Is human leukocyte antigen-DR and intercellular adhesion molecule-1 expression on human thyrocytes constitutive in papillary thyroid cancer? Comparative studies in human thyroid xenografts in severe combined immunodeficient and nude mice.

We have studied human leukocyte antigen (HLA)-DR and intercellular adhesion molecule (ICAM)-1 expression on thyroid epithelial cells (TEC) from papillary thyroid carcinoma (PTC) tissues xenografted into two different mouse strains [the severe combined immunodeficient (SCID) mouse, which accepts human tissue with lymphocytes; and the nude mouse, which accepts the tissue but destroys all passenger lymphocytes]. Human PTC [PTC/TIL (PTC with tumor infiltrating lymphocytes) and PTC/PTC (PTC without tumor infiltrating lymphocytes)], Graves' disease (GD), and normal thyroid (N) tissues were xenografted sc into 22 SCID and 21 nude mice. Blood samples were taken every 2 weeks for measurement of human IgG and thyroid antibodies. Seven weeks after xenografting, xenografted thyroid tissues were analyzed for thyrocyte HLA-DR and ICAM-1 expression. SCID mice xenografted with PTC/TIL (PTC/TIL-SCID) manifested IgG production for 6 weeks, but nude mice showed diminished and disappearing IgG production from these xenografts. Thyroperoxidase (TPO)-antibody (Ab)(TPO-Ab) was not detectable in PTC/TIL-SCID despite the presence of TPO-Ab in some donors. Thyroglobulin-Ab (Tg-Ab) was detectable in all mice of PTC/TIL-SCID. Thyrocyte HLA-DR expression from PTC-SCID was markedly increased, compared with that from nude mice xenografts or from N xenografts in SCID mice. In addition, thyrocyte HLA-DR expression from PTC-nude was markedly increased, compared with the expression seen in GD-nude and N-nude xenografts. ICAM-1 expression on TEC from PTC xenografts in the SCID mouse was markedly increased, compared with N xenografts. ICAM-1 expression on TEC from PTC did not show any difference between SCID and nude mice. ICAM-1 expression on TEC from PTC xenografts in the nude mice was markedly increased, compared with those from GD and N xenografts. In conclusion, TIL in PTC produce Tg-Ab but do not produce TPO-Ab. HLA-DR expression on TEC from PTC is strongly constitutive, but it is also affected by TIL. TIL might have some role in control of PTC through partial expression of HLA-DR on TEC. ICAM-1 expression on TEC from PTC seems to be entirely constitutive, and it is not affected by the presence of local lymphocytes, in contrast to autoimmune thyroid disease.

Differential effects of human interferon alpha and interferon gamma on xenografted human thyroid tissue in severe combined immunodeficient mice and nude mice.

We have studied the in vivo effects of human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) administration on human thyroid tissue xenografted into two mouse strains: severe combined immunodeficient (SCID) mice and nude mice. Human lymphocytes survive in SCID mice but are lysed in nude mice. Thyroid tissues from Graves' disease or Hashimoto's thyroiditis, or paranodular [normal, (N)] tissue was xenografted into SCID mice (0.8 g/mouse) pretreated with anti-asialo GM-1 antiserum and radiation and also into nude mice. One week after xenografting, SCID and nude mice were divided into three groups. Group A was treated with IFN-alpha intraperitoneally (2,000 units/mouse) three times weekly; group B was treated with IFN-gamma similarly; group C was treated with phosphate buffered saline (PBS) only (control). Autologous human peripheral blood mononuclear cells (PBMCs) were added to mice receiving N xenografts. Blood was taken every 2 weeks for levels of IgG and thyroid antibodies (TAb). After 6 weeks of treatment, mice were sacrificed, and xenograft thyrocyte histocompatibility leukocyte antigen (HLA-DR) and intercellular adhesion molecule (ICAM-1) expression were measured. In addition, thyrocyte cultures were stimulated in vitro with 200 units/ml of either IFN-alpha or IFN-gamma or PBS (control). SCID mice xenografted with autoimmune thyroid disease (AITD) in group A showed a significantly higher TAb production than group C, whereas in group B, TAb production was not statistically increased compared to control (group C). SCID mice xenografted with N did not produce TAb in any group, nor did nude mice xenografted with AITD. Thyrocyte HLA-DR expression was markedly increased in group A and B in SCID mice xenografted with Graves' disease, Hashimoto's thyroiditis, and N tissue compared to group C. In contrast, only group B (IFN-gamma) showed an increase in thyrocyte HLA-DR in nude mice. In the in vitro studies, only IFN-gamma (not IFN-alpha) stimulated thyrocyte HLA-DR and ICAM-1 expression in Graves' disease, Hashimoto's thyroiditis, and N tissues. We concluded that in SCID mice, IFN-alpha causes TAB production in AITD xenografts but not in N xenografts, while increasing thyrocyte HLA-DR expression in both. Also, IFN-gamma does not cause a statistically increased TAb in AITD xenografts in SCID mice, despite a sharp rise in thyrocyte HLA-DR expression. In addition, because IFN-alpha has no effect in nude mice or in vitro on thyrocyte HLA-DR expression, its effects in SCID mice must be mediated via local infiltrating lymphocytes. Finally, IFN-gamma has a direct effect on thyrocytes to increase HLA-DR expression (and, in vitro, ICAM-1 expression) but may not stimulate TAb production.

Activation of CD8+ T lymphocytes in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease directed against the insulin-secreting beta cells of the islets of Langerhans of the pancreas. We have previously shown that in organ-specific autoimmune diseases, Graves' disease (GD), and IDDM, the antigen that is specific for each of these disorders (i.e., TSH receptor for GD, glutamic acid decarboxylase-65 (GAD65) for IDDM) does not activate the disease-specific CD8+ cells as fully as CD8+ cells from normal persons. In order to identify the specific antigen responsible for triggering or maintaining autoimmunity in patients afflicted with the disease, we have studied the effects of islet (beta) cell-specific antigens GAD65, insulin, pancreatic antigen (P69), T cell epitope 69 (Tep69), and a milk-derived bovine serum albumin (BSA)-peptide-ABBOS (pre-BSA positions 157-169) on the activation of CD8+ T lymphocytes in IDDM patients. We compared the patterns of T cells activation with those mediated by an irrelevant peptide antigen, P348 (amino-terminal region of human cardiac myosin light chain-1), and also tetanus toxoid. We also studied the responses of CD8+ T lymphocytes to these IDDM-relevant and -irrelevant antigens in Hashimoto's thyroiditis patients (HT), rheumatoid arthritis patients (RA), and normal control subjects (N) to compare the pattern of responses in the other autoimmune diseases. Activation of lymphocytes was monitored by measuring the expression of the activation molecule-major histocompatibility complex class II antigen (HLA-DR) on the surfaces of CD8+ T lymphocytes by flow cytometry. Peripheral blood mononuclear cells (PBMC) obtained from 14 patients with IDDM, 14 N, 14 with HT, and 13 with RA were cultured for 7 days in the presense or absence of antigens. The stimulation index (SI) of activation of the lymphocytes was determined. When the response of CD8+ T lymphocytes of IDDM patients to each of the IDDM-relevant antigens was compared to that of the irrelevant antigen, only GAD65 and ABBOS showed a significantly reduced activation compared to P348 and tetanus toxoid. Other relevant antigens, insulin, P69, and Tep69, did not show any significant differences in their SI compared to those of the irrelevant antigens. In the N, HT, and RA groups, there was no significant difference in the SI of the responses of CD8+ cells to any of the relevant antigens compared to that of the irrelevant antigens. Moreover, CD8+ T lymphocytes of IDDM patients showed a significantly lower activation by GAD65 than those from N, HT, and RA. In conclusion, our data suggest that CD8+ T lymphocytes of IDDM patients but not those from N, HT, and RA groups have specifically reduced potential for activation in response to GAD65 but not to insulin, P69, and Tep69, whereas ABBOS exerts a less well-defined reductive effect on the activation of CD8+ lymphocytes of IDDM patients. Since CD8+ cells have been shown to contain suppressor activity, our data support the notion that a disease-specific defect in GAD65 autoantigenic induction of suppressor T lymphocytes may be important in the pathogenesis of IDDM.

Thyroid xenografts from patients with Graves' disease in severe combined immunodeficient mice and NIH-beige-nude-xid mice.

OBJECTIVE: To compare human thyroid xenografts from patients with Graves' disease in severe combined immunodeficient (SCID) mice and triple immunodeficient NIH-beige-nude-xid (NIH-3) mice to obtain an improved animal model for studying these xenografts. DESIGN: Animal study. PARTICIPANTS AND ANIMALS: Patients with Graves' disease; SCID and NIH-3 mice. INTERVENTIONS: Thyroid tissue from six patients with Graves' disease was xenografted to SCID and NIH-3 mice; in addition, peripheral blood mononuclear cells (PBMC) from 12 patients with Graves' disease were grafted intraperitoneally to separate SCID and NIH-3 mice. OUTCOME MEASURES: Levels of human immunoglobulin (IgG), thyroperoxidase antibodies (TPO-Ab), thyroglobulin (Tg-Ab), and expression of thyrocyte intercellular adhesion molecule-1 (ICAM-1) and histocompatibility leukocyte antigen (HLA-DR) in mice after xenografting. RESULTS: IgG was detected in all mice grafted with Graves' thyroid tissue and some mice grafted with PBMC; levels of human IgG peaked 6 to 10 weeks after xenografting. Human IgG levels reached a mean of 500 mg/L (standard error of the mean [SEM] 150 mg/L) in the NIH-3 mice with thyroid xenografts. This was similar to results in SCID mice with thyroid xenografts, which had a mean level of human IgG of 640 mg/L (SEM 230 mg/L). PBMC xenografting resulted in a mean IgG level of 1200 mg/L (SEM 250 mg/L) in NIH-3 mice, which was similar to the mean level of 1000 mg/L (SEM 280 mg/L) in SCID mice. The rate of rise in human IgG in the sera of the NIH-3 mice with thyroid xenografts was similar to that in the SCID mice. TPO-Ab were also detected in some mice with Graves' thyroid grafts and in a few mice injected with PBMC, with levels peaking 4 to 6 weeks after xenografting. TPO-Ab levels reached a mean 109.3 U/mL (SEM 57.2 U/mL) in the NIH mice with thyroid xenografts, which were similar to the mean level of 91.7 U/mL (SEM 34.2 U/mL) in the SCID mice. There were no significant differences in the Tg-Ab levels in each type of mice (13.9 [SEM 12.1] U/mL v. 17.9 [SEM 7.9] U/mL). Eight weeks after xenografting into mice, the expression of xenograft thyrocyte ICAM-1 decreased significantly in both the SCID and NIH-3 mice (from 43.4%, SEM 4.9%, to 35.9%, SEM 4.6%, in the NIH-3 mice, p < 0.05, and from 43.4%. SEM 4.9%, to 32.5%, SEM 5.2%, in the SCID mice, p < 0.05). However, the expression of thyrocyte HLA-DR did not change significantly in the NIH-3 mice (from 11.5%, SEM 3.3%, to 10.8%, SEM 3.3%), whereas it decreased significantly in the SCID mice (from 11.5%, SEM 3.3%, to 4.2%, SEM 2.0%, p < 0.02). CONCLUSIONS: Not only SCID mice but also NIH-3 mice may be useful as animal models for xenografted thyroid tissue, which will help us elucidate the pathogenesis of autoimmune thyroid disease. NIH-3 mice are superior to SCID mice in maintaining the expression of thyrocyte HLA-DR in Graves' thyroid xenografts at levels as high as those before xenografting; this maintenance of expression may be due to the lack of natural killer cells in NIH-3 mice.

The effect of adding a surfeit of autologous CD8+ T cells to SCID mice after secondary rexenografts of Graves' thyroid tissue.

To investigate the effect of adding a surfeit of CD8+ T cells as a potential immunoregulator in Graves' disease (GD), thyroid tissues from 4 patients with GD and 2 normal subjects (N) were initially xenografted into nude mice. Eight weeks after xenografting, the thyroid tissues, which were then devoid of lymphocytes and appeared normal, were retrieved from the nude mouse, and rexenografted (rexenografts) into severe combined immuno-deficient (SCID) mice; 20 x 10(6) of autologous peripheral blood mononuclear cells (PMBC) or 20 x 10(6) of CD8(+)-depleted PBMC ("non-CD8 cells," i.e., CD4-enriched PBMC) were simultaneously engrafted into SCID mice with thyroid rexenografts. In addition, 20 x 10(6) of CD8(+)-enriched PBMC ("CD8-doubled" cells, which were prepared to double the percentage of CD8+ T cells compared to that of PBMC) were engrafted into SCID mice with rexenografts from 2 GD and 2 N; finally, 20 x 10(6) of PBMC plus an extra 10 x 10(6) of CD8+ T cells ("extra-CD8 added" cells, total 30 x 10(6) of CD8-enriched cells) were engrafted into separate SCID mice with rexenografts from 2 GD. The reengraftment of GD rexenografts or N rexenografts alone did not result in the detection of thyroperoxidase (TPO)-antibodies (Abs), thyroglobulin (Tg)-Abs, thyroid-stimulating Ab (TSAb) production, human IgG, or lymphocytic infiltration in the xenografts. However, the engraftment of either autologous PBMC or non-CD8 cells from patients with GD and N into SCID mice with rexenografts caused human IgG to become detectable and then rise further in 10 of 17 SCID mice; when human IgG, TPO-Ab, Tg-Ab, and TSAb were quantitated, GD rexenografts plus non-CD8 cells engrafted into SCID mice showed a higher production of each antibody and human IgG than in GD rexenografts plus PBMC, or GD rexenografts plus CD8-doubled cells, or GD rexenografts plus extra "CD8-added" cells. Moreover, when CD8-doubled cells or extra CD8-added cells with rexenografts were engrafted to SCID mice with rexenografts, they showed generally lower production of human IgG and thyroid antibodies compared to SCID mice into which PBMC were engrafted with rexenografts, despite the fact that 50% more cells (30 x 10(6)) were engrafted in the preparations of extra CD8-added cells. In conclusion, CD8+ T cells from patients with GD appeared to suppress the induction of thyroid antibodies, TSAb, and human IgG. The CD8+ cells thus are acting as suppressor or regulatory T cells. Such cells might be important in the pathogenesis of autoimmune thyroid disease.

Antibody to gp39, the ligand for CD40 significantly inhibits the humoral response from Graves' thyroid tissues xenografted into severe combined immunodeficient (SCID) mice.

Experimental evidence suggests that interference with gp39-CD40 interactions may have therapeutic potential in prevention of certain autoimmune disorders (i.e., collagen-induced rheumatoid arthritis). The binding between CD40 expressed on mature B cells and CD40 ligand (CD40L, gp39) transiently expressed on activated T helper cells (Th) further stabilizes the interactions (between Th and B cells) and co-ordinates the responses of the interacting cells during antigen presentation, and is essential for thymus-dependent humoral immunity. Graves' disease is the most common form of hyperthyroidism, in which hyperactivity of the thyroid gland is due to an autoantibody directed against the thyrotropin receptor (TSHR). The main objective of our study was to determine the role of interactions between gp39 and CD40 in "an established" human Graves' disease (GD). Severe combined immunodeficient (SCID) mouse served as a vehicle for human Graves' thyroid tissue. This experimental setting allows us to study, observe, and immunomodulate human autoimmune tissue in so called in vivo condition. We studied the effects of ip administration of anti-gp39 mAb on humoral response, thyroid function tests, expression of adhesion molecules, and HLA-DR on human thyrocytes and histopathological changes from human GD thyroid tissue xenografts. GD thyroid tissue from 4 patients was xenografted into 20 SCID mice (0.8 g/mouse). Human immunoglobulin G (IgG) levels became detectable in SCID mice 1 week after xenograftment. Ten SCID mice were sequentially administered anti-gp39 mAb (250 micrograms/mouse/ dose) ip every 4 days until the end of the experiment. Ten control animals were injected with vehicle (PBS) in similar fashion. Blood samples were taken every 2 weeks from the tail veins for measurement of the humoral response [human IgG, thyroid-stimulating antibody (TSAb), antithyroperoxidase (anti-TPO), and antithyroglobulin (anti-Tg), Abs], and thyroid function tests. After 8 weeks, animals were sacrificed and thyroid tissue was examined histologically. The humoral response from the intrathyroidal lymphocytes was measured and the tissue morphology of GD was preserved during the 8-week period in phosphate-buffered saline (PBS)-treated SCID mice xenografted with GD xenografts. However, administration of anti-gp39 mAb completely blocked or significantly decreased the humoral response in all treated animals. On the other hand, no significant histological changes were associated with the administration of anti-gp39 mAb. The degree of lymphocytic infiltration in thyroid tissue xenografts was comparable in both groups. Serum thyroxine values were normal in both groups. In spite of a profound immunosuppressive effect on the humoral response by directly blocking CD40-gp39 interactions in vivo, this did not result in complete deletion of the responding Th in the thyroid specimens.

Expression of intercellular adhesion molecule-1 on human thyroid cells from patients with autoimmune thyroid disease: study of thyroid xenografts in nude and severe combined immunodeficient mice and treatment with FK-506.

It has been suggested that intercellular adhesion molecule-1 (ICAM-1) may play an important role in the initiation, localization, and perpetuation of autoimmune thyroid diseases (AITD). In an effort to clarify its role, we have investigated the expression of ICAM-1 on thyroid epithelial cells (TEC) of patients with AITD, patients with nontoxic goiter (NTG), and normal subjects (PN) by flow cytometric analysis under basal conditions and after modulation with cytokines, before and after 8 weeks of thyroid tissue xenotransplantation in nude athymic mice (which lyses all passenger lymphocytes), and in severe combined immunodeficient (SCID) mice where these cells survive. Before xenografting, ICAM-1 was expressed on 56% of TEC from Hashimoto's thyroiditis (n = 5), 54% of Graves' disease (n = 6), 15% of NTG (n = 5), and 12% of PN TEC. After the xenografts had been 8 weeks in nude mice, ICAM-1 expression decreased markedly in AITD TEC [from 56% to 10% in Hashimoto's thyroiditis (P < 0.001) and from 54% to 8% in Graves' disease (P < 0.01)], but did not change significantly in NTG or PN. After the xenografts had been 8 weeks in SCID mice, the expression of ICAM-1 was significantly higher on TEC of AITD compared with the same tissue in nude mice. When the SCID mice engrafted with AITD tissue were treated with the anti-CD4+ T (helper) cell agent FK-506, the expression of ICAM-1 was reduced significantly compared with that in the original tissue or that in nontreated mice engrafted with the same tissue. The proportion of TEC that were ICAM-1 positive was up-regulated in all cases by certain cytokines (e.g. interferon-gamma and tumor necrosis factor-alpha applied alone or in combination). We also detected the presence of ICAM-1 in AITD frozen tissues using an immunohistochemical technique. These data suggest a role for ICAM-1 in human AITD. However, the expression of ICAM-1 appears to be a secondary phenomenon in response to the immune assault, rather than a primary event. Our results support the idea that TEC may act as passive captives to immunological events in human AITD.

Effects of monoclonal antibody against CD45RB on peripheral blood mononuclear cell proliferation and on HLA-DR and adhesion molecule expression on thyrocytes of patients with autoimmune thyroid disease.

To evaluate the role of CD45 (especially that of the ectodomain region B) on immunocyte-thyrocyte signaling in patients with autoimmune thyroid disease (AITD), we have examined the in vitro and in vivo effects of a monoclonal antibody (mAb) against with CD45RB, termed MT3. MT3 was added to cultured peripheral blood mononuclear cells (PBMC) from patients with AITD and was additionally injected into severe combined immunodeficient (SCID) mice to which Graves' thyroid cells and intrathyroidal lymphocytes were engrafted. MT3 stimulated proliferation of PBMC when cultured for 2 to 3 days in patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD) and in normal controls (NC). However, when cultured for 7 days, the stimulation index [SI: counts per minute (cpm) with mAb/cpm without mAb] was lowered by MT3 in NC and GD patients. However, the mean SI was not lowered in patients with HT. In SCID mice, the concentrations of human immunoglobulin G, antithyroglobulin and antithyroperoxidase antibodies in sera were not significantly changed by injecting MT3. The expression of human leukocyte antigen (HLA)-DR and intercellular adhesion molecule (ICAM)-1 on engrafted human thyrocytes decreased after the tissues were engrafted into the control mice to which vehicle alone was injected. However, in the mice injected with MT3, HLA-DR and ICAM-1 expression remained high or up-regulated by the injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Homing of 51Cr-labeled human peripheral lymphocytes to Graves' thyroid tissue xenografted into SCID mice.

We have recently described a NUDE/SCID mouse model that has been useful for the study of human thyroid autoimmunity in in vivo conditions. The reappearance of lymphocytic infiltration in Graves' thyroid tissue and a humoral response in SCID mice (rexenografted with normalized thyroid tissues from NUDE mice) was detected only if autologous Graves' human peripheral lymphocytes (PBMC) were engrafted into the same animals. Therefore it was presumed that some autoreactive PBMC directed themselves to the thyroid. However, there was previously no direct evidence regarding the trafficking of the engrafted PBMC to the target tissue. To elucidate this point we have studied the migration of 51Cr-labeled PBMC in SCID mice. Human thyroid tissue from six Graves' disease (GD) patients and six patients with nontoxic nodular goiter were initially xenografted into NUDE mice for 8 weeks. The same tissues were retrieved and rexenografted into several "virgin" SCID mice, i.e., no previous xenografts. Autologous PBMC were isolated from blood of the same patients obtained at the time of the tissue rexenograftment and labeled with radioactive 51Cr. Twenty million labeled PBMC were engrafted into each SCID mouse. The distribution of labeled lymphocytes into mouse organs and trafficking into Graves' and normal xenografts was measured. A significant amount of radioactivity in Graves' xenografts was detected after 1 week with the peak of radioactivity at 2-3 weeks. This radioactivity was significantly higher than radioactivity in surrounding tissues (skin, muscle). In contrast, homing of autologous lymphocytes into normal paranodular thyroid tissue was very minimal; the radioactivity of GD thyroid xenografts with engrafted autologous lymphocytes was significantly higher than that of normal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of T lymphocyte subsets by synthetic TSH receptor peptides and recombinant glutamate decarboxylase in autoimmune thyroid disease and insulin-dependent diabetes.

We have postulated that a defect in specific antigenic induction of suppressor T lymphocytes may account for the immunoregulatory disorder in autoimmune thyroid disease. In this context, we have measured the proliferative responses of peripheral blood mononuclear cells (PBMC) to the synthetic peptides corresponding to the extracellular domain of the TSH receptor (TSHR) and recombinant glutamate decarboxylase (GAD65) by means of 3H thymidine incorporation. We have also studied the antigenic activation of CD4+ and CD8+ T lymphocytes by measuring human leukocyte antigen-DR (HLA-DR) expression on the cell surface by flow cytometric analysis. PBMC obtained from 47 patients with Graves' disease (GD) [including 19 hyperthyroid GD (hyper GD)], 18 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), 18 with insulin-dependent diabetes (IDDM), and 20 normal controls (N), were cultured for 7 days in the presence or absence of the pool peptides representing 3 different segments of TSHR or GAD65 at final concentration of 30 micrograms/mL or 10 micrograms/mL. The proportion of subjects whose PBMC gave a positive proliferative response with a stimulation index (SI) of over 2.3 (i.e. above the mean +2 SD for N) to TSHR peptides was significantly higher in the hyper GD group than among euthyroid GD (eu GD), HT, IDDM, and N group. The corresponding differences in mean SI provided analogous results, showing significant responses above normal in only hyper GD. The CD4+ T lymphocytes from hyper GD group were significantly more activated by TSHR peptides compared to eu GD, HT, IDDM, and N, and this induction correlated to their thyroid hormone levels. Quite differently, the activation of CD8+ T lymphocytes from both hyper GD and eu GD group in response to TSHR peptides was impaired compared to HT, IDDM, and the N group; in contrast to the findings with CD4+ T lymphocytes, this was independent of thyroid hormone levels. On the other hand, while the CD8+ T lymphocytes from GD and N groups were activated equally by GAD65, the activation of CD8+ T lymphocytes from the IDDM group by GAD65 was impaired compared to the GD and N groups. In conclusion, the activation of CD8+ T lymphocytes from GD and IDDM by relevant antigens (i.e. TSHR peptides for GD and GAD65 for IDDM) was impaired, but not by irrelevant antigens (i.e. GAD65 for GD and TSHR peptides for IDDM). There was also a modest stimulation of CD8+ T cells from all groups by tetanus toxoid and cardiac myosin light chain peptide, both irrelevant antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of interleukin-2 on suppressor T lymphocytes in autoimmune thyroid disease.

We have investigated the effects of interleukin-2 (IL-2) on the activation of suppressor T lymphocytes in autoimmune thyroid disease (AITD), with insulin-dependent diabetes mellitus (IDDM) as an autoimmune disease control; this was accomplished by measuring the expression of major histocompatibility complex class II (HLA-DR), CD25 (IL-2 alpha receptor (R)), and IL-2 beta R expression on their surfaces by flow cytometric analysis. Peripheral blood mononuclear cells (PBMC), obtained from 10 patients with Graves' disease (GD), 11 with Hashimoto's thyroiditis (HT), 9 with insulin-dependent diabetes mellitus (IDDM), and 10 normal persons (N), were cultured for 7 d in the presence or absence of IL-2 at a final concentration of 50 U/mL. CD8+ cells were isolated from cultured PBMC with immunomagnetic beads, and were stained with fluorescent-conjugated monoclonal antibodies (anti-CD11b, anti-IL-2 alpha R, anti-IL-2 beta R, and anti-HLA-DR); the activation of CD8+CD11b+ ("suppressor") T cells (Ts) by IL-2 was then analyzed on a flow cytometer. In the absence of IL-2, i.e., in the autologous mixed lymphocyte reaction (AMLR), Ts from patients with GD, HT, and IDDM showed significantly lower activation as compared to N when analyzed by HLA-DR expression, but were not significantly different when IL-2R expression was measured. We determined the Stimulation Index (SI) of the activation of T lymphocytes by IL-2 for comparison between N and patients. With stimulation of 50 U/mL of IL-2, SI of HLA-DR+ Ts was significantly (p < 0.05 to 0.01) lower in GD, HT, and IDDM as compared with N.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of human peripheral blood mononuclear cells (PBMC) from patients with subacute thyroiditis in severe combined immunodeficient (SCID) mice: less production of human interferon gamma than that seen for Graves' disease.

Human peripheral blood mononuclear cells (PBMC) from 2 patients with de Quervain's subacute thyroiditis (SAT), 2 with Graves' disease (GD), and 3 normal persons (N) were engrafted into severe combined immunodeficient (SCID) mice so as to study whether SAT PBMC would differ immunologically from GD PBMC in vivo. Human IgG was detected in all mice engrafted with PBMC from either group of patients or normal persons. Thyroid Stimulating Antibody (TSAb) was detected in the sera of mice with PBMC from SAT or GD patients, but not N. Thyroperoxidase (TPO)-antibody (Ab) and/or thyroglobulin (Tg)-Ab was detectable in the mice with GD PBMC only, but not in those with SAT or normal PBMC. The production of interferon gamma (IFN gamma) in mice engrafted with N PBMC was 8, 13 and 14 U/ml, similar to values found in sera of SCID mice with SAT PBMC (14 and 11 U/ml), i.e., much lower than that seen for GD PBMC (127 and 78 U/ml); this is consistent with the view that, compared to GD T lymphocytes, that there is probably a lower number of T lymphocytes sensitized to the thyrotrophin (TSH) receptor antigen in SAT patients. Another possibility is that the transient thyroidal antigenic release seen in the acute (hyperthyroid) phase may be insufficient for adequate T cell sensitization. Still other possibilities include the effect of more severe hyperthyroidism of GD on T cell sensitization, and CD4/CD8 cell ratios. In any event, these results are consistent with our previous view that antigenic release in SAT will not itself lead to autoimmune thyroid disease.

Effect of FK-506 on xenografted human Graves' thyroid tissue is severe combined immunodeficient mice.

OBJECTIVE: We studied the macrolide antibiotic FK-506, an immunosuppressive agent, in an attempt to ameliorate the lesion of autoimmune thyroid disease in human thyroid tissue xenografted into severe combined immunodeficient (SCID) mice. It was not felt appropriate to employ this agent directly in patients with autoimmune thyroid disease because adequate therapeutic modalities are available and the introduction of new, experimental agents could not be justified. Moreover, the study of the tissue before and after treatment could not have been undertaken directly in patients. DESIGN: Human thyroid xenografts from four patients with Graves' disease and two normal persons were xenografted into SCID mice. Two weeks after xenografting, human immunoglobulin G (IgG) was detectable in all SCID mice xenografted with Graves' thyroid tissue. Mice were divided into two groups with human IgG levels similar to each other. Mice in the first group were treated with FK-506 daily for 6 weeks; mice in the second (similar) group were given phosphate-buffered saline (PBS) only (control group). MEASUREMENTS: Blood samples were taken every 2 weeks from the tail veins for human IgG, thyroid stimulating antibody, thyroperoxidase antibodies, thyroglobulin antibodies, and interferon-gamma (IFN-gamma). After 8 weeks treatment, animals were sacrificed; thyroid tissue was examined histologically and for thyrocyte HLA-DR expression. FK-506 was also added to thyrocytes in in-vitro tissue culture conditions. RESULTS: After 4-6 weeks of FK-506 therapy, human IgG, all thyroid antibodies and IFN-gamma were suppressed, while the levels remained elevated in the control group. Lymphocytic infiltration virtually disappeared in the human thyroid tissue of the FK-506-treated mice and thyrocyte HLA-DR expression markedly declined; in the control mice, lymphocytic infiltration remained heavy and HLA-DR expression remained high. On the other hand, FK-506 added directly to thyrocytes in vitro (without lymphocytes) did not reduce thyrocyte HLA-DR expression. CONCLUSIONS: FK-506 appears to suppress the activation of intrathyroidal lymphocytes, but not thyrocytes. From these observations, it is concluded that this agent, by its action on intrathyroidal lymphocytes, is able to ameliorate the immunologically mediated histological and serological disturbance in human autoimmune thyroid disease, at least under these circumstances.

Effect of removing human Graves' thyroid xenografts after eight weeks in nude mice and rexenografting them into SCID mice.

Human thyroid xenografts from four patients with Graves' disease (GD) and two normal persons were initially xenografted into nude mice. Eight weeks after xenografting, the thyroid tissue appeared normal; indeed, thyroid infiltrating lymphocytes in the GD xenograft could no longer be identified when analyzed histologically. Thus, human immunoglobulin G (IgG), thyroperoxidase (TPO)-antibodies (Abs), thyroglobulin (Tg)-Abs, thyroid-stimulating antibodies (TSAb), and thyrocyte histocompatibility leucocyte antigen (HLA)-DR expression were undetectable. These same tissues were retrieved from the nude mouse and rexenografted into severe combined immunodeficient (SCID) mice (with no prior xenograft); autologous peripheral blood mononuclear cells (PBMC) or CD8-depleted PBMC (non-CD8 cells) were simultaneously injected into some of these SCID mice. Engraftment of a GD thyroid rexenograft (TH) alone did not cause IgG, TSAb, TPO-Ab, or Tg-Ab production, thyrocyte HLA-DR expression, or lymphocytic infiltration in thyroid grafts. Engraftment of GD PBMC or non-CD8 cells alone (i.e. without a thyroid xenograft) caused human IgG to rise, but only minimal titers of thyroid antibodies appeared. When TSAb, TPO-Ab, and Tg-Ab were quantified, GD TH plus PBMC-engrafted SCID mice showed significantly higher production of each antibody than that of GD PBMC alone, and this phenomenon was further enhanced by the removal of CD8+ cells. GD thyrocytes showed marked HLA-DR expression at human surgery; however, after 8 weeks' sojourn in nude mice, DR expression disappeared. After a further 8 weeks following rexenografting into SCID mice, TH plus PBMC resulted in a reappearance of DR expression only in GD but not in grafts from normal persons, and this was enhanced by the depletion of CD8 cells. These results were also in parallel with histological findings inasmuch as the normal tissue remained normal with no thyroid antibodies appearing with PBMC or CD8-depleted cells. In experiments from two GD patients, autologous skeletal muscle as well as thyroid tissue were xenografted into nude mice. Eight weeks after xenografting, these were rexenografted into SCID mice that contained prior autologous primary GD thyroid xenografts. Histological findings showed new lymphocytic infiltration in rexenografted thyroid tissues in the SCID mice but not in autologous skeletal muscle. This signifies that the immune assault in GD is specifically targeted to the thyroid tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Seroreactivity to bacterial antigens is not a unique phenomenon in patients with autoimmune thyroid diseases in Canada.

It has been suggested elsewhere that the enteric pathogen Yersinia enterocolitica (Y.e.) might be implicated etiologically in autoimmune thyroid disease (AITD). To reevaluate this hypothesis in the Canadian population, where the prevalence of anti-Y.e. antibodies in the general population is very low (< 1%), we have studied the occurrence of antibacterial reactivity (against Y.e. 0:3 and 0:9, Escherichia coli and Staphylococcus aureus) in the sera of patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), nontoxic nodular goiter (NTG), and autoimmune rheumatic diseases (ARD) as well as normal controls (C). Using the tube agglutination method, no single positive sample was detected in these subjects. No differences in the mean levels of anti-Y.e. 0:3 or 0:9 by ELISA were observed between various groups of patients. Immunoreactivity in the course of medical therapy during 5-12 months did not show significant changes in any of 12 ARD and AITD patients. Some serological reactivity to the plasmid containing strain of Y.e. 0:3 was demonstrated in all subjects by the Western blotting technique. However, weaker signals and fewer bands were noticed in these sera compared to sera from patients with acute yersiniosis. Analysis of the pattern of reactivity did not show any difference in reactivity to any protein between the groups of subjects. The immunodominant antigen in Y.e. 0:3 to which IgG reacted in almost all subjects was the plasmid encoded 240-kDa protein. Our study favors the view that there is a merely coincidental incidence of seroreactivity to bacterial antigens, which appears to be irrespective of diagnosis.

Studies of human thyroid xenografts from Hashimoto's thyroiditis in severe combined immunodeficient (SCID) mice: detection of thyroid stimulation-blocking antibody.

Human thyroid xenografts from 7 patients with Hashimoto's thyroiditis (HT) and 3 normal persons (N) were xenografted into severe combined immunodeficient (SCID) mice to study the intrathyroidal lymphocytes that were expected to survive in these animals. Human IgG was detected in all mice engrafted with HT thyroid tissue peaking at 6-10 weeks after xenografting. Thyroperoxidase-antibody (TPO-Ab) was also detected in all mice with HT thyroid grafts peaking at 4-6 weeks after xenografting, reaching up to 44% of donors' original concentrations. In contrast, maximal thyroglobulin (Tg)-Ab production in some SCID mice with HT thyroid grafts was higher than the donors' original level, and was detectable in mice with thyroid grafts from Tg-Ab-negative HT donors. Thyroid stimulation-blocking antibody (TSBAb) was found in 2 mice with thyroid xenografts from 1 HT patient whose original serum TSBAb and thyrotropin-binding inhibitor immunoglobulin (TBII) had been positive; the maximal TSBAb level in SCID mice exceeded the donor's original level. TSBAb production in SCID mice reached its peak at 10 weeks after xenografting, i.e., later than that of thyroid-stimulating antibody (TSAb) observed in our recent report, suggesting the existence of distinct intrathyroidal B cell autoreactive clones of different life span responsible for secreting TSAb or TSBAb. When autologous peripheral blood mononuclear cells (PBMC) were engrafted alone (without thyroid tissue), TSBAb was undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)

New animal models for human autoimmune thyroid disease. Xenografts of human thyroid tissue in severe combined immunodeficient (SCID) and nude mice.

We have been employing two mouse models for the study of human thyroid xenografts from patients with autoimmune thyroid disease (AITD). The first mouse strain is that of the athymic "nude" mouse which accepts human thyroid xenografts, but the passenger lymphocytes are lysed in this model over several weeks; in the second model, the severe combined immunodeficient (SCID) mouse, both the xenograft and its lymphocytes survive. The AITD thyroid xenograft returns to normal function and morphology in the nude mouse over several weeks whereas the same AITD thyroid xenografts undergo aggravation of their lesions in the SCID mouse. Normalized ("cleansed") thyroid tissue in the nude mouse, now bereft of the passenger lymphocytes, can be removed and then re-xenografted into SCID mice. There it will remain normal unless autologous peripheral blood mononuclear cells are added, whereupon the AITD lesion will be reproduced. Autologous "irrelevant" muscle tissue having undergone the same process will not show such lesions. There is thus no evidence for a primary thyroid cell disturbance in AITD, the abnormality appearing to be only in the immune system. Moreover peripheral blood mononuclear cells appear to contain sufficient memory cells to be able to mount an immune assault on the autologous normalized thyroid tissue (to which they had been previously sensitized) but not on irrelevant autologous (muscle) tissue.

Reduced activation of suppressor T lymphocytes by specific antigens in autoimmune thyroid disease.

In order to study the activation of suppressor T lymphocytes by thyroid-specific antigens in autoimmune thyroid disease (AITD), we have investigated the effects of the organ-specific antigens, thyroperoxidase (TPO), thyroglobulin (Tg), and thyroid microsomal antigen (TMc), as well as renal microsomes (RMc) as a control antigen, on the activation of suppressor T lymphocytes; this was accomplished by measuring major histocompatibility complex class II (HLA-DR) expression on their surfaces by flow cytometric analysis. Peripheral blood mononuclear cells (PBMC), obtained from 33 patients with Graves' disease (GD), 26 with Hashimoto's thyroiditis (HT), 5 with nontoxic nodular goiter (NTG), and 30 normal persons (N), were cultured for 7 days in the presence or absence of TPO, Tg, or RMc at final concentration of 10, 100, and 1000 ng/ml. Cultured cells were stained with fluorescent-conjugated monoclonal antibodies (anti-CD8, anti-CD11b, and anti-HLA-DR), and the activation of CD8+ and CD8+CD11b+ (pure suppressor) T cells by the antigens was analyzed on a flow cytometer. In the absence of antigen, i.e., the autologous mixed lymphocyte reaction (AMLR), CD8+ and CD8+CD11b+ T lymphocytes from patients with GD and HT showed significantly lower activation as compared to N. We measured the Stimulation Index (Sl) of activated T lymphocytes to compare antigen-specific activation between CD8+ and CD8+CD11b+ cells from normal persons and patients. With stimulation of 100 and/or 1000 ng/mL of TPO or Tg, Sl of activated CD8+ cells was significantly (p < 0.05 to 0.01) lower in GD and HT as compared with N.(ABSTRACT TRUNCATED AT 250 WORDS)