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Experimental Studies |
Repertoire in Hashimoto’s Thyroiditis: Evidence for the Restricted Accumulation of CD8+ T Cells in the Absence of CD4+ T Cell Restriction1
Department of Medicine, University of Sheffield, Clinical Sciences Center, Northern General Hospital, Sheffield, United Kingdom S5 7AU
Address all correspondence and requests for reprints to: Dr. Richard S. McIntosh, Department of Medicine, University of Sheffield Clinical Sciences Center, Northern General Hospital, Sheffield, United Kingdom S5 7AU. E-mail: r.s.mcintosh{at}sheffield.ac.uk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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family usage. No evidence was found for V family
restriction in the PBL, ITL, CD4-selected ITL, or CD8-selected ITL.
However, restriction was frequent in the CD8-selected ITL after
denaturation/reannealing of the PCR products followed by nondenaturing
PAGE; similar restriction was uncommon in PBL, CD8-selected PBL, ITL,
or CD4-selected ITL. V3 and V6 TCR chains from CD8-selected ITL
bands from one patient were cloned and sequenced. There was marked
sequence restriction, particularly within the ITL V6 TCR chains, in
which 14 of 15 homoduplex band sequences used the J4 segment and had an
identical V/N/J junction amino acid (but not nucleotide) sequence.
Sequence restriction was not detected in matched CD8-selected PBL
material. These data show that there is a marked restriction of V
chain usage in the CD8+ (but not CD4+) T cells
in the Hashimoto’s thyroid, with clonal expansion of some sequences. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Demonstration of TCR restriction typically involves PCR amplification-based analysis of the various TCR families present, using family-specific primers, anchored PCR, or inverted PCR (12, 13). V/(D)/J junction size determination is more precise and relies on high resolution separation of PCR products (14, 15) or directly sequencing V/(D)/J junctions. A more rapid method for the screening of TCR restriction in multiple clinical samples is, therefore, desirable (15). A further general problem with the analysis of TCR restriction in autoimmune infiltrates is the presence of a background population of bystander lymphocytes attracted to the autoimmune site by inflammatory cytokines (9, 10, 11).
There is currently controversy over the restriction of the TCR
repertoire in autoimmune thyroid disease. In Graves’ disease and
Hashimoto’s thyroiditis (HT), there have been reports of V, but not
Vß, restriction (16, 17), but this has not been confirmed in Graves’
disease (14, 18, 19). To determine the level of possible restriction,
we analyzed three patients with HT for evidence of restriction of the
V repertoire in unselected and CD4- and CD8-selected intrathyroidal
lymphocytes (ITL). We examined the relative degree of oligoclonality
using a denaturation/reannealing regimen followed by nondenaturing PAGE
(20), a technique first developed to detect small numbers of clonal
cells in leukemia patients (21). We confirmed the validity of this
latter technique using sequencing; in the CD8-selected ITL material
from a single patient, there was marked restriction in the V3 and
V6 sequences amplified.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Samples of blood and thyroid gland were obtained from three women (no. 1–3), aged 70, 29, and 56 yr, respectively, with HT undergoing subtotal thyroidectomy with informed consent. Goiter had been present for 6, 2, and 10 yr, respectively, before surgery and was enlarging in all cases. Thyroidectomy was performed because of equivocal fine needle aspiration biopsy results in patients 1 and 3 and for relief of compressive symptoms in patient 2 despite T4 treatment. None of the patients had detectable thyroglobulin antibodies, and patients 2 and 3 both had thyroid peroxidase antibodies. All blood samples were collected 1 day before thyroidectomy. Patients were euthyroid on T4 (TSH, 4.9–20.4 mIU/L before treatment) at the time of surgery. Thyroid histology confirmed uncomplicated HT with lymphoid follicles; prominent fibrosis was also present in patient 1. This study had local ethical committee approval.
Human leukocyte antigen (HLA) typing
Peripheral blood lymphocytes (PBL) were prepared from heparinized blood using Ficoll-Hypaque density gradient centrifugation. Genomic DNA was prepared from PBL using a Puregene DNA isolation kit (Gentra Systems, Research Triangle Park, NC), and HLA was typing performed as previously described (22).
Immunohistochemistry
Samples of thyroid tissue were snap-frozen in liquid nitrogen immediately after obtaining the gland. For immunohistochemical detection of CD3+, CD4+, and CD8+ cells in the tissue sections, frozen sections were cut and stained with anti-CD3 (clone UCHT1, Serotec, Oxford, UK), anti-CD4 (clone Q4120, Sigma, Poole, UK), and anti-CD8 (clone UCHT4, Sigma), and matched slides were stained with isotype-matched negative antibody (clones B-Z1 and B-Z2, Serotec). Detection of antibody binding was performed with an alkaline phosphatase/antialkaline phosphatase kit (Dako, High Wycombe, UK), using new Fuchsin substrate (Dako).
Preparation of PBL and ITL populations
An aliquot of 2 x 106 PBL was used for
ribonucleic acid (RNA) extraction. ITL were prepared from thyroidectomy
specimens by collagenase/dispase digestion as described previously
(23). An aliquot of ITL (
5 x 106 cells) were used
for RNA extraction. To select CD4+ and CD8+ T
cells, ITL or PBL were incubated for 30 min at 0°C with magnetic
beads coated with mouse antihuman CD4+ following the
manufacturer’s protocols (Dynal, Oslo, Norway). After magnetic removal
of the beads/cells using a magnetic particle concentrator (Dynal), the
remaining cells were incubated for 30 min at 0°C with magnetic beads
coated with mouse antihuman CD8+ (Dynal), and
CD8+ cells were isolated. For both positive separations, a
bead/target cell ratio of 4:1 was used. Yields were typically 1–5
x 105 cells, of 95–99% purity (as judged by subsequent
staining and flow cytometry), of which all were used for RNA
extraction.
Preparation of total RNA and complementary DNA (cDNA) synthesis
Total RNA was prepared from lymphocytes using RNAzol B (Biogenesis, Bournemouth, UK) following the manufacturer’s protocol. Two to 4 µg total RNA were preincubated with 50 µg/mL oligo(deoxythymidine)12–18 (Pharmacia, Uppsala, Sweden) in 50 µL H2O for 10 min at 70 C. First strand cDNA was then synthesized in 0.1 mg/mL BSA (nuclease free; Pharmacia), 0.5 mmol/L deoxy-NTPs (Promega, Southampton, UK), 70 U RNAguard ribonuclease inhibitor (Pharmacia), and 800 U Superscript II ribonuclease H- reverse transcriptase (Life Technologies, Paisley, UK) with the supplied 5x and 0.1 mol/L dithiothreitol buffers (Life Technologies) in a 100-µL reaction volume for 60 min at 37 C. cDNA was ethanol precipitated and resuspended in 200 µL autoclaved H2O; 1 µL cDNA was used in each PCR amplification.
PCR amplification
First strand cDNA was amplified with V family-specific
primers (18 in total) and a common C primer as described previously
(14, 16, 18) for 35 or 40 cycles (94 C at 1 min, 56 C at 2 min, and 72
C at 3 min). Negative controls were carried out frequently. Amplified
products were resolved on 2% agarose gels and blotted onto
Hybond-N+ membrane (Amersham, Aylesbury, UK). Membranes
were hybridized with a 32P-labeled internal C
oligonucleotide (14), and densitometry was carried out using the
Gelbase Image Analysis System (UVP, Cambridge, UK). For each cDNA,
relative expression was calculated as the optical density for each
hybridized band as a percentage of the total optical density for all
bands (14).
Analysis of V gene oligoclonality and sequencing of V PCR
products
After chloroform extraction, PCR amplification products were
denatured at 94 C for 5 min and reannealed at 50 C for 1 h under
mineral oil (20). After chloroform extraction, reannealed products were
kept on ice until loaded. Samples were resolved on a 7-cm PAGE
apparatus, using a 6% polyacrylamide nondenaturing gel in 1 x
TBE (Tris-borate-EDTA) buffer at 25 volts for 500 volt-hours. Bands
were visualized by soaking the gel in 0.5 µg/mL ethidium bromide in
TBE buffer. DNA was recovered by cutting out the homoduplex and
heteroduplex bands, crushing them, and incubating them overnight at
4°C in 200 µL TE buffer. The recovered DNA was ethanol precipitated
and resuspended in 200 µL TE buffer, and 1 µL was reamplified using
20 cycles of the original PCR conditions to eliminate mismatches that
would otherwise preclude cloning. PCR products were cloned using a TA
kit (Invitrogen, San Diego, CA), individual colonies were picked, and
Miniprep DNA was prepared using a Wizard Miniprep kit (Promega). After
confirming the presence of an insert using an EcoRI
digestion, Miniprep DNA was sequenced with the original PCR
amplification primers using Sequenase version 2.0 (Amersham) with
[-35S]deoxy-ATP (Amersham). TCR V and J regions were
assigned using the GCG analysis package with the EMBL database (24); J
region nomenclature conforms to that of Koop et al.
(25).
Statistical analysis
Statistical analysis was determined using the 
2
test with Yates’ correction and 1 degree of freedom.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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HLA specificities were determined by PCR-SSP (22) as follows; patient 1, A2, A3, B44, B60, DR1, DR4, DQ5, DQ7; patient 2, A3, A24, B8, B55, DR3, DR14, DQ2, DQ5; and patient 3; A3, A24, B7, B16, DR4, DR15, DQ6, DQ7.
Immunohistochemical determination of the intrathyroidal CD4+:CD8+ T cell ratio
Analysis of anti-CD3-, anti-CD4-, and anti-CD8-stained thyroid slides indicated that the ratio of CD4+:CD8+ cells was approximately 2:1 in all three patients. CD8+ T cells were observed predominantly in two areas: in the T cell-dependent areas of intrathyroidal germinal centers and as a diffuse infiltrate throughout the gland.
TCR V expression in the thyroid
There was no evidence of restriction of the V repertoire
present in ITL relative to that in PBL (Table 1
).
Furthermore, there was little difference in the pattern of
amplification between these two populations and the CD4- and
CD8-selected subpopulations of ITL (Table 1), although slightly fewer
families were amplified from CD4-selected ITL (n = 12–13
families) and CD8-selected ITL (n = 11–13) than from total ITL
(n = 13–16).
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Analysis was carried out on the V families in which
amplification products from the CD8-selected ITL were clearly visible
on agarose gels, with matched PBL, ITL, and CD4-selected ITL.
Nondenaturing PAGE was performed after first denaturing and reannealing
the PCR amplification products (20). This method allows separation of
the resulting matched DNA pairs (homoduplex band) from mismatched DNA
pairs (heteroduplex band); the latter ran slower than the former.
A substantial oligoclonal component was found more frequently in the
material amplified from CD8-selected cDNA than from the other cDNAs
(Fig. 1). Heteroduplex material was detected in all PBL,
unfractionated ITL, and CD4-selected ITL tested (four of four, seven of
seven, and five of five samples) and in most CD8-selected ITL (four of
four, six of seven, and five of five samples). Homoduplex material was
detected in all CD8-selected ITL tested (four of four, seven of seven,
and five of five samples), but in less than half of the PBL (one of
four, four of seven, and two of five samples), unfractionated ITL (one
of four, zero of seven, and two of five samples), and CD4-selected ITL
tested (one of four, two of seven, and zero of five samples).
2 analysis for the presence of a homoduplex band showed
significant differences among CD8-selected ITL and PBL
(P = 0.0017), unselected ITL (P =
1.56 x 10-5), and CD4-selected ITL
(P = 1.56 x 10-5).
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Sequencing of intrathyroidal TCR products
To validate the heteroduplex method and to study the TCR sequences
present at the site of autoimmune attack, nucleotide sequences from the
V3 and V6 homoduplex and heteroduplex bands from patient 1 were
determined. These families were chosen for analysis as the V3
product had a substantially weaker homoduplex band than heteroduplex
band, whereas the V6 product had a stronger homoduplex band than
heteroduplex band (Fig. 1
). Junctional sequence information was derived
from between 15–26 independently derived bacterial colonies (Tables 2 and 3).
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3 sequences were identical to
published sequences (25, 28). Although there was no predominance of a
single TCR sequence, several sequences were detected frequently in both
the homoduplex and heteroduplex bands, representing 26 of 26 sequences
detected from the homoduplex band and 14 of 25 sequences from the
heteroduplex band (Table 2). The heteroduplex band also contained 9
unique sequences, which were each detected only once or twice. The most
frequent J regions detected in the homoduplex band were J12 and
J18, representing 9 of 26 and 7 of 26 sequences, respectively (Table 2).
The V6 V region sequences were identical to one of two published
V6 family sequences, V6.2 and V6.4 (29, 30, 31); J regions were
identical to previously published sequences (25). Unlike the V3
material, there was a single predominant TCR species in the homoduplex
band. Two distinct nucleotide sequences (both J4, but using
different V6 V region subfamily members and dissimilar V/N/J
nucleotide sequences), which resulted in an identical amino acid
sequence at the V/N/J junction, accounted for 10 of 15 and 4 of 15
observed sequences (Table 3). These sequences were also present in the
heteroduplex band, in which they represented 7 of 22 and 4 of 22
sequences, respectively (Table 3). The heteroduplex band also contained
5 unique sequences, which were detected between 1–3 times each (Table 3).
Sequencing of peripheral blood TCR products
To determine whether the TCR sequences found in the thyroid were a
result of PBL contamination, V3 and V6 sequences were determined
from CD8-selected PBL from patient 1. After PCR product denaturation
and PAGE analysis, no homoduplex bands were detected in either the CD4-
or CD8-selected PBL samples. Because of this, V3 and V6 PCR
products from CD8-selected PBL were cloned directly, and 10–11
examples, respectively, of each were analyzed by sequencing. Each of
the sequences detected in PBL was detected only once; however, 2 of the
V3 sequences detected were identical to 2 of the sequences detected
in the ITL homoduplex and heteroduplex bands (P3.01 was identical to
306, and P3.13 was identical to 303). None of the V6 sequences
detected in CD8-selected ITL was detected in the CD8-selected PBL.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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/ß TCR usage in autoimmune thyroid
disease. V restriction in interleukin-2 (IL-2)/phytohemagglutinin
(PHA)-expanded ITL from three HT patients and whole thyroid tissue from
four additional patients has been reported, with between 2–8 V
families expressed (16, 17). In an analysis of fine needle aspirate
thyroid biopsies, between 6–16 V families were detected in samples
from 5 patients (33).
In the present study, there was no V family restriction in
unselected, CD4-selected, or CD8-selected ITL from HT patients. Our
previous studies had also failed to detect restriction in unselected
and CD25-selected ITL from Graves’ patients. Due to the relative
rarity of surgical specimens from HT patients, we have only been able
to analyze ITL subpopulations from three patients. However, there was
notable restriction in the CD8-selected ITL upon heteroduplex analysis.
V families 1, 2, and 3 were amplified from more than one
CD8-selected ITL sample with a sufficient intensity to allow
heteroduplex analysis; in particular, V3 amplification was detected
in all three CD8-selected ITL samples, and in two it formed a dominant
band. In addition, V families 6, 8, and 10 were amplified in all
three CD8-selected samples, although in only one case from each was it
sufficiently strong to allow heteroduplex analysis. Dominant V1 and
V3 expression was reported in several fine needle aspirate samples
of unfractionated ITL, in which there was also expression of V
families 2, 5, 6, 8, 12, and 15 (33), and dominant V1, -2, -7, -10,
and -11 expression was reported in three IL-2/PHA-expanded ITL samples
(16). Our results from CD8-selected ITL, therefore, suggest that this
apparent restriction could have arisen through preferential
amplification of CD8 TCR chains, for instance via more rapid expansion
of CD8+ T cells during IL-2/PHA expansion.
Several methods have been described to allow more detailed analysis of
TCR restriction in clinical samples. Monoclonal antibodies to human TCR
families give no information on TCR junction structure and are
available for few V families. Analysis of the TCR junction structure
(CDR3) uses differences in CDR3 lengths between family members (14, 15), junction heterogeneity (20, 21), or determination of the actual
junction sequence. Using CDR3 length determination, it is difficult to
unambiguously detect oligoclonality in the presence of a polyclonal
background (14), whereas direct sequence analysis is not suited to
rapid analysis of multiple samples. The heteroduplex analysis method
used in this study allows separation of the oligoclonal and polyclonal
TCR species, thus facilitating rapid analysis of oligoclonality in
clinical samples.
The analysis of junction heterogeneity carried out in this study using the heteroduplex technique was supported by sequencing homoduplex and heteroduplex bands (20). There is a substantial overlap of sequences between the homoduplex and heteroduplex bands. Oligoclonal sequences are not partitioned in an either/or fashion, but are enriched in the homoduplex band, which arises when the sequence forms a sufficient fraction of the material present (0.5–5%) (20) at the expense of the heteroduplex band. Thus, when the PCR products reanneal, which is a stochastic process, the most abundant species reanneal in many cases to nonmatching complementary strands, thus resulting in their presence in the heteroduplex band. An adaptation of this technique, using single stranded nuclease treatment to remove heteroduplex material, has recently been used to study the T cell repertoire in rheumatoid synovia (34).
Fifty-one V3 and 37 V6 sequences from homoduplex and heteroduplex
bands derived from the CD8-selected ITL from a single HT patient were
analyzed. The presence of two distinct V6 subfamily members allowed
detailed analysis of TCR CDR3 oligoclonality. In particular, two groups
of TCR chain present in the V6 bands, typified by clones 601 and
602, had the same TCR CDR3 amino acid sequence, which had arisen
independently. This implies that this V/N/J amino acid sequence has
been actively selected in vivo and argues against any
involvement of superantigens in the selection event (16, 17). The
greater intensity of the heteroduplex band than the homoduplex band in
the CD8-selected ITL V3 analysis is readily explained by the number
(6) of individual oligoclonal TCR species present; during reannealing,
these species would be more likely to reanneal with mismatched than
matched partners. In contrast, only two major oligoclonal species were
present in the V6 material, resulting in a far stronger homoduplex
band.
Although the expression of V families in the CD8-selected ITL
overlapped, there were, nevertheless, distinct patterns of expression
in each patient. The accumulation of CD8+ cells expressing
different V chains in the thyroids of different patients has several
potential explanations, the most obvious being that the patients are
expressing different class I HLA alleles. In addition to the direct
effects of selection by different class I alleles, the naive
CD8+ repertoire differs between individuals (35), and there
can be a heterogeneous TCR response for single HLA-peptide complexes
(31).
The CD8+ T cell population contains clonally expanded
populations in normal subjects (26, 27), so that the oligoclonal
CD8+ populations detected in this study could simply
reflect clonal expansion present in the peripheral blood. The families
chosen for analysis were examples of those in which there was no
evidence of PBL oligoclonality, even after selection of
CD4+ and CD8+ PBL. However, a comparison of
CD8-selected PBL sequences was also carried out to assess the overlap
between the PBL and ITL repertoires. A degree of overlap was evident
only in the V3 sequences; two of the V3 sequences most frequently
detected in the CD8-selected ITL sample (303 and 306) were detected
once each in the CD8-selected PBL material. These sequences were
detected at frequencies of 6 in 26 (23%) and 4 in 26 (15%) in the ITL
V3 homoduplex band, and 3 in 25 (12%) and 2 in 25 (8%) in the
heteroduplex band, suggesting an actual frequency in the PCR-amplified
ITL material between these figures. Only 2 of the 6 possible V3
sequences detected in the ITL V3 homoduplex band were detected in
the PBL material; these sequences made up all 26 (100%) of the
homoduplex sequences and 14 of 25 (56%) of the heteroduplex sequences,
but only 2 of 10 (20%) of the PBL sequences. In addition, the lack of
a detectable homoduplex band in the CD8-selected PBL indicates that the
sequences detected do not individually represent 10% of the
CD8-selected V3 TCR amplification products. It would, therefore,
appear that the V3 sequences were present at a greater frequency in
the ITL than in PBL from the patient, indicating thyroid-specific
accumulation with substantial leakage of thyroid-specific T cells into
the peripheral blood.
The data available do not support the hypothesis that the V3
sequences (and by inference other V families) detected in ITL are
simply a direct result of PBL contamination of the thyroid material
(36). This is confirmed by the fact that none of the V6 sequences
detected in the ITL were detected in PBL. Four other results also
suggest that this is not the case. Homoduplex (i.e.
oligoclonal) material was detected in several V families in each ITL
sample; there were several distinct clonal expansions detected in the
V3 and V6 sequences; there were two distinct V6 sequences that
formed 14 of the 15 homoduplex sequences, but had identical amino acid
junction sequences; and no evidence for a CD8+ expansion
was detected in the majority of PCR products amplified from PBL.
The CD8+ T cell population has been studied in several other autoimmune diseases. In polymyositis, evidence has been found of clonal expansion of the CD8+ T cells most intimately associated with the site of autoimmune attack (37). The predominance of CD8+ T cells in this disease contrasts with HT, in which there is a generally an excess of CD4+ infiltration (32). Restriction of CD8+ TCR Vß expression has also been reported in psoriatic lesion biopsies (38). The present results suggest that there is an accumulation of clonally restricted intrathyroidal CD8+ T cells in the thyroid of patients with HT, inferring that they play an important role in the pathogenesis of the disease.
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Acknowledgments |
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Footnotes |
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Received August 16, 1996.
Revised November 19, 1996.
Accepted December 27, 1996.
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/C) region: organization, sequence
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