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Experimental Studies |
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, and the University of California, San Francisco, California 94121
Address all correspondence and requests for reprints to: Dr. Sandra McLachlan, Veterans Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.
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Abstract |
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light chain variable region genes, spliced by
overlap PCR to a mammalian signal peptide, were transferred to
expression vectors for human IgG1, IgG4, and L chains. Plasmids
containing the IgG1 (or IgG4) heavy chain and the L chain were
cotransfected into SP2/0 mouse myeloma cells. Cells secreting TPO
autoantibodies were cloned, and IgG1-SP and IgG4-SP were affinity
purified from medium using protein G. Their subclass specificities were
confirmed by enzyme-linked immunosorbent assay and fluorometry after
binding to Chinese hamster ovary cells expressing cell surface TPO.
Further confirmation of SP1.4 Fab conversion to full-length molecules
was the ability of protein A to precipitate IgG1-SP and IgG4-SP
complexed to [125I]TPO. IgG1-SP1.4, IgG4-SP1.4, and Fab
SP1.4 had similar high affinities for TPO (Kd = 
2
x 10-10 mol/L). Complexes of [125I]TPO and
IgG1-SP (but not IgG4-SP) bound to peripheral blood mononuclear cells
(PBMC), but not to a B cell line. Flow cytometry demonstrated Fc
receptors Fc
RI, FcRII, and FcRIII on PBMC, but only FcRII
on the B cell line. Together, these data indicate that IgG1-SP/TPO
complexes bind to either FcRI on monocytes or RIII on natural killer
cells. In assays for antibody-dependent cytotoxicity using PBMC,
51Cr release was higher for thyroid cells preincubated with
IgG1-SP (13.4%) than with IgG4-SP (2.5%) or with culture medium alone
(-0.7%). No specific 51Cr release was observed when
either fibroblasts or Chinese hamster ovary cells expressing cell
surface TPO were used as target cells. In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.
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Introduction |
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Besides complement-induced cytotoxicity, damage can also be mediated by
an antibody-dependent cell cytotoxic mechanism (ADCC). In this process,
effector cells (macrophages or natural killer cells), via their Fc
receptors (FcR) engage and kill target cells coated with antibody.
Indeed, thyroid autoantibodies can damage cultured thyroid cells by
ADCC (11). However, unlike complement-mediated thyroid autoantibody
damage, the autoantigen(s) involved in ADCC is not well established.
Thus, an association between thyroid microsomal/TPO autoantibodies and
ADCC has been found in some studies (11, 12, 13), but not in others
(14, 15, 16).
As is well known, antibodies are bifunctional molecules. The Fab region binds to antigen. The Fc region, which differs in immunoglobulin molecules of different classes and subclasses, is responsible for the biological effects of the antibody (reviewed in Ref.17). For example, IgG1 and IgG3 antibodies to red blood cells are more efficient than IgG2 or IgG4 antibodies of the same specificity in lysing erythrocytes by ADCC (18). This subclass difference is pertinent to the ability of thyroid autoantibodies to mediate ADCC, because TPO autoantibodies are predominantly IgG1 and IgG4, with IgG2 in some patients and little if any IgG3 (19, 20). Based on the red blood cell model, it is possible that ADCC would be more effective with IgG1, rather than IgG4, TPO autoantibodies in patients’ sera.
We have previously isolated a panel of human monoclonal TPO autoantibodies, expressed as Fab, by cloning immunoglobulin genes from patients’ intrathyroidal B cells (reviewed in Ref.21). One of these Fab, SP1.4, interacts with a TPO epitope recognized by the sera of all patients with autoimmune thyroid disease (22). In the present study, we have converted Fab SP1.4 to full-length IgG molecules of two different subclasses, IgG1 and IgG4. Data obtained with these two antibodies, with identical antigen-binding regions, support the likelihood that TPO autoantibodies of subclass IgG1 mediate thyroid cell damage in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of plasmids pAH4604-SP-G1 and pAN4621-SP-K for the
expression of the TPO autoantibody SP1.4 full-length IgG1 heavy (H)
chain and light (L) chain have been described previously (23) (Fig. 1
). In brief, because the SP1.4 H and L chains in the
bacteriophage 
vector (Immunozap, Stratagene, La Jolla, CA)
contained bacterial signal peptides, in pAH4604-SP-G1 and pAN4621-SP-K,
these signal peptides were replaced with the human TSH receptor signal
peptide (24). The full-length IgG4 version of the SP1.4 H chain was
generated by inserting the EcoRV-NheI fragment
containing the VH, D, and J regions of SP1.4 from pAH4604-SP-G1 into
the same sites in the H chain mammalian cell vector with human IgG4
constant regions (pAH4801; kindly provided by Dr. Sherie Morrison,
University of California-Los Angeles). The new SP1.4 IgG4 H chain
plasmid was called pAH4801-SP-G4 (Fig. 1).
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Plasmids pAN4621-SP-K (2 µg) and either pAH-SP-G1 or pAH-SP-G4 (20 µg), linearized with PvuI, were stably transfected into SP2/0 cells by electroporation and neomycin (G418) selection, as previously described in detail (23). Screening of 96-well plates for TPO antibody production was performed by enzyme-linked immunosorbent assay (ELISA) or [125I]TPO binding (see below). Cells from positive wells were cloned by limiting dilution. Two clones (IgG1-SP and IgG4-SP) that secreted high levels of IgG1 and IgG4 class TPO autoantibodies, respectively, were expanded to provide about 1 L medium. IgG was affinity purified using a protein G column (Pharmacia Biotech, Piscataway, NJ).
ELISA for IgG1-SP and IgG4-SP
TPO secreted into culture medium from Chinese hamster ovary
(CHO) cells (25) was used to coat ELISA plates, as previously described
(22). TPO autoantibodies IgG1-SP and IgG4-SP were detected using murine
monoclonal antibodies to human IgG1, IgG4, or chains (NL16, RJ4,
and QE11, respectively; Recognition Sciences, Birmingham, UK) and a
horseradish peroxidase detection system (22).
TPO autoantibody binding to CHO cells expressing TPO (CHO-TPO cells)
IgG1-SP or IgG4-SP binding to CHO-TPO cells (25) was performed as previously described (26). After incubation in IgG1-SP or IgG4-SP, cells were exposed to biotin-conjugated, mouse monoclonal anti-human IgG1 or anti-IgG4 (Caltag, San Francisco, CA) and subsequently incubated with streptavidin-R-phycoerythrin (Caltag). Fluorescence was determined using a Perkin-Elmer fluorimeter (Norwalk, CT; emission, 575 nm; excitation, 488 nm).
Protein A precipitation of [125I]TPO binding by IgG1-SP or IgG4-SP
The assay was performed on duplicate aliquots using
125I-labeled recombinant TPO, as previously described
in detail (22). Background binding (5% of the total counts),
determined using normal human serum diluted 1:20, was subtracted from
the values for IgG1-SP or IgG4-SP when calculating the percentage of
[125I]TPO bound. Data obtained in the presence of
increasing concentrations of unlabeled TPO were used to determine
affinities by Scatchard analysis (27). In some assays, precipitations
were performed using monoclonal antihuman chains (QE11, Recognition
Sciences), as previously described for Fab SP1.4 (22).
Binding by lymphoid cell populations of [125I]TPO/IgG1-SP or IgG4-SP complexes
Peripheral blood mononuclear cells (PBMC) from a normal donor
were separated by density centrifugation (Histopaque-1077, Sigma
Chemical Co., St. Louis, MO). Before use in binding studies (see
below), PBMC were incubated for 1 h at 37 C in petri dishes to
elute IgG/antigen complexes bound in vivo to
monocytes/macrophages. Lymphoblastoid B cell lines (EBVL) were obtained
by transformation of Graves’ lymphocytes with Epstein-Barr virus as
previously described (23). FcR expression was analyzed by flow
cytometry. Aliquots of PBMC and EBVL (106 cells) were
incubated (1 h, 4 C) with fluorescein isothiocyanate (FITC)-conjugated
murine monoclonal antibodies as follows: FcRI, anti-CD64 (clone
10.1); FcRII, anti-CD32 (clone 2003); and FcRIII, anti-CD16
(clone 3G8; all from PharMingen, San Diego, CA). Controls included
unstained cells and FITC-conjugated isotype controls (IgG1 and IgG2b;
PharMingen). Cells were analyzed (10,000 events) using the Becton
Dickinson FACScan-CELLQuest system (Mountain View, CA).
Binding of [125I]TPO complexed with IgG1- or IgG4-SP to
PBMC or EBVL was performed as follows. Aliquots (400 µL) of culture
medium containing IgG1-SP or IgG4-SP were incubated overnight at 4 C
with [125I]TPO (200,000 cpm; 5 ng) in the absence or
presence of excess unlabeled TPO (1 µg). As controls, radiolabeled
TPO was incubated in aliquots of fresh culture medium with or without
unlabeled TPO. The following day, PBMC or EBVL (duplicate aliquots,
5 x 106 cells) were resuspended in the
antibody-antigen complexes. After incubation for 2 h at 4 C, the
cells were washed twice with 3 mL buffer B (phosphate-buffered saline
and 2% BSA), and [125I]TPO bound to the pelleted cells
was determined by -counting.
ADCC
Effector cells in ADCC assays were PBMC from a normal donor (see
above). As targets, we used 1) Graves’ thyroid cells (28), 2) CHO-TPO
cells (25), and 3) human fibroblasts of thyroid tissue origin. To
restore TPO expression by thyroid cell primary cultures, cells were
cultured for 3–4 days with TSH (10-7 mol/L) (29).
Cytotoxicity assays were performed according to previously described
protocols (11, 13, 30). Target cells were detached from culture dishes
by light trypsinization, and aliquots (3 x 105
cells) were incubated (4 C, 30 min) in RPMI 1640 with 10% FCS and
IgG1-SP or IgG4-SP or (as a control) in culture medium alone.
Subsequently, 75 µCi 51Cr (sodium chromate, DuPont,
Boston, MA) was added, and incubation was continued (45 min, 37 C, 5%
CO2). After washing twice with culture medium, labeled
target cells were distributed (3 x 103 cells/well)
in 96-well U-bottomed culture plates. Target cells (triplicate wells)
were incubated with effector cells (PBMC; 1.5 x
105 cells/well; effector/target ratio, 40:1). Triplicate
aliquots of the same target cells were used to determine
51Cr release without PBMC and total 51Cr
release by the addition of culture medium or detergent (5% Triton
X-100; Sigma), respectively. The culture plates were centrifuged (2
min, 55 x g) and incubated for 18 h (37 C, 5%
CO2). After centrifugation (5 min, 550 x
g), aliquots (100 µL) of the supernatant were removed for
-counting. Cytotoxicity was expressed as specific 51Cr
release (mean ± SE of triplicate wells) calculated as
follows: specific 51Cr release = [(cpm with PBMC) -
(cpm without PBMC)]/total cpm released. Differences in specific
51Cr release were analyzed by Student’s t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SP2/0 mouse myeloma cells cotransfected with pAN4621-SP-K (SP1.4
L chain) and pAH4604-SP-G1 (SP1.4 IgG1 H chain; Fig. 1
) secreted
TPO antibody detectable by ELISA with antihuman IgG1, but not with
anti-human IgG4 (Fig. 2, left panel).
Conversely, SP2/0 cells cotransfected with the same L chain plasmid and
pAH4801-SP-G4 containing the SP1.4 IgG4 H chain gene (Fig. 1) produced
TPO antibody detectable with anti-IgG4, but not anti-IgG1 (Fig. 2, right panel). Both IgG1-SP and IgG4-SP could also be
detected with monoclonal anti-human . The subclass specificity
demonstrated by ELISA using soluble TPO was confirmed by fluorometry
(Fig. 3) using full-length TPO expressed on the surface
of CHO cells (25).
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Further confirmation of the conversion of the SP1.4 Fab into
full-length IgG1 and IgG4 was the ability of protein A to precipitate
IgG1-SP and IgG4-SP complexed to [125I]TPO.
Competition for this binding by increasing amounts of unlabeled TPO
(Fig. 4A) permitted Scatchard analysis (27) of the
IgG1-SP and IgG4-SP affinities for TPO. The affinities
(Kd) of 1.7 and 1.9 x 10-10
mol/L for IgG1-SP and IgG4-SP, respectively (Fig. 4, B and C), were
essentially identical to that previously observed for Fab SP1.4
(2.2 x 10-10 mol/L) (22).
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The above experiments were performed using IgG affinity purified
with protein G from medium containing FCS. Despite its very low
concentration in this medium, some bovine IgG was copurified with the
TPO autoantibodies. Consequently, we could not determine the absolute
concentrations of IgG1-SP and IgG4-SP. By ELISA, using antihuman ,
the activity of IgG1-SP was approximately 10-fold greater than that of
IgG4-SP (Fig. 2). Similarly, 10-fold more IgG4-SP than IgG1-SP (1:30
vs. 1:300 dilution) was required to attain comparable
(23.9% vs. 23.8%) [125I]TPO binding as
detected by precipitation with the same anti- antibody. Thus, both
methods provided similar estimates of the relative functional
concentrations of the two autoantibodies. Therefore, for all functional
assays (below), IgG4-SP and IgG1-SP were used at a 10:1 ratio.
FcR binding of recombinant IgG1-SP and IgG4-SP
The functional activities of the IgG1 and IgG4 Fc regions
introduced into the SP1.4 Fab can be assessed by their ability to bind
to lymphoid cells via FcR. PBMC, which include T cells, B cells,
monocytes, and natural killer cells, bound higher levels of
[125I]TPO/IgG1-SP than [125I]TPO/IgG4-SP
immune complexes (Fig. 5A). Inclusion of excess
unlabeled TPO during complex formation reduced radiolabel binding by
PBMC to background levels, namely culture medium without TPO
autoantibody. In contrast, no specific binding to Epstein-Barr
virus-transformed B cells (EBVL) could be detected (Fig. 5B). Flow
cytometry of PBMC revealed the presence of all three forms of FcR,
namely FcRI (CD64), FcRII (CD32), and FcRIII (CD16), on the
surface of some cells in the population (Fig. 6, left panels). In contrast, EBVL had only FcRII (CD32) on
their surface (Fig. 6, right panels). Taken together, the
data in Figs. 5 and 6 indicate that IgG1-SP/TPO complexes bind to
lymphoid cells via either their FcRI or FcRIII.
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We examined the relative abilities of IgG1-SP and IgG4-SP to
mediate ADCC using human thyroid cells in primary culture. In three
separate experiments, IgG1-SP-mediated 51Cr release (mean,
13.4%) was higher than that for IgG4-SP (mean, 2.5%) as well as that
for medium without antibody (mean, -0.7%; Fig. 7, upper panel). The significance of the difference between
IgG1-SP and IgG4-SP in the individual experiments (each with triplicate
values) was P < 0.001, P < 0.001, and
P = 0.05, respectively. No specific 51Cr
release was observed when either fibroblasts or CHO-TPO were used as
target cells (Fig. 7, lower panel). Indeed, PBMC reduced the
spontaneous release of 51Cr in these cells regardless of
the presence or absence of IgG1-SP or IgG4-SP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 x
10-10 mol/L) (22).
Binding of IgG to FcR requires glycosylation (reviewed in Ref.17).
Consequently, PBMC binding of IgG1-SP/[125I]TPO complexes
confirms that the mammalian SP2/0 cells secrete glycosylated IgG
molecules. Unlike PBMC, no binding of complexes was observed to EBVL.
Like other B cells, the EBVL only express FcRII. PBMC, on the other
hand, include monocytes, B cells, and natural killer cells that express
FcRI, FcRII, and FcRIII, respectively (reviewed in Ref. 17 and
32). Consequently, PBMC binding of IgG1-SP/TPO complexes must occur
either via the monocyte FcRI and/or the natural killer cell
FcRIII.
The differential binding of IgG1-SP and IgG4-SP to PBMC is consistent
with other observations on preferential IgG1 antibody binding to Fc
receptors (reviewed in Refs. 17 and 32). FcRI have a higher affinity
for IgG than do FcRIII (32). Consequently, it is more likely that
the binding we observed by PBMC of IgG1-SP/TPO complexes involves
monocytes. However, the possibility that natural killer cell binding is
also involved cannot be excluded, as the affinity of FcRIII is
higher for IgG complexes than for monomeric IgG. Further, the
monoclonal antibodies used to characterize FcRI and -RIII do not
interfere with binding of receptors (17). Regardless of whether our
findings reflect binding to monocytes and/or natural killer cells, both
cell types can mediate ADCC.
The most important finding in our study was that ADCC mediated by monoclonal IgG1-SP was higher than that mediated by the same autoantibody containing a different constant region (IgG4-SP). This ADCC was evident with human thyroid cells. The lack of ADCC with human fibroblasts is consistent with the findings of other studies using nonclonal serum autoantibodies (13). We cannot explain the puzzling lack of ADCC using CHO-TPO cells, whose surface TPO is readily detectable by fluorometry (present study) and by flow cytometry (26). However, other studies have demonstrated that immortalized cell lines are resistant to killing, for example via complement (33). We conclude, therefore, that a human monoclonal TPO autoantibody that interacts with an epitope on TPO recognized by serum autoantibodies in all patients can mediate ADCC against human thyroid cells. Since the completion of our study, Rodien et al. (34) have demonstrated that affinity-purified polyclonal TPO autoantibodies can mediate ADCC in porcine thyroid cells.
Although our finding of preferential ADCC to thyrocytes with TPO autoantibodies of IgG1 subclass is consistent with data obtained in other systems, our study involves a number of important features. Thus, two major types of approach have been used previously to examine IgG subclass difference in relation to ADCC. First, differential IgG1- and IgG4-mediated ADCC has been studied with polyclonal antibodies and nonnucleated erythrocytes as target cells (18). This system can be influenced by the presence in polyclonal sera of antibodies of different affinities and to different epitopes (18). The second type of investigation involves ADCC by monoclonal antibodies of different subclasses to an identical epitope, usually a small molecule (hapten), and target cells chemically coated with the hapten (35). Our study of the role of IgG subclass in ADCC involved 1) nucleated cells (such as thyrocytes), which are less sensitive to lysis than are nonnucleated erythrocytes (30); 2) a large, naturally expressed protein antigen, as opposed to a small hapten chemically coupled at high density to target cells; and 3) human monoclonal autoantibodies of identical affinity and epitopic specificity. A comparison has been performed of ADCC mediated by IgG1 and IgG4 antibodies with identical V regions specific for the CDw35 antigen on T lymphocytes (36). With this exception, our study appears to be one of the few performed in which all three of these features have been present simultaneously.
The small extent of ADCC that we observed is lower than might be anticipated for a high affinity monoclonal antibody. The efficacy of an antibody in mediating ADCC cytotoxicity is influenced not only by antibody isotype, but also by epitopic density (reviewed in Ref.17). In particular, it has been suggested that for therapeutic purposes, combinations of different murine monoclonal antibodies to different epitopes on the same antigen may be more effective than one antibody alone (reviewed in Ref.17). Future studies will indicate if multiple monoclonal TPO autoantibodies to nonoverlapping epitopes are more effective.
The preferential effect of IgG1-SP relative to IgG4-SP in mediating ADCC is consistent with clinical observations on TPO autoantibodies in patients during the postpartum period. During this period, some TPO autoantibody-positive women develop symptoms of thyroid dysfunction suggestive of thyroid damage (for example, Ref.37). Consequently, it is of interest that in two studies (38, 39), but not in a third (40), an association has been noted between IgG1 (but not IgG4) TPO autoantibodies and hypothyroidism.
In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.
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Acknowledgments |
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genes in mammalian cells, and Dr.
Morrison and her colleagues for their advice on plasmid construction
and expression. |
Footnotes |
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2 Recipient of a University of California-San Diego Molecular
Medicine Training Program Fellowship Award.
Received August 23, 1996.
Revised October 31, 1996.
Accepted December 3, 1996.
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