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Original Studies |
Endocrinology Research Laboratory, Departments of Medicine (K.K., E.R., T.E., R. V.) and Pathology (V.F), The Wellesley Hospital, University of Toronto, Toronto, Ontario, M4Y1J3, Canada.
Address all correspondence and requests for reprints to: Professor Robert Volpé, Endocrinology Research Laboratory, The Wellesley Hospital, University of Toronto, 160 Wellesley Street East, 112D Jones Building, Toronto, Ontario, M4Y1J3, Canada.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and may actually be protective (9, 10, 11, 12, 13, 14). Thyrocytes from PTC may also express HLA-DR and ICAM-1 without coexisting AITD (6, 8, 15). It has thus been suggested that such expression in neoplastic tumor tissue may be linked to oncogene expression (6). However, mechanisms involved in the participation of the immune system in PTC are still unknown. Is thyrocyte HLA-DR and ICAM-1 expression in PTC constitutive, or secondary, as in AITD ? Why does lymphocytic infiltration so often accompany PTC? What is the exact role of HLA-DR and ICAM-1 expression on PTC thyrocytes? To investigate these questions, we have studied thyrocyte HLA-DR and ICAM-1 expression on TEC from PTC tissue xenografted into two different mouse strains, namely, severe combined immunodeficient (SCID) and nude mice. SCID mice have a defect in the recombinase system for antigen receptor genes resulting in a lack of mature T and B cells (16). Both the thyroid xenografts and its lymphocytes survive in the SCID mice (17, 18). In contrast, nude mice accept the solid tissue xenograft but lyse passenger human lymphocytes because of the presence of functional murine natural killer (NK) and B lymphocytes (17). The aim of this study was to evaluate the immunological aspects of PTC in vivo in the presence (in the SCID mice) or absence (in the nude mice) of the immune environment, thus comparing the response of the thyroid tissue under these two different conditions. We also compared these responses with those observed in AITD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SCID male (C.B-17, scid/scid) mice and athymic nude mice (BALB/c, nu/nu) were obtained from Taconic Farms, Inc. (Germantown, NY) and the Wellesley Hospital, University of Toronto. Mice were 6–10 weeks old at the time of xenografting. The institutional standards of animal care guidelines were observed.
Xenotransplantation of human thyroid tissue
Xenotransplantation of human thyroid tissue was performed as
previously described (18). Briefly, 2.5–15.3 g human thyroid tumors
from 3 patients with PTC/TIL (PTC with tumor infiltrating lymphocytes)
and 2 patients with PTC/PTC (PTC without tumor infiltrating
lymphocytes), 2.3–28.9 g human thyroid tissues from 4 patients with
hyperthyroid Graves’ disease (GD), and 5 samples from normal persons
(paranodular tissues) were obtained at surgery after receiving informed
consent (Table 1
). These tumors and
tissues were cut into small pieces and xenografted sc into 22 SCID and
21 nude mice (total weight: 0.8 g per mouse) within 2 h after
surgery. SCID mice were pretreated, 1 day before thyroid xenograftment,
with a single dose of antiasialo GM 1 antiserum (anti-ASGM 1)
antibodies (Wako Chemicals, Dallas, Texas): lyophilized antibody was
resuspended in 1 mL of PBS and 20 µl was given ip. Anti-ASGM 1
consists of rabbit polyclonal antibodies (Abs) that recognize murine NK
cells and deplete NK activity when injected ip into the mice (19). In
addition, immediately before thyroid xenograftment, SCID mice were
irradiated; a dose of 3 Gy -radiation was administered from a
137Cs source. The use of Anti-ASGM 1 and irradiation
depletes NK activity from the SCID mice (20).
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One week after xenografting, blood samples were obtained from the lateral tail vein of the SCID and nude mice, and this was repeated every 2 weeks for the measurements of human IgG, TPO-Ab, and Tg-Ab. Human IgG was quantitated by the single radial immunodiffusion method using immunodiffusion plates (NOR-Partigen IgG-MC and LC-Partigen IgG; Behringwerke AG, Marburg, Germany). TPO-Ab and Tg-Ab were assayed in each donor serum in duplicate by a hemagglutination kit (Thymune, Wellcome Diagnostics, Dartford, England) and in each mouse serum in duplicate RIA kits (Kronus, Dana Point, CA) (sensitivity 0.3 U/mL).
Thyrocyte HLA-DR and ICAM-1 expression
Seven weeks after thyroid tissue xenotransplantation, mice were killed, and the xenografts were analyzed for thyrocyte HLA-DR and ICAM-1 expression and also for histological studies. Measurements of thyrocyte HLA-DR and ICAM-1 expression were carried out by flow cytometric analysis, as described (8). Briefly, specimens were minced with scissors and digested with 2 g/L collagenase (type II, Sigma, St.Louis, MO) in PBS at 37 C and 5% CO2:95% air in RPMI-1640 medium with 10% (vol/vol) FCS (GIBCO Gaithersberg, MD). The adherent thyroid cells were washed the next day and cultured for 1–2 days until a monolayer was obtained. To assess HLA-DR and ICAM-1 expression, cultured cells (1 x 106) were allowed to incubate for 45 min at 4 C with 10 µL phycoerythrin-conjugated antihuman HLA-DR monoclonal antibody (mAb) (Becton Dickinson Immunocytometry Systems, Mountain View, CA) or with 10 µL fluorescein isothiocyanate (FITC)-conjugated antihuman ICAM-1 mAb (CD54; Serotec, Toronto, Canada). Mouse IgG FITC and phycoerythrin of the same isotype were used as negative controls. After incubation, cells were analyzed on a flow cytometer (FACScan, Becton Dickinson) and with a computer system (Lysis II, Becton Dickinson). The percentage of HLA-DR+ or ICAM-1+ thyrocytes was expressed as the number of positive thyrocytes per total thyrocytes x 100%. To rule out the existence of contaminating lymphocytes in this culture system, antihuman CD45-FITC-mAb (human lymphocyte marker, Becton Dickinson) was used; no CD45 positive molecules were identified.
Histopathology
The thyroid tissues before and after xenografting were fixed in neutral formalin and embedded in paraffin and then submitted to the Department of Pathology for routine light-microscopic studies.
Statistics
Data were shown as mean ± SE, and comparison between means was performed by Student’s t test. A P value < 0.05 was chosen as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
Figure 1 shows the IgG production of the individual mice
xenografted with thyroid tumor or tissue [PTC/TIL, PTC/PTC, GD, normal
thyroid (N)]. There was an immediate sharp increase of IgG production
in SCID mice xenografted with PTC/TIL tumor (PTC/TIL-SCID). In
contrast, IgG production in SCID mice xenografted with GD tissue
(GD-SCID) gradually increased. IgG production in SCID mice xenografted
with PTC/PTC tumor (PTC/PTC-SCID) and N tissue (N-SCID) remained very
low throughout. Nude mice xenografted with PTC/TIL tumor
(PTC/TIL-nude), PTC/PTC tumor (PTC/PTC-nude), GD tissue (GD-nude), and
N tissue (N-nude) showed diminished and disappearing IgG
production.
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)
TPO-Ab was not detectable in PTC/TIL-SCID despite the presence of
TPO-Ab in some donors [PTC/TIL (1, 2, 3)] (Table 1
). TPO-Ab was
detectable in eight of eight mice of GD-SCID. TPO-Ab was not detectable
in mice of PTC/TIL-nude, PTC/PTC-SCID, PTC/PTC-nude, GD-nude, N-SCID,
and N-nude.
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Thyrocyte HLA-DR expression of thyroid xenografts (see
Fig. 3)
Thyrocyte HLA-DR expression from PTC (PTC/TIL + PTC/PTC)-SCID and GD-SCID was markedly increased, compared with those from nude mice xenografts (34.9 ± 4.45 vs. 9.1 ± 2.6, P < 0.05; 24.5 ± 3.83 vs. 2.4 ± 0.21%, P < 0.01), but thyrocyte HLA-DR expression from N did not show any difference between the SCID and nude mice (4.12 ± 1.01 vs. 2.3 ± 0.165). Thyrocyte HLA-DR expression from PTC-nude was markedly increased, compared with the expression seen in GD-nude and N-nude (9.1 ± 2.6 vs. 2.37 ± 0.21, P < 0.01; 9.1 ± 2.6 vs. 2.3 ± 0.17, P < 0.05). In addition, thyrocyte HLA-DR expression from PTC/TIL-SCID was higher than that from PTC/PTC-SCID, but the difference between them did not reach statistical significance (41.9 ± 5.6 vs. 26.0 ± 3.6, P = 0.07). Thyrocyte HLA-DR expression from PTC/TIL-SCID and PTC/PTC-SCID were higher than that from PTC-nude, respectively (41.9 ± 5.6 vs. 9.6 ± 2.6, P < 0.01; 26.0 ± 3.6 vs. 9.6 ± 2.6, P < 0.01).
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)Thyrocyte ICAM-1 expression from PTC and N did not show any difference between the SCID and nude mice (35 ± 4.6 vs. 38.12 ± 3.9, 15.8 ± 3.2 vs. 13.8 ± 3.6). Thyrocyte ICAM-1 expression from GD-SCID was markedly increased, compared with that from nude mice xenografts (26.9 ± 3.2 vs. 15.0 ± 2.9, P < 0.05). Thyrocyte ICAM-1 expression from PTC-SCID was markedly increased, compared with that from N-SCID (35 ± 4.6 vs. 15.8 ± 3.2, P < 0.05). In addition, thyrocyte ICAM-1 expression from PTC-nude was markedly increased, compared with the expression seen in GD-nude and N-nude (38.12 ± 3.9 vs. 15.0 ± 2.87, P < 0.01; 38.12 ± 3.9 vs. 13.8 ± 6.32, P < 0.05). There was no significant difference among PTC/TIL-SCID, PTC/PTC-SCID, and PTC-nude, in terms of thyrocyte ICAM-1 expression (28.6 ± 4.5 vs. 43.0 ± 3.9 vs. 38.12 ± 3.9).
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Light-microscopic photomicrographs of the thyroid tissue before
and after xenografting are shown in Fig. 5. Figure 5A shows PTC/TIL
thyroid tumor before xenografting. There is a moderate lymphocytic
infiltration, and PTC/TIL shows the typical branching fronds of
papillary carcinoma. After a 7-week sojourn in the SCID mice, the
xenografts (PTC/TIL-SCID; Fig. 5D) shows a severe intrafollicular
lymphocytic infiltration, and lymphocytes seem to be attacking TEC. TEC
are severely hypertrophic and show some variability in size, with large
nuclei. In contrast, after a 7-week sojourn in the nude mice, the
xenografts (PTC/TIL-nude; Fig. 5G) show no demonstrable lymphocytic
infiltration. However, TEC are still severely hypertrophic, with large
nuclei. The xenografts show evidence of malignancy in both PTC/TIL-SCID
and PTC/TIL-nude. Before xenografting, histology of the Graves’
thyroids was typical GD (Fig. 5B). As we previously reported (17, 18),
after a 7-week sojourn in the SCID mice, the histology of xenografts
(GD-SCID) had changed to HT, with moderate lymphocytic infiltration
(Fig. 5E). In the nude mice, the xenografts (GD-nude) showed no
demonstrable lymphocytic infiltration, and the follicles seemed normal
(Fig. 5H) (17). With respect to normal tissue (Fig. 5C), the histology
of xenografts [N-SCID (Fig. 5F) and N-nude (Fig. 5I)] had maintained
a normal appearance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, IgGs had shown a rapid increase in PTC/TIL in SCID, compared with GD in SCID. In other words, lymphocytes from PTC responded to antigen quickly, in comparison with lymphocytes from AITD, suggesting that antigenicity of the neoplasm might be stronger than that of AITD. TIL in PTC produced Tg-Ab but did not produce TPO-Ab, in contrast to AITD. The character of TIL from PTC thus seems to be different from the character of TIL from AITD. It is suggested that infiltrating lymphocytes from patients with PTC may be predominantly cytotoxic T cells with NK or lymphokine-associated killer activity acting as carcinoma cell killers and secreting cytokines, such as interleukin-1, that inhibit thyroid carcinoma cell growth (22, 23). In addition, Tg is used as a marker for recurrent or metastatic disease after PTC treatment (24). Several reports suggest a loss of antigenicity or immunogenicity in thyroid cancers as these tumors progress (summarized in Ref.3). Thus, TIL, especially those recognizing Tg as a tumor antigen, might participate in limiting cell growth; moreover, Tg from PTC might be one of the important antigens promoting a tumor-related immune response system, rather than TPO or TSH receptor, in relation to AITD.
Second, thyrocyte HLA-DR expression from PTC-SCID and GD-SCID was
markedly increased, compared with those from nude mice xenografts. In
addition, thyrocyte HLA-DR expression from PTC-nude xenografts was
markedly increased, compared with that seen in GD-nude and N-nude
xenografts. Goldsmith et al. (25) reported that the
expression of DR antigens on malignant thyrocytes did not correlate
with the degree of thyroidal lymphocytic infiltration. Our results
suggest that HLA-DR expression on TEC from PTC is quite strongly
constitutive but is also affected by TIL. In addition, thyrocyte HLA-DR
expression from PTC/TIL-SCID was higher than that from PTC/PTC, but the
difference did not reach statistical significance, perhaps because of
the small numbers available. In our results, thyrocyte HLA-DR
expression from PTC/PTC-SCID had been still higher than that from
PTC-nude. It is suggested that PTC/PTC-SCID must have a few human
lymphocytes capable of producing cytokines. Lahat et al.
showed that tumor necrosis factor alpha (TNF-
), either alone or
synergistically with -interferon, enhanced class II HLA-DR
expression in human thyroid cancer cell lines (26) as well as AITD (4).
The expression of DR antigen in PTC is not only a primary event that
might have occurred during the neoplastic transformation of thyroid
cells (27), but also seems to be partly a secondary event that occurred
by secretion of cytokines (TNF- and/or IFN-) from TIL.
Third, the expression of ICAM-1 can be also up-regulated on somatic
cells by various cytokines, such as TNF- and IFN- (8, 28).
However, ICAM-1 expression on TEC from PTC did not show any difference
between SCID and nude mice or between PTC/TIL and PTC/PTC. ICAM-1
expression on TEC from PTC xenografts in the nude mice was markedly
increased, compared with those from GD and N xenografts. Thus, our
results demonstrate that ICAM-1 expression on TEC from PTC may be
entirely constitutive, and it is also not affected by the presence of
TIL, in contrast to AITD (8).
It is possible that our collagenase method might be affected by de novo effects in this environment. In addition, it would be anticipated that thyrocyte HLA-DR expression would radically diminish in two-day cultures (8, 9, 13). To clarify these points, we had compared PTC and normal tissues under the same conditions. Moreover, others have shown that PTC TEC expressed HLA-DR strongly (6, 24). To have the proof that HLA-DR and ICAM-1 expression is truly constitutive on human thyrocytes in PTC, it will be necessary to perform more experiments, i.e. immunofluorescence studies with anti-HLA-DR antibodies and anti-ICAM 1 antibodies on sections of xenografted tissues after removal.
We had recently reported that lymphocytic infiltration was associated with a good prognosis in PTC (2). This phenomenon might be caused by a specific immune response to thyroid antigens (2). Baker has raised an interesting question regarding the immune response of the individual, which might not recognize the tumor, thus allowing it to grow; or alternatively, is there a change in the tumor itself that makes it less immunogenic and more aggressive (27)? In addition, the actual significance of the aberrant HLA-DR and ICAM-1 expression on TEC is still considered controversial (3, 12, 13, 14, 29, 30, 31). On neoplastic thyroid cells, reduced DR expression could be harmful, because expression of HLA-DR antigens generally (but not always) is considered an element-favorable event to tumor rejection (29). Pfitzenmaier et al. (30) reported that DR-antigen-positive cells induced cytotoxity in the autologous mixed lymphocyte/tumor cell cultures, but negative cells did not. On the other hand, it has been suggested recently that the failure to express costimulatory signals, such as ICAM-1 and B7, by endocrine cells may explain their inability to stimulate T cells and suggests that an alternative role for aberrant class II expression may be to induce peripheral tolerance (12). That is to say, the aberrant expression of thyrocyte HLA-DR may induce peripheral tolerance in AITD (12, 13, 14). Class II MHC (HLA-DR in human) antigen expression on cancer cells may induce tolerance, through partial stimulation of CD4 cells, in the absence of costimulation molecules such as ICAM-1 and B7 (31).
Thus, the aberrant expression of thyrocyte HLA-DR may contribute specific immune tolerance to the thyroid cancer, by evasion of the immune system (3).
However, our data demonstrated that ICAM-1 expression (i.e. costimulatory signals) on TEC from PTC seems to be constitutive, in contrast to AITD. Therefore, in such circumstances, aberrant HLA-DR expression on TEC from PTC might activate T cells (at least, not induce peripheral tolerance) in our mouse model, as reported by Todd et al. (32). In addition, several studies have shown that MHC class I antigens, expressed on tumor cells, are more important in inducing tumor cells, by inducing cytotoxic T cells (3, 33). However, tumor cells present tumor-associated antigen in the context of MHC class II antigen to CD4+ helper cells, so as to activate CD4 T cells to produce various lymphokines, which are necessary for the differentiation of the precytotoxic T lymphocytes (pre-CTL) (34). Autologous tumor killer cells, induced in mixed lymphocyte/tumor cell cultures against tumor cells expressing both HLA class I and II antigens, showed not only MHC class I restriction but also class II restriction in the effector phase, indicating that the CTL population consists of both CD4+ and CD8+ CTL (32). These papers suggest that HLA-DR expression on TEC might participate in cell cytotoxicity, in addition to MHC class I. Thus, our results suggest that HLA-DR expression on TEC from PTC might have some role on limiting cell growth. We also demonstrated that TIL partially controlled HLA-DR expression on TEC from PTC. Immune control of thyroid cancer may be important in limiting PTC growth. Immunotherapy may be one of the new forms of treatment in thyroid cancer.
In conclusion, HLA-DR expression on TEC from PTC seems to be strongly constitutive, but it is also affected by TIL. In contrast, ICAM-1 expression on TEC from PTC seems to be entirely constitutive.
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Acknowledgments |
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Footnotes |
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2 Fellows of The Wellesley Hospital Research Institute.
Received January 7, 1997.
Revised April 17, 1997.
Revised September 11, 1997.
Accepted September 23, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-interferon response of human colon carcinoma cells:
inhibition of proliferation and modulation of immunogenicity as
independent effects of -interferon on tumor cell growth. Cancer Res. 45:3503.This article has been cited by other articles:
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  K. Fujarewicz, M. Jarzab, M. Eszlinger, K. Krohn, R. Paschke, M. Oczko-Wojciechowska, M. Wiench, A. Kukulska, B. Jarzab, and A. Swierniak A multi-gene approach to differentiate papillary thyroid carcinoma from benign lesions: gene selection using support vector machines with bootstrapping Endocr. Relat. Cancer, September 1, 2007; 14(3): 809 - 826. [Abstract] [Full Text] [PDF]
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