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Production, Rather than Antibody, Dominates the Immune Response in Mice
Autoimmune Disease Unit, Cedars-Sinai Research Institute and UCLA School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to TSH receptor
antigen. In conclusion, DNA vaccination is less effective at inducing
TSH receptor antibodies than the Shimojo approach, but it permits the
future characterization of TSH receptor-specific T cells generated
without adjuvant. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genetic differences among mouse strains can influence the outcome of immunization, as illustrated for DNA vaccination (above) and as previously reported for injection of TSHR+, MHC class II+ fibroblasts (3). In addition, different immunization protocols within a particular mouse strain may affect the characteristics of the immune response. Both the Shimojo approach (2, 6, 7) and naked TSHR-DNA vaccination (4, 8) induce TSHR antibodies with TSH binding inhibition (TBI) activity. On the other hand, examination of these different studies suggests that thyroid-stimulating antibodies (TSAb) arise more frequently in the Shimojo model than after DNA vaccination. However, there has been no direct comparison of TSHR antibodies induced by these two protocols. Such information is important for future studies using mouse models to understand the immune response to the TSHR.
In the present study, we directly compared the responses of inbred mice to vaccination with TSHR-DNA vs. injection with TSHR+, MHC class II + fibroblasts (Shimojo approach). Because the RT4.15HP fibroblasts (9) used in the Shimojo model express the MHC class II molecule I-Ak, this model is limited to mouse strains (such as AKR/N) with the same I-A allele. On the other hand, data on TSHR-DNA vaccination of an inbred strain are only available for BALB/c mice (4), which differ from AKR/N mice in terms of MHC class II (I-Ad) and background genes. We, therefore, performed a three-way comparison of immune responses in 1) AKR/N and 2) BALB/c mice vaccinated with TSHR-DNA and 3) AKR/N mice injected with TSHR and MHC class II expressing fibroblasts.
We now demonstrate that the TSHR antibody response (measured by
TBI and TSAb assays) is much lower after TSHR-DNA vaccination than
after injection of TSHR+, MHC class II+ fibroblasts. Moreover, rather
than antibody production, the characteristic immune outcome of TSHR-DNA
vaccination is an antigen-specific lymphocyte response characterized by
production of the Th1 cytokine interferon- (IFN-).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animal studies were approved by the Institutional Animal Care and Use Committee and performed in accordance with the highest standards of humane care in a pathogen-free facility.
Purified TSHR antigen
TSHR-289 is a TSHR ectodomain variant corresponding to the
A subunit (13). This protein, expressed in Chinese
hamster ovary (CHO) cells, contains two conformationally different
forms (active and inactive) with respect to their ability to be
recognized by human TSHR autoantibodies (14). Inactive
TSHR-289 was isolated from culture medium by affinity chromatography
using mouse mAb 3BD10. Active TSHR-289 not captured by the column was
then recovered using an affinity column with a mAb to histidine
residues in the C-terminus (15). Purity and concentrations
were determined by SDS-PAGE. Before use as immunogen (see below), in
ELISA or in lymphocyte cultures, both forms of TSHR-289 were dialyzed
against 10 mM Tris, pH 7.4, 50 mM NaCl.
To provide positive controls for serum antibody binding in ELISA and flow cytometry, BALB/c mice (The Jackson Laboratory) were immunized with purified TSHR-289 (50 µg) emulsified in Complete Freund’s Adjuvant (Calbiochem). For this purpose we used inactive TSHR-289 because the active form is quite labile and will be converted into the inactive form in the immunization procedure. Mice were boosted with the same antigen (50 µg) in Incomplete Freund’s Adjuvant (Calbiochem) after 3 wk and again after 4 wk before tail bleeding.
Serum binding to purified TSHR antigen
ELISA wells coated with TSHR-289 (inactive form; 1 µg/ml in 10
mM Tris, pH 7.4, 50 mM NaCl) were incubated
with test sera (diluted 1:100 for DNA vaccinated mice and 1:10,000 for
purified TSHR + adjuvant immunized mice). Antibody binding was detected
with horse radish peroxidase conjugated mouse anti-IgG
(Sigma), the signal developed with
O-phenylenediamine and
H2O2 and OD read at 490
nm.
Flow cytometry for serum binding to TSHR-expressing cells
Mouse sera (diluted 1:50) were examined for antibody binding to
CHO cells expressing high numbers of TSH receptors (
2 x 10
6 TSHR/cell) (16) and detection with
fluorescein isothiocyanate (FITC)-conjugated, affinity-purified goat
antimouse IgG (Caltag Laboratories, Inc.,
Burlingame, CA) as previously described (17). All assays
included cells incubated with second antibody alone and with normal
mouse serum. Flow cytometry was performed (10,000 events) using a
FACScan with Cellquest Software (Becton Dickinson and Co.,
San Jose, CA).
Inhibition of 125I-TSH binding to its receptor
(TBI)
TBI (18) was measured using a kit
(Kronus, Boise, ID). Duplicate serum aliquots (50 µl;
undiluted) were incubated with detergent solubilized porcine TSHR;
125 I-TSH (bovine) was added and the
TSHR-antibody complexes were precipitated with polyethylene glycol. The
TBI values are calculated from the formula:
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TSAb assay
TSAb activity was assayed following the approach of Costagliola
et al. (8). CHO cells expressing approximately
150,000 TSHR/cell (11) were grown to confluence in 96-well
plates and incubated (4 h at 37 C) with test sera diluted 1/30 in
hypotonic buffer (19) containing 10
mM HEPES, pH 7.4, 1 mM
isobutylmethylxanthine and 0.3% BSA. Supernatants (500 µl diluted
1:400) were acetylated (20 µl triethylamine and 10 µl acetic
anhydride) and cAMP levels were measured by RIA using
2'-O-succinyl-[125 I] iodotyrosine
methyl ester (NEN Life Science Products, Boston, MA) and a
rabbit anti-cAMP antibody (Fitzgerald, Concord, MA). Results are
expressed as % basal cAMP released in the presence of normal mouse
serum.
Proliferation in response to purified TSHR antigen
Splenocytes (quadruplicate aliquots; 4 x
105 cells; 200 µl) were incubated in 96-well
round bottomed plates in the presence or absence of TSHR 289 (see
above, 4 or 20 µg/ml). Culture medium was RPMI 1640, 10% heat
inactivated FBS, 2 mM glutamine, 1 mM sodium
pyruvate, 50 µg/ml gentamycin, 50 µM
ß-mercaptoethanol, and 100 U/ml penicillin. After 6 days (37 C, 5%
CO2), 150 µl medium was removed from each well
to determine cytokine production (see below). Each well was then
supplemented with the same volume of fresh medium with or without
murine IL-2 (20 ng/ml, R & D, Minneapolis, MN). After approximately
8 h incubation, 1 µCi (3) H-thymidine
(3H-TdR; NEN Life Science Products)
was added to each well and cultures were harvested approximately
18 h later for scintillation counting using a Tomtec Harvester 96
(Orange, CO). This approach for using the same set of cultures
to determine both proliferation and cytokine production is based on the
protocol of Hafler and colleagues (20). The data for
lymphocyte proliferation are reported as 3H-TdR
incorporated (mean cpm + SEM for
quadruplicate wells).
Cytokine production in response to TSHR antigen
Supernatants from quadruplicate aliquots were pooled,
centrifuged to remove cell debris and stored (-80 C). Duplicate
aliquots (100 µl) were assayed for IFN- and IL-4 by ELISA using
capture and biotinylated detection-antibodies from BD
PharMingen (San Diego, CA) and following the
manufacturer’s protocol. Cytokine production was expressed as pg/ml
using standard curves of recombinant murine INF- and IL-4
(PharMingen).
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Results |
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Serum IgG antibody binding was studied using ELISA wells coated with a
truncated TSHR ectodomain variant, TSHR-289, secreted by mammalian
cells (13). We used the inactive form of TSHR-289
(15) because coating on ELISA wells converts the active to
the inactive form (data not shown). Sera from only a few BALB/c and
AKR/N mice vaccinated three or four times with TSHR-DNA gave ELISA OD
values 2 SD greater than the mean for control DNA
vaccinated mice (Fig. 1
). As expected,
the positive signals obtained for these sera (1:100 dilution) were very
low compared with the high values obtained for sera from BALB/c mice
conventionally immunized with TSHR-289 protein and adjuvant (1:10,000
dilution) (Fig. 1).
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2 x
106/ cell) (16). However, only a
small proportion of TSHR-vaccinated BALB/c and AKR/N mice had serum IgG
antibody that exceeded the median fluorescence values for sera from
vector-treated mice (Fig. 2). There was
no correlation between serum antibody positivity by flow cytometry
and ELISA (not shown). As with TSHR-289 coated ELISA wells,
conventional immunization with TSHR protein and adjuvant was more
effective than DNA vaccination in generating TSHR antibodies that
recognize the full-length TSHR (Fig. 2).
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). In contrast, as previously reported,
sera from approximately 60% AKR/N mice injected with TSHR-transfected
fibroblasts were TBI positive compared with untransfected
fibroblast-injected controls. Finally, TSAb was undetectable in
sera of BALB/c or AKR/N mice vaccinated with TSHR-DNA (Fig. 4). In contrast, sera from all three
AKR/N recipients of TSHR-fibroblasts had TSAb activity, consistent with
their elevated T4 levels (of 8.2, 9.6, and 12.6 µg/dl), as previously
reported (17).
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Splenocyte proliferation in response to TSHR antigen
Several studies indicate that DNA vaccination is more effective at
immune priming than inducing antibody production (for example, see Ref.
21). Therefore, it was possible that, despite low or
absent TSHR antibody levels, lymphocytes from TSHR-vaccinated mice
would proliferate when exposed in vitro to TSHR antigen. To
investigate this possibility, we incubated spleen cells with purified
TSHR-289. Initially, we used the active form of TSHR-289. Cell
proliferation was measured by 3H-thymidine
incorporation after 6–7 d incubation. One day before the end of these
incubations medium was taken for cytokine analysis (see below) and was
replaced with medium containing IL-2. We focused on BALB/c mice because
previous investigations of TSHR-DNA vaccination were performed with
this inbred strain (4).
Variable splenocyte thymidine incorporation from individual mice was
observed in the absence of TSHR-289 (Fig. 5, clear bars in
left and right panels). TSHR-289 (4 and/or 20
µg/ml) induced a significant proliferative response in splenocytes
from all 5 TSHR-vaccinated BALB/c mice (Fig. 5, left panel;
P < 0.05). Splenocyte proliferation was unrelated to
serum TSHR antibody levels. In contrast, splenocytes from only one of 4
vector-vaccinated mice showed a significant response to TSHR-289 (Fig. 5, right panel). Inactive TSHR-289 was as effective as the
active form at stimulating splenocyte proliferation, regardless of
whether IL-2 was, or was not, added to the culture (Fig. 6A).
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; note the
ordinate values compared with Fig. 6A). In the absence of IL-2, there
was no response to either active or inactive TSHR-289 (Fig. 6B).
However, proliferation was decreased in IL-2 supplemented cultures
containing TSHR-289 (active or inactive). The reason for this apparent
suppression is unclear. It was unrelated to cytokines (see below) and
may simply reflect erratically high proliferation levels seen for
splenocytes from Shimojo mice.
Cytokine production by splenocytes cultured with TSHR-289
We determined the effect of TSHR-289 on the production by spleen
cells of the major Th1 and Th2 cytokines, IFN- and IL-4. As
described above, supernatants from splenocyte cultures were removed
after 6 d and assayed by ELISA. Addition of TSHR-289 to splenocytes
from TSHR DNA-vaccinated BALB/c mice greatly increased IFN-
production from undetectable basal levels (Fig. 7A). Similar responses were induced with
active and inactive TSHR-289 (data not shown). Control mice vaccinated
with the empty vector showed no increase in IFN- production when
challenged with TSHR-289. Overall, IFN- production in BALB/c mice
reflected the proliferative response to TSHR-289 (Fig. 5), but the
cytokine response provided a more specific indicator of prior TSHR
sensitization by DNA vaccination (Fig. 7).
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in the absence of TSHR-289
(compare open bars in Fig. 7, A and B). Moreover, TSHR-289
did not significantly increase IFN- production by splenocytes from
mice injected with TSHR-expressing or nonexpressing fibroblasts (Fig. 7B). The Th2 cytokine IL-4 was undetectable in splenocytes cultures from DNA vaccinated mice, as well as from Shimojo model mice, regardless of the presence or absence of TSHR-289.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Low or undetectable levels of TSHR antibody in DNA vaccinated mice did
not preclude the possibility of lymphocyte sensitization to the TSHR.
Indeed, splenocytes from TSHR-DNA vaccinated mice proliferated and
produced large amounts of IFN- in response to challenge with
purified TSHR-289, an ectodomain variant corresponding to the TSHR A
subunit (14, 15). Splenocyte proliferation and INF-
production were unrelated to TSHR antibodies in the donor mouse’s
serum. In these studies we used BALB/c mice because they had been
previously used in DNA vaccination (4), as well as in
numerous TSHR immunization studies (5, 22, 23). Our
finding of TSHR sensitization following DNA vaccination is consistent
with the studies of Many et al. (24) in which
splenocytes from TSHR DNA-vaccinated mice could be reactivated in
vitro with TSHR antigen before adoptive transfer to naive mice. In
the Shimojo model, the generalized immune activation, particularly
INF- production, precluded our ability to detect in vitro
responses to TSHR antigen.
Our finding of IFN-, but not IL-4, production in TSHR-vaccinated
mice is perhaps surprising for two reasons: 1) BALB/c mice generally
display a bias toward Th2 responses, at least with respect to
Leishmania infection (reviewed in Ref. 25) and 2)
TSHR-primed T cells generated by DNA vaccination and transferred to
naive BALB/c recipients induced thyroiditis characterized by a Th2 type
response, with cells containing IL-4 and IL-10 (24). On
the other hand, our data are consistent with numerous other
observations that im DNA vaccination against diverse antigens induces
Th1-type responses as reflected in IFN- production and antibodies of
subclass IgG2a (21, 26, 27). Moreover, as discussed by
Costagliola et al. (4), their isolation of
three IgG2a mAb following TSHR-DNA vaccination is also in agreement
with the Th1 bias of im DNA vaccination.
The proliferative and cytokine responses to the two alternately folded
forms of native TSHR-289 antigen provide additional insight into the
outcome of TSHR-DNA vaccination. Active TSHR-289 is recognized by
patients autoantibodies and not by a mouse mAb (3BD10), whereas the
inactive form is recognized by the mAb but not by patients’
autoantibodies (14, 15). Unlike recognition by human
autoantibodies, both active and inactive TSHR-289 induced proliferation
and IFN- cytokine production by splenocytes from TSHR-DNA vaccinated
mice. This lack of discrimination between active and inactive TSHR-289,
at least at the T cell level, is likely to be in accordance with the
low or absent levels of antibody responses in the TSHR-DNA vaccinated
mice.
Important observations in our study were the very poor TSHR antibody response and absence of thyroiditis following TSHR DNA-vaccination. These data are in striking contrast to those of Costagliola et al. (4). We can only speculate about the basis for these differences. We used the same mouse strain and the same immunization protocol. We pretreated our mice with cardiotoxin, the agent found by Costagliola et al. (4) to provide the strongest enhancement of genetic immunization. In addition, both studies were performed using BALB/c mice. However, the animals were derived from different breeding colonies, Charles River in the study by Costagliola et al. (4) and The Jackson Laboratory in our case. Another clue to the difference between our findings may be the marked variance in diabetes in NOD mouse colonies around the world, which arises despite similar breeding protocols and may reflect environmental factors including housing conditions (28). Our animal facility is pathogen-free. From discussions at a workshop at the International Thyroid Meeting, Kyoto, Japan (October 2000), it was clear that facilities used by a number of researchers (apparently including that used by Costagliola et al.) are not pathogen-free. Obviously, humans do not live in barrier conditions, and for that unforeseen (and unchangeable) reason our facility may be less well suited than others for the development of an animal model resembling Graves’ disease in humans. Nevertheless, some environments are less hygienic than others. For example, infectious mononucleosis occurs in older teenagers in Europe but not in undeveloped rural Africa owing to continual exposure from infancy to the causative agent (Epstein-Barr virus) in the latter. Moreover, there is evidence that infectious organisms may play a role in the manifestation of human thyroid autoimmunity (reviewed in Ref. 29). It is possible, therefore, that infections could contribute to the expression of experimentally induced Graves’ disease.
In conclusion, DNA vaccination is much less effective at inducing TSHR antibodies than the Shimojo approach (injecting fibroblasts coexpressing the TSHR and MHC class II), at least under pathogen-free conditions and with this particular inbred colony. The Shimojo approach has the disadvantage of generalized nonspecific immune activation. In contrast, DNA vaccination offers the opportunity for future characterization of TSHR-specific T cells generated without adjuvant.
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Acknowledgments |
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Footnotes |
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Abbreviations: CHO, Chinese hamster ovary; IFN, interferon; MHC, major histocompatibility complex antigen; TBI, TSH binding inhibition; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.
Received January 31, 2001.
Accepted for publication April 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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