T. Morita

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 (SI) of activated T lymphocytes to compare antigen-specific activation between CD8+ and CD8+CD11 b+ cells from normal persons and patients. With stimulation of 100 and/or 1000 ng/mL of TPO or Tg, SI of activated CD8+ cells was significantly (p<0.05 to 0.01) lower in GD and HT as compared with N. Furthermore, when results with CD8+CD11b+ (pure suppressor) T lymphocytes were analyzed, this became even more significant (p<0.001). The SI of activated CD8+ and CD8+CD11b + T lymphocytes correlated significantly (r= 0.86, p<0.02). On the other hand, both normal and patients’ suppressor T lymphocytes were activated equally by RMc antigens. In conclusion: i) Impaired AMLR reactivity of suppressor T lymphocytes was observed in GD and HT patients; ii) Activation of AITD suppressor T lymphocytes was significantly reduced by thyroid-specific antigens but not control antigen, and iii) There thus appears to be a defect in specific antigen induction of support T lymphocytes which may be important in the pathogenesis of AITD.

Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD)

Anti‐CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti‐human OKT3 MoAb (IgG1) alone or in combination with IL‐2. human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H‐TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG‐secreting cells and specific anti‐TPO/anti‐Tg‐secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimotos thyroiditis (HT). 40 Graves’ disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25–250 ng/ml, IL‐2 15 U/ml. Tg and TPO (I νg/ml). Then cultures were pulsed with 0.2 μCi 3H‐TdR/well and incorporation was measured after 18 h. IgG and anti‐TPO/Tg‐secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL‐2 alone increased these responses markedly, but equally in all groups. IL‐2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG‐secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.

Thyroid lymphomas in human thyroid tissue with autoimmune thyroid disease xenografted in severe combined immunodeficient mice

Malignant lymphoma of the thyroid (MLT) frequently arises in patients with a background of Hashimoto’s thyroiditis (HT); however, the mechanisms underlying this chain of events are unknown, and there has been no experimental model. Recently, the development of malignant lymphoma has been reported to occur in peripheral blood lymphocytes engrafted into severe combined immunodeficient (SCID) mice. We xenografted human thyroid tissue from patients with HT or Graves disease (GD) into SCID mice to determine the frequency and nature of MLT in these grafts. Human thyroid tissues ( 12 HT, 1 GD, and 15 from normal [paranodular] tissue) were xenografted into 72 mice (43 mice with HT or GD tissue) within 2 hours after human surgery. Human peripheral blood mononuclear cells (PBMC; 4 autologous HT, I allogeneic HT, and 1 allogeneic GD) were injected intraperitoneally into 6 of the latter 43 mice. In addition, 16 additional SCID mice received normal PBMC injections (alone). The mice were killed 6 to 20 weeks after xenografting. In 4 of 33 SCID mice bearing HT thyroid grafts (without addition of PBMC), MLT developed in the HT graft between 8 and 16 weeks after xenografting. In addition, one spleen of a mouse xenografted with GD tissue alone developed a human malignant lymphoma, although the xenografted thyroid in that mouse did not manifest lymphoma. One additional mouse xenografted with HT thyroid tissue and allogeneic HT PBMC developed malignant lymphoma of both the xenografted thyroid and the mouse spleen. In this mouse, the clonality of these lesions in the two organs was different: the thyroid showed restricted expression of immunoglobulin A (IgA) kappa, whereas the spleen exhibited lambda light chain restriction. One human MLT was removed from a SCID mouse, and equal halves were rexenografted into a nude mouse and another SCID mouse. Thyroid antibodies and IgG levels increased in the second SCID mouse, and the MLT survived; in the nude mouse, however, thyroid antibodies and IgG gradually disappeared, and the MLT regressed, virtually to normal. No MLTs were found in the normal human thyroid xenografts. In SCID mice receiving normal PBMC alone, lymphomas tended to develop when more than 35 x 106 cells were engrafted (a number similar to that of the lymphocytes in the HT xenografts); thus, the MLTs may reflect merely the numbers (and perhaps density) of human lymphocytes present in the xenografts. It is possible that committment of many of the HT-infiltrating lymphocytes to the thyroid might add an additional factor. However, whether this model will prove useful to study the possible transition of HT to MLT remains problematic.