown) and cytolytic activities mediated by
CD8+ CTLs (Fig. 6). Because T cells from the Tg
mice can respond normally to allogeneic Ags, our results suggest a
defect in the Ag-presentation function of APCs in the Tg mice. It is
not clear what causes the reduced ability of Ag presentation in
splenocytes of the 4-1BBL-Tg+ mice. DCs are
believed to be the predominant APCs in MLR assays (25).
However, the number of DCs of spleen and LNs in young and aged Tg mice
are in the normal range based on immunohistochemistry and FACS analyses
using anti-CD11c Ab (data not shown). Nevertheless, this result
does not exclude the possibility that there may be a functional defect
in DCs from our Tg mice.
In summary, the generation of 4-1BBL-Tg mice revealed a loss of humoral immune responses to Ags caused by progressive depletion of mature B cells in peripheral lymphoid organs. In addition, splenocytes from the Tg mice appear defective in their capacity to stimulate allogeneic T cell responses. Therefore, 4-1BBL may regulate humoral immune responses by the control of the survival of B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lieping Chen, Department of Immunology, Mayo Clinic, Mayo Graduate and Medical Schools, 2000 First Street, Southwest, Rochester, MN 55905. E-mail address: chen.lieping{at}mayo.edu
3 Abbreviations used in this paper used in this paper: 4-1BBL; 4-1BB ligand; KLH, keyhole limpet hemocyanin; DC, dendritic cell; LN, lymph node; LtM, nontransgenic littermates; Tg, transgenic.
Received for publication April 24, 2001. Accepted for publication July 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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