Key words
TSH receptor blocking autoantibodies - cell-based bioassay - binding assay - Hashimoto’s
thyroiditis - Graves’ disease
Abbreviations
AA:
Amino acids
ATD:
Antithyroid drugs
Ab:
Autoantibodies
anti-TSHR-Ab:
Autoantibodies to the TSHR
AITD:
Autoimmune thyroid disease
bTSH:
Bovine TSH
CHO:
Chinese hamster ovary
CREB:
cAMP response element-binding protein
cAMP:
Cyclic adenosine 3′,5′-monophosphate
FRTL-5:
Fisher rat thyroid line-5
FDA:
Food and Drug Administration
GD:
Graves’ disease
HT:
Hashimoto’s thyroiditis
IgG:
Immunoglobulin G
L-T4:
Levothyroxine
LH-CG:
Luteinizing hormone-choriongonadotropin
MAb:
Monoclonal antibody
Mc4:
Mutant chimeric 4
RAI:
Radioactive iodine
RIA:
Radio-immunoassay
TBII:
TSHR-binding inhibitory immunoglobulin
TBAb:
TSHR-blocking autoantibodies
TSAb:
TSHR-stimulating autoantibodies
TSH:
Thyroid-stimulating hormone
T4:
Thyroxine
T3:
Triiodothyronine
TSHR:
TSH receptor
wt:
Wild-type
Introduction
Autoimmune thyroid diseases (AITD) are characterized through increased familial clustering,
reduced DNASE1 gene expression, CTLA-4 polymorphisms, and are strongly associated
with the major histocompatibility (MHC) complex [1]
[2]
[3]
[4]. However, autoantibodies to various thyroid antigens are the most important biomarkers
that are used to differentiate AITD from other thyroid conditions. Whereas antibodies
to thyroid peroxidase (TPO) and thyroglobulin (Tg) neither play a major nor casual
role in the pathophysiology of AITD, antibodies to the TSHR play a unique role in
the development of autoimmune hyper- and hypothyroidism. Autoantibodies to the TSHR
(anti-TSHR-Ab) are directly involved in the pathophysiology of Graves’ disease (GD)
and Hashimoto’s thyroiditis (HT). GD is caused by TSHR-stimulating antibodies (TSAb),
which act as agonists by stimulating thyroid growth and thyroid hormone synthesis
in an unregulated manner [5]
[6]
[7]. In contrast, blocking anti-TSHR-Ab (TBAb) acts as TSHR antagonists, which block
the action of the thyroid-stimulating hormone (TSH) and can cause the hypothyroidism
of HT. Anti-TSHR-Ab can be detected either with immunoassays, which measure TSHR-binding
inhibitory immunoglobulins (TBII) [8] or with cell-based bioassays, which measure either TSAb or TBAb [9]. The advantages and disadvantages of binding assays versus functional bioassays
for anti-TSHR-Ab, with special emphasis on TBAb, are shown in [Table 1].
Table 1 Advantages and disadvantages of binding (TBII) Assays versus functional bioassay
for TSHR blocking autoantibodies.
|
Advantages
|
Disadvantages
|
|
Binding Assays (TBII)
|
International standardization with reference material
|
Do not discriminate the functional antibody type
|
|
Easy handling and performance
|
Only measure antibody binding to the TSHR
|
|
M22 and KSAb1, human and mouse thyroid stimulating mAbs respectively, have replaced
bTSH in newer automated binding assays
|
TBII levels reflect total anti-TSHR-Ab
|
|
Commercially automated assays are available
|
|
|
Functional Bioassay
|
Measure the net sum of the functional activity
|
Absence of an international standard
|
|
Discriminate the functional antibody type, specifically identifying TBAb*
|
More time consuming than TBII
|
|
Higher analytical sensitivity than binding assays (TBII)
|
Requires experienced laboratory technician
|
|
Newly developed bioassays: minimal handling of the cells, no IgG purification, no
serum starvation, no serum concentration
|
Not widely available
|
|
Predict fetal/neonatal risk for hypothyroidism in pregnant women with active or treated
AITD
|
Not automated yet
|
* According to references [23]
[25] and JP Banga, personal communication.
Bioassays involve the measurement of signal transduction pathways mediated by binding
of a ligand to the TSHR [10]
[11]. Compared to TBII binding assays, cell-based bioassays are more sensitive in detecting
low anti-TSHR-Ab concentrations and exclusively differentiate between the anti-TSHR-Ab
functionality [12]
[13]. Historically bioassays that measure TSAb were based on measurement of cAMP levels
in cells using radio-immunoassays (RIA) [14]
[15]
[16]
[17]
[18]
[19]. More recently, non-radioactive methods to measure cAMP have been used. In addition,
cell lines that contain a cAMP-inducible reporter gene such as luciferase have been
employed to measure TSAb [20]
[21]
[22]
[23]. Bioassays that measure TSHR-blocking activity are based on the same cell-based
systems, but they detect the ability of patient antisera to block TSH or TSAb-stimulated
cAMP levels or luciferase expression [23]
[24]
[25].
Most reviews of anti-TSHR-Ab have focused primarily on TSAb. In the present, paper
we will focus on TBAb. Compared to TSAb [26]
[27]
[28], the measurement and clinical utility of functional TBAb have been less well investigated.
Many reports have studied the occurrence of TBAb in various autoimmune diseases associated
with hypothyroidism such as autoimmune-induced atrophic thyroiditis or primary myxedema
[29]
[30]
[31]
[32]
[33]
[34]. The prevalence of TBAb in these various conditions is still controversial. Other
investigations have focused on the role of TBAb in congenital and neonatal hypothyroidism
associated with maternal AITD [31]
[35]
[36]
[37]
[38]. The importance of TBAb in congenital hypothyroidism, however, has been questioned.
Ever since it has been known that anti-TSHR-Ab exhibit different functional activities
it has been suspected that certain patients might contain both TSAb and TBAb, and
that this may explain certain clinical presentations. Recent evidence has proven that
a patient can have both TSAb and TBAb by isolating separate monoclonal antibodies
(MAb) with stimulatory and blocking activity from the lymphocytes of the same patient
[39]
[40]. In addition, there has been speculation that patients with GD may shift between
stimulating and blocking antibodies as they transition from hyperthyroid to hypothyroid
and vice versa. The mechanism of transition from TSAb to TBAb is most probably due
to a different ratio between the two functional autoantibodies; while the exact antigenic
sites of the TSHR against TBAb and TSAb are strongly overlapping and not completely
defined. In the present paper, we will review the historical and currently used assays
used for the measurement of TBAb activity and discuss their clinical applications.
Pathophysiology of Hashimoto’s Thyroiditis
Pathophysiology of Hashimoto’s Thyroiditis
Hashimoto’s thyroiditis is the major cause of autoimmune hypothyroidism and GD is
the primary cause of autoimmune hyperthyroidism. Whereas the hyperthyroidism of GD
is exclusively due to TSAb, there are several mechanisms, by which hypothyroidism
develops in HT. The primary pathological basis of decreased thyroid hormone production
is related to immune-mediated apoptosis and cytolysis of thyrocytes. Normal thyroid
epithelial cells express a variety of death receptors including Fas and activation
of the Fas-ligand-Fas signaling system contributes to the follicular cell destruction
characteristic of HT. In addition, cytokine stimulation from antigen-presenting cells
and Th1 cells (IL-1) can induce functional Fas and also Fas ligand on thyroid follicular
cells which can lead to self-apoptosis [41]. Furthermore, the accumulation of activated T cells expressing Fas ligand may induce
apoptosis of thyrocytes directly by interacting with Fas on the cells [42].
The characteristic histopathological abnormalities of HT are profuse lymphocytic infiltration,
lymphoid germinal centers, and destruction of thyroid follicles. Intrathyroidal lymphocytes
are both T and B cells. Recent insight into the development of intrathyroidal germinal
centers and lymph vessels suggests the importance of local production of chemokines
[43]. B cells from thyroid tissue of patients with HT are activated, as indicated by
their ability to secrete thyroid antibodies spontaneously in vitro. Thus, the thyroid
gland may be a major site of thyroid Ab secretion. Anti-Tg and anti-TPO Ab of the
appropriate IgG subclass have the potential to fix complement, and thus antibody-mediated
complement-dependent cytotoxicity (CDC) may contribute to thyroid damage in some patients
with HT [44]. More important may be the role of thyroid Ab secreting B cells in presenting thyroid
antigen to the T cells, which react with processed thyroid antigens and peptides derived
from these antigens. These activated T cells secrete cytokines, which themselves activate
a variety of other immune cells. T cells have two roles in this disease: a role in
Ab production (a Th2 type of function) and a role in the apoptotic destruction of
thyroid cells by activating cytotoxic T cells (a Th1 function) [45]. The Th1 CD4+ lymphocytes, when stimulated by antigen, secrete interleukin-2 (IL-2),
interferon gamma, and tumor necrosis factor-beta. In contrast, Th2 cells, when stimulated
by antigens, secrete IL-4 and IL-5. Both types of T cells are found in thyroid tissue
of patients with HT, but Th1 cells predominate [46]
[47]. A T-cell clone that caused cytolysis of autologous thyroid cells has been reported
in a patient with HT [48]. Finally, in addition to the various mechanisms of thyroid cell death, decreased
thyroid hormone production in HT may be caused by TBAb which rather than causing thyroid
cell death, bind to the TSHR and interfere with the ability of thyrocytes to respond
to TSH. The relative contribution of all of these immunopathophysiological mechanisms
in HT varies from patient to patient.
Bioassays for TSHR-Blocking Autoantibodies
Bioassays for TSHR-Blocking Autoantibodies
Definition of blocking activity
Anti-TSHR-Ab that bind to the TSHR and neither activate the cyclic AMP pathway nor
stimulate thyroid hormone synthesis, but rather act as TSHR antagonists inhibiting
the activation of signal transduction pathways, are defined as blocking anti-TSHR-Ab
or TBAb [49]
[50].
Historical methodology
Early bioassays for the detection of TBAb were based on the same cell-based systems
used for TSAb, including thyroid plasma membranes, thyroid tissue slices, and eventually
cultured thyroid cells [51]
[52]
[53]. In contrast to TSAb bioassays, TBAb bioassays detected the ability of patient sera
to block various concentration of bovine TSH (bTSH) or TSAb-stimulated cAMP levels.
Alternative TBAb assay systems were sought to address the limitations associated with
using thyroid tissue or primary thyroid cells. A major advance in the field was the
isolation of a cell line from normal rat thyroid (Fisher rat thyroid cell line, FRTL-5),
which endogenously expressed the TSHR and was TSH-dependent for growth [54]. Importantly, FRTL-5 cells retained the main features of normal thyroid functions,
such as hormone response, iodide uptake and thyroglobulin synthesis and could be maintained
over the relatively long culture period which used by several investigators [33]
[49]
[54].
Further improvement in the detection of blocking activity was achieved with a luminescent
bioassay for TBAb utilizing the CHO-K1 cell line (named lulu*) stably transfected
with the human TSHR and a luciferase gene under control of a promoter with a cAMP
responsive element [24]. Using CHO lulu cells and a luminescent assay for TBAb, twelve samples, previously
shown to contain TBAb by an established method quantifying cAMP by RIA, were also
positive by the luciferase-based assay [24]. The specificity of this assay was excellent in that 20/20 patients with systemic
lupus erythematosus, 13/14 with rheumatoid arthritis and 12/12 with multinodular goiter
were negative for TBAb. Bioassays using such engineered CHO cells have the advantage
of expressing the human TSHR and they require less cumbersome procedures for cell
culture [55].
Another CHO cell line was generated for the measurement of TBAb that stably expressed
a TSHR/LH-CG receptor construct that was reported to have a reduced response to most
TSAb [56]. This Mc1+2 TSHR has amino acid (AA) residues 8 to 165 from the extracellular region
of the TSHR substituted by the residues 10 to 166 of the LH-CG receptor. The CHO Mc1+2
cell line was responsive to TSH as measured by cAMP induction [57]
.> In a follow up study the CHO Mc1+2 cell line was compared with a wild type human
TSHR CHO cell line, W25, for the measurement of TBAb [56]. A chimeric TSHR was also used in a coated tube assay for the detection of TBAb
employing an adaptation of the TSH binding-inhibitory format [58]. This method utilized HEK-293 cells in which the wild-type TSHR of the TBII assay
was exchanged for a chimeric receptor in which supposed TSAb epitopes (AA 8–165) were
replaced by a TSH/Luteinizing hormone-choriongonadotropin (LH-CG) residues resulting
in a chimeric receptor (chimera A) [57]. In studies using this assay sera from patients with GD and AITD were grouped according
to their activity in a TBAb bioassy. At the decision threshold, the chimera assay
had a sensitivity of 78% for TBAb with a specificity of 90.2%, but correlation with
the bioassay results was only moderate (r=0.46). The technique was purported to allow
the differentiation between sera containing only TBAb, both TBAb and TSAb, only TSAb,
or sera without any bioactivity. Despite such efforts to map and remove stimulation-specific
epitopes and incorporate chimeric receptors into assays for exclusive detection of
TBAb, it is not clear that these efforts have been successful.
Currently available TSHR-blocking antibody assays
A bioassay is commercially available in Japan (Yamasa, Corp., Chosi, Chiba, Japan)
that utilizes porcine thyroid gland cells for the measurement of TBAb with a cAMP
RIA [59]
[60]. RSR Limited (Cardiff, UK) offers a research-use only service to measure TBAb. This
assay uses CHO cells expressing the human TSHR and TBAb is detected by inhibition
of porcine TSH-induced cAMP [61]. In another assay, autoantibodies inhibit the binding of M22 labeled with biotin
to TSHR-coated enzyme-linked immunosorbent assay (ELISA) plate wells [62]. Recently, a bioassay for the TBAb measurement based on luciferase expression was
described [23]
[25]. The assay uses the same Mc4 cell line, that is used in the FDA-cleared TSAb bioassay
[22]. The chimeric construct in the Mc4 cells amino acid (AA) residues 262–368 of the
human TSHR are substituted with the AA residues 262–334 from the rat LH-CG receptor
[25]. There is no cell line available expressing a modified TSHR that binds exclusively
to TSAb or TBAb including the Mc4 TSHR which was originally purported to have TBAb
epitopes removed. The TBAb bioassay using CHO Mc4 cells was shown to provide excellent
analytical performance and a high level of reproducibility [25]. A schematic of the TBAb bioassay principle is shown in [Fig. 1]. Early in the development of this TBAb bioassay, CHO cells expressing either the
chimeric human TSHR (Mc4) or the wild-type (wt) TSHR were compared side by side. Both
CHO cell lines were induced with different concentrations of bTSH. The half maximal
inhibitory concentration of K1-70 MAb in CHO Mc4 cells was more than five-fold lower
compared to the CHO wt cells. The CHO Mc4 cell line showed several advantages over
the CHO wt cell line including a broader linear range in response to bTSH, higher
sensitivity in detecting blocking activity of the purely human blocking MAb K1-70
as well as detecting higher levels of blocking activity of TBAb positive sera from
patients with AITD [23]
[25].
Fig. 1 Schematic of reporter gene-based functional bioassay for TBAb. CHO cells are engineered
to constitutively express the human TSHR and to express luciferase following induction
of cAMP. The binding of bovine (b) TSH to the TSH receptor on the surface of the cells
induces a signaling cascade that leads to an increase of intracellular cAMP and subsequently
to the luciferase expression. However, the presence of blocking anti-TSHR-Ab (TBAb)
in serum of patients with autoimmune thyroid diseases inhibits the bTSH stimulation
of the luciferase reporter gene. The TBAb level is correlated with blocking activity
which is defined as percent inhibition of luciferase expression relative to induction
with bTSH alone.
Another TSAb/TBAb bioassay was reported that utilizes a unique cAMP detection method.
CHO cells were engineered to express the TSHR and a calcium-activated bioluminescent
protein [63]. Following TSHR activation, increased intracellular cAMP levels activate the cyclic
nucleotide-gated calcium channel. The subsequent influx of calcium results in bioluminescence
of aequorin which is quantified with a luminometer [63]. As with other TSAb bioassays, TBAb can be measured with minor modifications of
the protocol [63].
[Table 2] summarizes first generation (cAMP RIA) and second generation (cAMP/luciferase) bioassays
described for the measurement of TBAb. Although head-to-head comparisons are limited,
the variable methodologies used to detect TBAb do not give identical results and thus
it is difficult to compare studies on the prevalence of TBAb [24]. Finally, it is important to note that when sera contain both TSAb and TBAb, bioassays
measure the net activity of stimulating and blocking antibodies and dilutional analysis
of serum may be required to detect both activities [13]
[24]. At the time of publication there are no TBAb bioassays approved by U.S. or European
regulatory agencies. Although second generation cAMP and luciferase assays using CHO
cells have simplified the TBAb assay procedure, the availability of TBAb bioassays
remains limited. Hopefully, this will change in the near future with increasing recognition
of the importance of TBAb in evaluating and managing patients with AITD.
Table 2 First generation (cAMP RIA assays)and second generation (cAMP/luciferase assays)
for TSHR blocking autoantibodies.
|
Year
|
Species
|
Assay time
|
Comments
|
Reference
|
|
FIRST generation assays (cAMP radio-immunoassay)
|
1978
|
Human
|
4 days
|
Human thyroid plasma membranes, adenyl cyclase activity
|
[51]
|
|
1980
|
Human
|
4 days
|
Human thyroid plasma membranes, IgG preparation by column chromatography through DEAE-Sephadex
|
[52]
|
|
1983
|
Human
|
4 days
|
Thyroid adenoma cells, IgG fractions preparation by DEAE-Sephadex column chromatography
|
[29]
|
|
1985
|
Human
|
4 days
|
Human thyroid plasma membranes, IgG preparation by DEAE-cellulose column chromatography
|
[53]
|
|
1987
|
Rat
|
4 days
|
FRTL-5 cells, IgG preparation by DEAE Sephadex
|
[33]
|
|
1989
|
Rat
|
1–2 weeks
|
FRTL-5 cells, IgG preparation by polyethylene glycol (PEG) precipitation
|
[68]
|
|
1989
|
Rat
|
1–2 weeks
|
FRTL-5 cells, IgG extraction by affinity chromatography on columns of protein A-Sepharose
|
[66]
|
|
1990
|
Rat
|
1–2 weeks
|
FRTL-5 cells, IgG dialyzed in hypotonic buffer
|
[49]
|
|
1999
|
Porcine
|
2 days
|
Porcine thyroid cells, IgG preparation by PEG precipitation
|
[59]
|
|
1999
|
Hamster
|
2 days
|
CHO cells (clones JP02 and JP26)
|
[73]
|
|
2000
|
Hamster
|
3 days
|
CHO cells (clone JP26)
|
[55]
|
|
SECOND generation assays (cAMP / luciferase)
|
1994
|
Hamster
|
4 days
|
CHO JP09 cells transfected with the recombinant human TSHR, IgG dialyzed and diluted
in hypotonic buffer
|
[17]
|
|
2001
|
Hamster
|
1 day
|
CHO cells (clone JP09) stably transfected with the TSHR (clone lulu*)
|
[24]
|
|
2004, 2009
|
Hamster
|
20–24 h
|
CHO cells expressing the wild-type human TSHR, use of serum, spectrophotometric cAMP
immunoassay
|
[62]
[83]
|
|
2013
|
Hamster
|
20 h
|
Fresh frozen vials of CHO cells expressing the chimeric human TSHR Mc4 and a luciferase
reporter gene, use of serum, luminescence (cAMP-dependent luciferase expression)
|
[23]
[25]
[76]
|
|
2015
|
Hamster
|
8 h
|
CHO cells expressing the wild-type human TSHR, use of serum, no sterile conditions
necessary, luminescence (cyclic nucleotide-gated calcium channel and aequorin)
|
[63]
|
Clinical Relevance and Clinical Applications of TBAb
Clinical Relevance and Clinical Applications of TBAb
Studies on TBAb in primary hypothyroidism
One of the first studies that identified TBAb in patients with AITD reported on the
inhibition of TSH-induced cAMP increase by IgG from patients with primary myxedema
[29]. IgG fractions prepared from sera of patients with primary myxedema, goitrous HT,
and controls, respectively, were tested for their ability to alter TSH stimulation
of cAMP production in cultured human thyroid cells and the binding of TSH to its receptor.
When compared with the cAMP increase induced by bTSH in the presence of normal IgG,
cAMP accumulation was significantly inhibited by IgG from patients with primary myxedema.
TSH-induced cAMP accumulation was not affected by IgG from patients with goitrous
thyroiditis. These authors further studied TSH-stimulated cAMP response inhibitory
immunoglobulins in patients with atrophic (primary myxedema) and goitrous HT and found
TBAb in 40.5% and 17% patients with atrophic and goitrous thyroiditis, respectively
[64]. In another study the prevalence of TBAb was also evaluated in hypothyroid patients
with either goitrous or atrophic thyroiditis [30]. IgG was incubated with porcine thyroid cells in the presence of bTSH and the results
were expressed as the percent inhibition of TSH-stimulated production of cAMP, as
compared with production in a pool of IgG from normal subjects [30]. TBAb were detected in 9% and 25% of the patients with goitrous and atrophic thyroiditis,
respectively [30]. In a case report TBAb were detected in a pre-pubertal severely hypothyroid boy
(baseline serum TSH 254 mU/l) with atrophic HT and decreased growth velocity [65].
Potent blocking type anti-TSHR-Ab was observed in the majority of patients with primary
myxedema using a FRTL-5 cell-based bioassay in which the prevalence of TBAb in primary
myxedema was 75% versus 0% in goitrous HT [66]
[67]. In line with this, inhibition of TSH-stimulated RAI uptake in FRTL-5 cells by crude
Ig fractions was reported for patients with goitrous and atrophic HT as well as from
patients with overt hypothyroidism [16]
[68]. TSH-stimulated RAI uptake was inhibited by the Ig fractions from TBII-positive
patients with atrophic thyroiditis due to their TBAb positivity. The mean inhibition
of TSH-stimulated RAI correlated with the ability of the Ig fractions to inhibit TSAb-stimulated
RAI uptake (r=0.882) and TSH-stimulated cAMP accumulation (r=0.929) [16]. TBAb were also present in 6/41 (14.6%) patients with hypothyroidism [68].
Other groups also evaluated Ab blocking the TSH-dependent cAMP production in FRTL-5
cells [33]. TBAb were evaluated in patients with primary autoimmune hypothyroidism and were
detectable in 15/23 patients with untreated idiopathic myxedema, and in 2/15 patients
under L-T4 treatment. IgG from normal subjects or from 10 patients with non-autoimmune
hypothyroidism did not cause any significant effect on the TSH-stimulated cAMP production.
No correlation was found between TBAb and the thyroid microsomal Ab. These authors
further studied the incidence of Ab blocking the TSH effect in vitro in patients with
euthyroid or hypothyroid HT [49]. TBAb were detected in 12/26 (46%), 1/27 (3%), 3/32 (9.4%), and in 20/55 (36%) patients
with atrophic thyroiditis, euthyroid HT, HT + subclinical hypothyroidism, and in hypothyroid
HT, respectively. The prevalence of TBAb was higher in atrophic thyroiditis vs. all
other collectives. Mean TBAb levels in atrophic thyroiditis were higher than those
in hypothyroid HT and subclinical HT. An inverse correlation was found between TBAb
levels and estimated goiter weight.
The detection of Ab blocking TSH effects using CHO cells transfected with the cloned
human TSHR was reported [17]. All IgGs producing an inhibition greater than two standard deviations from the
mean of controls (>25%) were considered positive for TBAb. TBAb were detected in 1/8
(12.5%), 7/30 (23.3%), and 16/47 (34%) patients with subclinical hypothyroid HT, hypothyroid
HT and atrophic thyroiditis, respectively. This group used the same bioassay to demonstrate
that humoral thyroid autoimmunity was not involved in the pathogenesis of myxedematous
endemic cretinism [69]. Further and unlike TSAb, which are restricted to the IgG1 subclass suggesting an
oligoclonal origin, the IgG subclass distribution of TBAb in primary hypothyroidism
are not restricted to a single subclass and are therefore likely to have a polyclonal
origin [70].
Transient TBAb
The often transient nature of TBAb was noted in a report that described the disappearance
of TBAb and the spontaneous recovery from hypothyroidism in a patient with autoimmune
thyroiditis [32]. In another study, 21 TBAb-positive patients were followed for 6–11 years. TBAb
disappeared in 15 patients (group 1), and persisted in six patients (group 2) [32]. Six patients in group 1 remained euthyroid, and nine became hypothyroid again within
three months [32]. In contrast, all six patients with persistent TBAb positivity remained hypothyroid
[32]. These authors, using a bioassay measuring porcine thyroid cell cAMP production,
reported on their long-term follow-up (>10 years) of TBAb-positive patients with hypothyroidism
associated with atrophic and goitrous thyroiditis [30]. TBAb disappeared in 15/34TBAb-positive patients with hypothyroidism. The disappearance
of TBAb correlated with recovery from hypothyroidism in 13 (87%). All 10 TBAb-positive
goitrous patients recovered from hypothyroidism while 19/24 (79%) TBAb-positive patients
with atrophic thyroiditis continued to be hypothyroid. Two of the 34 TBAb-positive
patients with hypothyroidism developed TSAb-positive Graves’ hyperthyroidism. In another
study changes in stimulating and blocking Ab were evaluated in a patient undergoing
three cycles of transition from hypo- to hyperthyroidism and back to hypothyroidism
[71]. Throughout the entire course of variation in thyroid function, TSAb were consistently
present, but TBAb appeared and disappeared. Monitoring such activity indicated that
the emergence of TBAb heralded the onset of hypothyroidism. A change in activity from
TSAb to TBAb was also observed in GD patients during pregnancy and may have contributed
to the remission of GD during pregnancy [72]. Median TSAb levels decreased during pregnancy while TBAb increased. The increase
of TBAb was observed among those who were in clinical remission before pregnancy.
A negative correlation was observed between TBAb activity and free T4 levels during
pregnancy.
TBAb in GD
A number of investigators have evaluated the prevalence of TBAb in GD. One study used
CHO cell lines expressing different TSHR numbers (JP09 and JP26) and found that TBAb
were frequently detected in GD [73]. Thirty-four (40%) of 86 TSAb-negative GD patients were positive for TBAb. Differences
between anti-TSHR-Ab bioactivity and inhibition of radioactive iodine (RAI)-labeled
bTSH binding were noted in that only 78% of TBAb-positive sera detected with JP26
cells exhibited inhibition of RAI labeled bTSH binding [74]. Using JP09 CHO cells and unfractionated human serum, TBAb were detected in 4/24
(17%) TSAb-negative, TBII-positive hypothyroid GD patients [19]. Blocking type anti-TSHR-Ab were detected by a radio-receptor bioassay in nine of
30 GD sera (30%) using unsolubilized porcine TSHR and FRTL-5 cells [75]. In 1079 unselected, consecutive patients with AITD and 302 healthy controls, TBAb
was found in 4.2% of patients with GD [76]. Twenty-eight hyperthyroid GD patients on methimazole were followed for 13 years
[77]. Eight of 28 (29%) developed TBAb. Patients with TBAb responded well initially to
ATD and showed earlier normalization of the serum T4 level compared to patients without
TBAb.
Transplacental transfer of TBAb
In the first report on TBAb in neonatal hypothyroidism, IgGs inhibiting the cAMP response
to TSH in human thyroid membranes were demonstrated in three infants with transient
neonatal hypothyroidism suggesting that transplacental transfer of TBAb caused the
hypothyroidism in these infants [78]. Further, transient neonatal hypothyroidism was found in a daughter of a mother
who was receiving treatment for primary hypothyroidism due to HT. IgG from maternal
serum blocked TSH-stimulated cAMP response, and cAMP-stimulated iodine uptake and
organification in cultured thyroid cells [31]. Maternal TBAb were detected in mothers of babies with either congenital [37] or neonatal [79] hypothyroidism. The levels of TBAb, based on the ability of patient serum to inhibit
TSH stimulated 3H-cAMP production following incubation of FRTL-5 or CHO-JPO9 cells
with 3H-adenine, decreased to control levels in the child with neonatal hypothyroidism
within two months of birth but remained elevated in the mother’s serum. Also, an infant
with severe neonatal hypothyroidism due to transplacental passage of TBAb was described
[36]. TBAb were detected using a cell line, which stably expresses the human TSHR and
a cAMP-responsive luciferase reporter by their ability to inhibit TSH-stimulated luciferase
expression. High levels of TBAb were detected in maternal serum and initially in the
infant’s serum, but TBAb decreased in the infant to within the reference range by
3–4 months of age, illustrating the transient nature of this condition. Surprisingly,
the thyroid function of this child did not return to normal on withdrawal of L-T4
therapy at 16 months of age when he developed transient compensated hypothyroidism.
Isolation and in vivo Effects of Human Blocking Monoclonal Antibodies
Isolation and in vivo Effects of Human Blocking Monoclonal Antibodies
Human MAb against the TSHR were isolated from EBV-immortalized B-cells of patients
with AITD both to understand the mechanism of the TSHR activation by Ab in patient
serum and for the possible development of new treatments [40]. The serum of patients with AITD contains a mixture of polyclonal Ab whereas it
was hypothesized that MAb, by virtue of their binding to a single epitope on the TSHR,
could exhibit either pure stimulating or pure blocking activity in bioassays. This
turned out to be the case and there have been several purely stimulatory and purely
blocking MAb reported. Interestingly, a blocking MAb and a separate stimulating MAb
were isolated from the same patient with GD [40].
Two human TSHR blocking MAb 129H8 and 122G3 were isolated from patients with GD that
did not exhibit stimulatory activity in assays of thyroid function, but rather showed
blocking activity in a bioassay [80]. A TSHR blocking MAb, 5C9 was also generated from the peripheral blood mononuclear
cells of a female patient with post-partum thyroiditis and high serum TBII levels
(260 U/l). This patient’s serum inhibited the stimulating activity of TSH, a stimulating
human MAb (M22), mouse TSHR stimulating MAb and TSAb from human serum. 5C9 IgG bound
with high affinity (4×1010 l/mol) to the TSHR and inhibited the binding of TSH and M22 to the receptor. Similar
to the patient’s serum, 5C9 IgG preparations inhibited the cAMP stimulating activities
of TSH, M22, serum TSAb, and TSHR-stimulating mouse MAb [81].
The human TSHR blocking MAb K1-70 was isolated in 2010 [40]. B cells were obtained from the peripheral blood of a 54-year old woman with hypothyroidism
and elevated TBII (160 U/l). The patient had an eight year history of AITD and she
initially presented with hyperthyroidism, which was treated with methimazole. She
then developed hypothyroidism and received levothyroxine. Evaluation of her serum
showed both TSAb and TBAb with stimulating activity seen up to a dilution of 1/100
and blocking activity of 82% inhibition at 1/10 dilution. Lymphocytes were infected
with EBV and fused with a mouse/human hybrid cell line (K6H6/B5). Using a TBII assay
supernatants of twelve thousand hybridoma clones were screened to identify the K1-70
clone which stably expresses around 40 mg/l of IgG. K1-70 IgG was purified from culture
supernatants using protein A affinity chromatography. K1-70 inhibited cAMP induction
by TSH, a stimulating MAb, and TSAb-positive sera in TSHR-expressing CHO cells [40]. K1-70 IgG1 lambda showed high binding affinity (4×1010 l/mol) to the TSHR and bound to leucine-rich repeats 1–10 [82]. Furthermore, K1-70 was able to inhibit the stimulating activity of M22 in CHO cells
expressing the human TSHR [82].
Human MAb produced against the TSHR were utilized to study their effects in vivo in
laboratory animals [83]
[84]. K1-70 was a potent inhibitor in vivo and was able to block the stimulating activity
of M22. Furthermore K1-70 inhibited thyroid hormone secretion in rats following the
intramuscular injection of 10–200 μg per animal [84]. In rats treated with K1-70 alone, the total T4 and free T4 decreased in a dose-dependent
manner. These results demonstrate that the activity of these MAb observed in bioassays
correlates with their in vivo activities. Further, it provides strong evidence that
the bioassays in which the activity of these anti-TSHR MAb were measured, though contrived
for convenient measurement, are nevertheless physiologically relevant.
Clinical Indications for TBAb and Concluding Remarks
Clinical Indications for TBAb and Concluding Remarks
Our laboratory uses a TBAb bioassay based on the CHO/Mc4 luciferase cells used in
an FDA-cleared TSAb assay. When evaluating serum samples of approximately 2000 unselected,
consecutive patients with AITD, seen and followed at our institution, TBAb were prevalent
in both subjects with HT (11%) as well as in those with GD (8%). The results of the
TBAb measurement in the first 1400 patients have been reported [25]
[76]. It is becoming clear that in addition to the well-known T-cell-mediated cytotoxicity
and the involvement of CDC in HT, thyroid dysfunction and hypothyroidism can be induced
by the occurrence and presence of TBAb. We and others have noted the occurrence of
TBAb in patients with GD post radioactive iodine therapy and/or during treatment with
antithyroid drugs [85], in patients with HT of recent onset, in AITD during pregnancy, as well as patients
with non-thyroidal autoimmune disorders, that is, type 1 diabetes [38]
[86]
[87]
[88]
[89]. The occurrence of TBAb during or after antithyroid drug treatment can impact the
course of GD with TBAb-positive patients going into early remission or becoming spontaneously
hypothyroid [85]
[88]. Also, the majority of TBAb-positive subjects with HT were hypothyroid in contrast
to TBAb-negative subjects who were predominantly euthyroid [76]. Regarding pregnancy and AITD, there is a real risk for the fetus in pregnant women
with TSHR autoantibodies since both stimulating and blocking anti-TSHR-Ab can cross
the placenta. Further, the risk for fetal/infant hyper- and hypothyroidism correlates
with TSAb/TBAb levels [90]
[91]. Therefore, and as listed in [Table 3], there are several potential clinical indications for the measurement of TBAb in
patients with AITD. Further, and as illustrated in [Fig. 2], in patients with abnormally elevated basal serum TSH and suspected AITD, the measurement
of TBAb can help diagnose humoral immunity-induced hypothyroidism in which thyroid
dysfunction is caused by TBAb. However, and as previously acknowledged in the introduction,
when compared to the abundant literature pertaining to the clinical applications of
TSAb, more prospectively designed, controlled studies are warranted to gain more insight
and daily experience regarding both the clinical utility of measuring TBAb as well
as their potential implications for diagnosis, differential diagnosis, management
and follow-up of patients with AITD.
Fig. 2 Diagnostic flowchart for TSHR blocking antibody-induced hypothyroidism.
Table 3 Potential indications for the measurement of thyrotropin receptor blocking antibodies.
-
Graves‘ disease: post radioactive iodine treatment and/or during therapy with antithyroid
drugs
-
Autoimmune Hashimoto‘s thyroiditis
-
Autoimmune thyroid disease in pregnancy
-
Neonatal hypothyroidism
-
Postpartum thyroid disease
-
Differential diagnosis of thyroiditis
-
Differential diagnosis of hypothyroidism
-
Down Syndrome*
-
Non-thyroidal autoimmune diseases (SLE, RA, type 1 diabetes, myasthenia)
|
* According to reference [92]; SLE: Systemic lupus erythematodus, RA: Rheumatoid arthritis.
