Endocrinology Vol. 138, No. 11 4868-4875
Copyright © 1997 by The Endocrine Society
Preservation of Functioning Human Thyroid "Organoids" in the scid Mouse. IV. In Vivo Selection of an Intrathyroidal T Cell Receptor Repertoire1
A. Martin2,
N. Matsuoka,
J. Zhang,
A. Zhou,
M. Nakashima,
P. Unger,
A. E. Schwartz,
E. W. Friedman,
L. D. Shultz and
T. F. Davies3
Departments of Medicine, Pathology (P.U.), and Surgery (A.E.S.,
E.W.F.), Mount Sinai School of Medicine, New York, New York 10029; and
The Jackson Laboratory (L.D.S.), Bar Harbor, Maine 04609
Address all correspondence and requests for reprints to: Dr. Andreas Martin, Box 1055, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029. E-mail:
amartin{at}smtplink.mssm.edu
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Abstract
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To study the in vivo influence of thyroid cells on the T
cell receptor repertoire in human autoimmune thyroid disease, we mixed
lymphocyte-free thyrocytes (
1.2 x 106) from
patients with Graves’ disease with autologous peripheral blood
mononuclear cells (PBMC; 1.5 x 106) and
transplanted this mixture sc into scid mice while
suspended in a basement membrane gel (0.4 ml). Controls included
mice that received either thyrocytes only or PBMC only. The resulting
artificial mixed cell thyroid organoids were explanted after 5 weeks,
and their T cell receptor repertoire was examined. Of a total of 63
organoids constructed, 60 were recovered (95.2%). Total RNA was
extracted and then analyzed by reverse transcription-PCR primarily for
human T cell receptor (hTcR) Vß gene expression using 21 hTcR Vß
amplimers. A restricted pattern of hTcR Vß gene expression was found,
with 6 Vß genes (Vß5, 6, 7, 8, 13.1, and 18) predominantly
expressed [P < 0.05, by ANOVA on ranks and
Student-Newman-Keul’s (SNK) test]. PBMC and control organoids showed
no preferential selection of particular hTcR V gene-expressing T
cells.
This reductionist, mixed cell, thyroid model reflected earlier
observations in human and murine autoimmune thyroid diseases in which a
bias in hTcR V gene family expression had been observed. The model
permitted in vivo T cell selection and/or enrichment of
potentially disease relevant human T cells.
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Introduction
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THE HUMAN autoimmune thyroid diseases
(AITD), encompassing Graves’ disease and Hashimoto’s thyroiditis, are
examples of organ-specific autoimmune diseases. C.B-17
scid/scid mice have provided an opportunity to study human
autoimmune processes in an in vivo environment, including
the study of AITD (1, 2, 3, 4). Recently, we reported the establishment of
artificial human thyroids (organoids) in scid mice in which
human thyroid monolayer cells, suspended in a liquid basement membrane
preparation, were observed to reorganize into thyroid neofollicles in a
TSH-independent manner (5, 6). These organoids secreted human
thyroglobulin into the follicular lumen and into the mouse circulation
and remained responsive to recombinant human TSH stimulation for over 3
months (5). This neofollicular model differed from traditional intact
thyroid tissue transplants by showing greater histological homogeneity,
by its reproducibility, and by being potentially reductionist in
concept. Furthermore, the convenient experimental procedure relied on
cryopreserved thyroid monolayer cells and lymphocytes for organoid
construction.
Organoids constructed from human thyroid monolayer cells derived from
patients with Graves’ disease contained small numbers of human T
cells that were bound to the thyroid monolayer cells and remained
adherent during organoid construction. Analysis of these attached
lymphocytes for human T cell receptor (hTcR) V gene expression showed a
bias in the T cell repertoire and evidence of in vivo clonal
expansion (7), data reminiscent of our studies in human thyroid tissue
(8). We hypothesized, therefore, that organoid T cells may represent
thyroid antigen-specific cells. To investigate the direct influence of
thyrocytes on the autologous human T cell repertoire, we reconstituted
organoids containing autologous peripheral blood mononuclear cells
(PBMC) from patients with Graves’ disease. We prepared thyroid
monolayer cells, removed any bound T cells using complement-mediated
lysis, reconstituted thyroid organoids with autologous PBMC, and
examined the resulting T cell receptor repertoire.
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Materials and Methods
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Mice
C.B17-scid/scid mice were bred at the Jackson
Laboratory (Bar Harbor, ME) and housed at the Mount Sinai School of
Medicine (New York, NY) in MicroIsolator cages (Lab Products, Inc.,
Maywood, NJ). All mice were maintained on sterilized food and water and
on a 3/7-day cycle of antibiotics (trimethoprim/sulfamethoxazole) as
previously described (9). Mice used in all experiments were at least 2
months old. Five groups of mice were implanted with thyrocyte/T cell
mixtures, they were labeled G11–G15 (patient G), L2–L5 (patient L),
O2–O3 (patient O), Q1–Q4 (5-week organoids, patient Q), and Q6–Q10
(5-month organoids, patient Q). Missing numbers are due to unnecessary
controls (made earlier in the study) or absence of the hTcR C region.
Thus, all mixed organoids with available data are illustrated.
Patients, cells, and tissues
Human thyrocytes were prepared and cultured as monolayers as
described previously (10) with minor modifications. Thyroid tissues
(n = 4) were obtained from patients undergoing surgery for
Graves’ disease or thyroid surgery for nonautoimmune disease. Briefly,
thyroid tissues were digested in collagenase (Worthington Biochemical
Co., Freehold, NJ) in three cycles of approximately 45 min in a 37 C
shaker water bath and cultured in 10% FBS (HyClone Laboratories,
Logan, UT) and RPMI 1640 (Life Technologies, Grand Island, NY),
supplemented with antibiotics. Nonadherent cells were removed after
24 h (two washes with PBS without Ca2+ and
Mg2+). After approximately 5 days, the monolayers were
trypsinized, and thyrocytes were cryopreserved for subsequent thyroid
organoid construction.
Lysis of T cells in thyroid monolayers
Thawed thyroid monolayer cells (up to 3 x 107)
were incubated with 100 µg anti-CD52w monoclonal antibody (11)
(Campath-1, Serotec USA, Washington DC) for 45 min, followed by
incubation with 50% rabbit complement (Low-Tox-H, Cedarlane, Hornby,
Canada) in Dulbecco’s PBS (with calcium and magnesium, Life
Technologies) for 90 min. For all mixed cell experiments, thyroid cell
preparations were checked for the absence of hTcR C region messenger
RNA (mRNA; see below).
PBMC
Autologous PBMC were obtained from the same patients (or as
indicated) and were separated from heparinized blood by Ficoll density
gradient centrifugation (12) and cryopreserved in 80% RPMI, 10%
autologous plasma, and 10% dimethylsulfoxide.
Organoid construction
Thyrocytes were suspended in a liquid basement membrane extract
(Matrigel, Collaborative Biomedical Products, Bedford, MA) consisting
of a solubilized basement membrane preparation from the
Engelbreth-Holm-Swarm mouse sarcoma. Matrigel was used at 4 C, and an
average of 0.42 ± 0.02 ml (range, 0.25–1.0 ml) containing a
suspension of 1.2 x 106 thyroid cells and 1.7 x
106 PBMC, either singly or mixed (mixed cell organoid) was
injected sc into the dorsum of each mouse. Mice were bled and killed 5
weeks later (except for five mice that were killed after 5 months;
Table 1
).
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Table 1. Origin of thyrocytes and peripheral blood
mononuclear cells used for organoid construction and their combinations
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Histology and immunohistochemistry
Thyroid organoids were removed; one third fixed in 10% formalin
for standard paraffin sections, and the remaining two thirds were
frozen in liquid nitrogen. Hematoxylin-eosin staining of the organoids
was performed according to routine procedures. Immunohistochemistry was
performed using the immunoperoxidase technique. Monoclonal antibodies
to human markers included antileukocyte antigen (anti-CD45, Dako,
Carpinteria, CA), UCHL1 (anti-CD45R0, Dako), and anti-B-cell L26
(anti-CD20, Dako).
Complementary DNA (cDNA) preparation
Total cellular RNA was extracted from the frozen human thyroid
organoids using guanidinium thiocyanate and phenol [RNAzol B
(Cinna/Bioteck Laboratories International, Friendswood, TX) or
Trireagent (Sigma Chemical Co., St. Louis, MO)]. cDNA transcripts were
prepared from 1 µg cellular RNA using oligo(deoxythymidine) priming
(1 µg/20-µl volume) and avian reverse transcriptase (30 U/20 µl;
Life Sciences, St. Petersburg, FL) in the presence of RNAsin (40 U/20
µl; Promega, Madison, WI) as previously described (13).
Reverse transcription-PCR (RT-PCRs)
For hTcR V gene expression, we used a series of 18 specific hTcR
V
and 21 Vß oligonucleotides (14, 15) together with hTcR C- and
Cß-specific primers, respectively. The PCR fragment-containing
agarose gels were transblotted onto nitrocellulose membranes
(Schleicher and Schuell, Keene, NH), baked, prehybridized, and
hybridized using 32P-labeled C or Cß oligonucleotides
(14, 15) prepared using T4 polynucleotide kinase. A
semiquantitative estimate of hTcR V gene expression was derived from
the intensity of the hTcR V genes present, as assessed by laser
densitometry (14). Contributions greater than 5% of the total activity
were considered evidence of a significant contribution. We have
previously demonstrated the human specificity of these RT-PCRs in the
mouse (7). To obtain an overall assessment of hTcR activity, we used an
RT-PCR for the hTcR Cß region as previously described (7). To detect
the presence of thyroid cell differentiated function, we employed an
RT-PCR for hTg gene expression, also as previously described (7).
Radiolabeled PCR
The radiolabeled RT-PCR was based on the differing lengths of
the hTcR complementarity-determining region 3 (CDR3). This region is
subject to random nucleotide additions and deletions. Hence T cell
clones have CDR3 regions of different lengths that can be visualized as
distinct bands within each hTcR V gene family. The radiolabeled PCRs
were performed with the same 18 V and 21 Vß oligonucleotides as
forward primers, but with 32P-labeled C region
oligonucleotides as reverse primers (8). The PCR products were then
separated on standard sequencing polyacrylamide gels and exposed. Films
were assessed by computerized densitometry and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). Data were expressed as the percent
contribution to total activity for each individual sample.
Statistical analyses
Nonparametric analysis was performed because the data were not
normally distributed. To give the individual organoid contributions
equal weight in our overall analysis, we graded all of their hTcR V
gene family percentages within each organoid. The highest grades were
given for the highest percentage contribution and vice
versa. Thus, grades were assigned from the top down, so that the
hTcR V gene family with the maximum percent contribution within each
organoid received the highest grade regardless of its absolute value,
including those less than 5%. Grades were then analyzed using ANOVA on
ranks (Kruskal-Wallis), followed by Student-Newman-Keuls test to
isolate significantly different expression of hTcR V gene families.
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Results
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Intrathyroidal hTcR V gene expression
The expression of hTcR V gene families was biased in the
samples of intrathyroidal lymphocytes from patients with Graves’
disease (Fig. 1A). In comparison, the
full repertoire was seen in the PBMC samples examined (not
illustrated), but a restricted number of V genes was present in the
mixed cell organoids similar to the patient’s intrathyroidal T cell
repertoire (Fig. 1B). Five organoids from series G that were examined
for hTcR V expressed 11 of 18 V gene families tested (not
shown).
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Figure 1. A, An example of hTcR Vß gene family expression
by intrathyroidal lymphocytes obtained from a patient with Graves’
disease (patient L); B, a 5-week organoid (L2) from the same patient
showing a restriction similar to that in A. Data are illustrated as
Southern analyses as described in Materials and Methods.
Peripheral blood T cells showed a normal hTcR V gene repertoire (not
shown).
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Evaluation of T cell lysis by RT-PCR
Campath-1 treatment of thyroid monolayer cells effectively removed
human T cells, as evidenced by negative RT-PCR for the hTcR constant
(C) region (Fig. 2). Although hTcR mRNA
was still detectable after 45 min of lysis, a prolonged incubation with
complement for 90 min left no detectable hTcR mRNA. All thyroid cells
were, therefore, subsequently subjected to the 90-min procedure. There
was loss of thyrocytes in some experiments, presumably due to CD52w
expression on thyrocytes, but useful numbers of thyrocytes were rescued
after each lysis. Thyrocyte viability was evident by thyroid follicle
formation in the organoids similar to the findings of previous studies
(Fig. 3A) (5) and by the demonstration of
thyroglobulin mRNA expression (Fig. 3B) as previously described
(7).
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Figure 2. Influence of lymphocyte lysis on thyroid
monolayer cell hTcR V gene expression. Graves’ thyroid monolayer cells
were treated with anti-CD52w (Campath-1 monoclonal antibody) as
described in Materials and Methods. Cells were analyzed
for any remaining attached lymphocytes by RT-PCR for hTcR Cß region
expression. Data are shown after 45 and 90 min of lysis. Note that
after 45 min, hTcR C region mRNA (arrow) was still
present due to incomplete lysis of lymphocytes. In contrast, after 90
min, no hTcR mRNA was detectable (0.1, 0.5, 1.0, and 2.0 µg RNA were
used for RT).
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Figure 3. A, Histology of a 5-week mixed cell thyroid
organoid derived from a patient with Graves’ disease (L3).
Note thyroid neofollicle and adherent lymphocyte
(arrow; hematoxylin-eosin staining; original
magnification, x400). B, Detection of human thyroglobulin mRNA by
RT-PCR using human thyroglobulin-specific primers (predicted fragment
size, 148 bp) in thyrocyte-only organoids (series G; n = 5)
following explantation after 5 weeks. Abundant hTg gene expression
indicated the maintenance of differentiated thyroid cell function.
Lanes 1–5 show control thyrocytes after anti-CD52w treatment. Lanes
11–15 show thyrocytes mixed with autologous PBMC. Lane B is blank, and
lane I is a sample from the original thyroid monolayer cells derived
from patient G.
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Histology
Human thyroid organoids showed typical thyroid follicular
structures (Fig. 3A
), and there was marked lymphocytic infiltration in
some thyroid/PBMC mixed cell organoids (Fig. 4).
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Figure 4. Immunohistochemistry of a 5-week mixed cell
thyroid organoid derived from a patient with Graves’ disease (organoid
L3). Note the lymphocytic infiltrate showing dark membrane staining
with antibody to leukocyte common antigen (LCA). Thyroid neofollicles
are indicated by arrows (original magnification,
x400).
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Human T cell receptor V gene expression in mixed cell thyroid
organoids
The mixed cell organoids demonstrated hTcR repertoires (Table 2, A and B) that were
also biased compared with PBMC and reminiscent of previous
intrathyroidal analyses (14, 16). Overall, there was similarity in the
5-week organoids between the intrathyroidal T cell V genes and the hTcR
Vß expression in the organoids (Table 2A). The average number of hTcR
Vß gene families expressed was only 4.6 ± 0.4 (>5%
contribution) after 5 weeks, and this was further reduced to 2.8
± 1.3 in the few samples available after 5 months. Overall, T cells
expressing hTcR Vß 5, 6, 7, 8, 13.1, and 18 were more prevalent (by
ANOVA on ranks and Student-Newman-Keuls test, P <
0.05) when all of the organoids from the different patients were
analyzed for hTcR V gene family contribution to total activity. Certain
hTcR V gene families were more prevalent in mixed cell organoids
constructed from the same patient (e.g. Vß3 in L1–L5 or
Vß4 in G12–G15; Table 2A). In the series of mixed cell organoids
(series G) that was analyzed for hTcR V gene expression, a similar
bias was observed compared with PBMC. hTcR V1 was the predominant V
gene in this series (data not shown). Control experiments were
conducted on PBMC to rule out that template was limiting. In earlier
serial dilution experiments, we showed that in a constant region PCR
the amount of template was sufficient (more than adequate), and V
region PCR on PBMC cDNA with RNA concentrations at 10, 5, 1, and 0.1
µg/ml showed detectable V genes (with only Vß7 and Vß8 not
amplified) at the highest dilution.
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Table 2B. Percent expression of hTcR Vß genes in patient Q
intrathyroidal lymphocytes (upper panel, Q-ITL), mixed cell
organoids at 5 weeks (middle panel, Q1–Q4), and 5 months
(lower panel; Q6–Q10)
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Human T cell receptor V gene expression in control organoids
Controls included PBMC-only organoids, organoids constructed from
normal nonautoimmune thyrocytes mixed with autologous PBMC, and
Graves’ disease thyrocytes mixed with allogeneic normal PBMC (Table 3). All cells were cryopreserved before
testing. The survival of lymphocytes in PBMC-only organoids was
sporadic and unpredictable, and organoid lymphocytes did not usually
survive unless large numbers of noncryopreserved PBMC were used (data
not shown). Likewise, normal mixed cell thyroid organoids were
unremarkable, with only sparse T cell survival as judged by RT-PCR for
the hTcR Cß region. In addition, one experiment with Graves’ disease
thyrocytes and allogeneic PBMC showed no specific lymphocyte survival,
although thyrocytes from the same patient supported T cell survival
with the patient’s autologous PBMC (Table 2, series G). Taken
together, these data showed that survival of PBMC within organoids in
the absence of presumed antigenic stimulation by autologous thyrocytes
was poor and unpredictable.
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Table 3. The control data showed that only a few hTcR V genes
were expressed in the thyrocyte-only and PBMC-only organoids; in
addition, the allogeneic studies showed a marked contrast to the
autologous data
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Evidence for clonal expansion
Five organoids from one series (series G) that showed strong
expression of hTcR Vß4 and Vß5 were examined using radiolabeled
PCR, as described in Materials and Methods. Two of five
examples showed expanded bands of different sizes when examined for
Vß4 (Fig. 5), and four of five
organoids tested positive for Vß5, suggesting the expansion of
different T cell clones within each organoid.
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Figure 5. Radiolabeled RT-PCR for hTcR Vß4 in the five
mixed cell organoids in series G. Lane I, Intrathyroidal T cells; lane
P, PBMC. Note the normal pattern of CDR3 lengths in lane P and the
enhanced band in lane I suggesting clonal expansion. An enhanced band
of the same CDR3 length is seen in mixed cell thyroid organoid G12
(lane 12). Another enhanced band of different CDR3 length is seen in
mixed cell organoid G13 (lane 13). All organoids came from the G series
(mice G11–G15), and thus, the thyroid cells (and the T cells) were
derived from patient G.
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Discussion
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In the present study the original intrathyroidal T cells from
patients with Graves‘ disease, analyzed as a source of thyrocytes for
the formation of thyroid organoids, exhibited a restricted hTcR
repertoire, data similar to our previous reports and those of others in
humans and animal models (14, 16, 17, 18, 19). We also showed that it was
possible to prepare human thyroid cell preparations free of
contaminating T cells using complement-mediated T cell depletion with
anti-CD52w. Furthermore, such thyrocytes formed the expected
neofollicles when incorporated into organoids. Using these T cell-free
thyrocytes, we found that the addition of the peripheral blood T cell
repertoire produced a mixed cell organoid with a highly biased hTcR
repertoire over a period of 5 weeks. These mixed cell organoids
appeared to reflect the hTcR V gene family restriction found in the
original intrathyroidal T cells. These data suggested that we had
imitated the in vivo situation and that the peripheral blood
of patients with Graves’ disease contained a reflection of the
intrathyroidal T cell repertoire; a fact better known from direct
testing of such T cells with thyroid antigens (20) (Martin, A., M.
Nakashima, A. Zhou, D. Aronson, A. J. Werner, and T. F. Davies, in
press). In contrast, these observations could not be replicated using
allogeneic T cells. Hence, the experiments suggested that an
intrathyroidal autoimmune response had been perpetuated within the
artificial thyroid organoids. It was likely, therefore, that the T
cells surviving within the organoids were survivors of antigen-specific
T cells from the patients’ peripheral blood. We also demonstrated the
presence of clonal expansion of some of the T cells surviving in such
an environment. However, different T cell clones appeared to be
expanding within different organoid samples from the same patient and
even within the same V gene family. This suggested that each organoid
may have had its own natural autoimmune history (see below). The
variability in the organoids derived from the same patient was
unexpected and suggests a stochastic selection of T cell receptors or
perhaps outgrowth of certain clones due to minute variations in the
organoid microenvironment.
The use of particular hTcR V gene families in particular patients
pointed to an HLA-associated antigen in the autoimmune process
underlying AITD. This observation together with the absence of
significant stimulation of T cells in allogeneic mixed cell organoids
argued against the influence of a superantigen in the development of
the biased T cell repertoires (5, 8, 16). In the overall analysis, six
hTcR V gene families were significantly different from other V gene
families; these data were reminiscent of the restriction observed
previously in vivo (14, 16). Although the survival of the
human lymphocytes appeared to be dependent on the presence of
autologous thyrocytes, the survival of particular hTcR V gene families
varied from patient to patient, no doubt dependent on their HLA
haplotypes (21, 22). This was further apparent from the control studies
with PBMC-only and allogeneic mixtures, which showed unpredictable and
erratic T cell survival.
Of particular interest in these experiments was the evidence in favor
of T cell clonal expansion within the mixed cell organoids. This was
observed by the same length of the hTcR amplification products
(suggesting similarity of many CDR3 regions), shown previously in
vivo by direct sequencing. These data suggested that selection of
some of the T cells was an ongoing intraorganoid phenomenon and argued
against cDNA template being a limiting factor (as was also suggested by
earlier serial dilution studies in which template was still detectable
at high dilutions). It is likely that the T cells surviving in the
organoid represented autoreactive T cells that were selected in the
patient and were present in the patient’s peripheral blood.
Furthermore, there was evidence that the same T cell clones were
expanding within the organoids as those found in the original
intrathyroidal T cell repertoire. For example, in the case of hTcR
Vß4, we found an enhanced intraorganoid band of exactly the same
length as in the intrathyroidal T cells, suggesting that the T cell
population expressing hTcR Vß4 was indeed related to the autoimmune
disease process in that particular patient. In one of our studies we
allowed the mixed cell organoids to develop for 5 months. With time,
different hTcR Vß gene families were expressed. However,
xenoreactivity in the long term organoids also needs to be
considered.
In conclusion, we were able to observe a reconstruction of a
restricted T cell repertoire in vivo. This was achieved by
using components of the disease process, i.e. thyroid cells
and autologous lymphocytes, within a basement membrane gel under the
skin of the scid mouse. The resulting thyroid organoids
induced a selection and/or enrichment of cotransplanted peripheral
blood T cells and showed a restricted T cell repertoire similar to that
seen within the thyroid glands of patients with Graves’ disease. This
model provides a basis for functional T cell analysis and immune
intervention in vivo.
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Acknowledgments
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We thank M. Zeffren for photography.
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Footnotes
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1 This work was supported in part by Grants DK-28242 and DK-35674 from
the NIDDK (to T.F.D.) and AI-30389 from NIAID (to L.D.S.). 
2 Supported in part by Grant NAG 9–816 from NASA.
3 Florence and Theodore Baumritter Professor of Medicine.
Received May 5, 1997.
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Abstract
Introduction
