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To assess the hypothesized imbalances of T-cell subsets in Hashimoto's disease (H.D.), we have evaluated human T-lymphocytes subpopulations defined by various markers (receptors: Fcγ or Fcμ; antigens recognized by monoclonal antibodies: OKT4 and OKT8, BT 2 9 , i.e., an anti-DR, MLR3, MLR4, and 4F2). In addition we employed a new monoclonal antibody named 5 9 , recently proved to define helper T cells in vitro. Using these reagents we have studied 11 patients with H.D. No imbalances of TG, TM, OKT4+, and OKT8+ cells were detected in these subjects. The increased levels of DR+, MLR4+, and 4F2+ T cells observed suggest an activation state of circulating T lymphocytes. In addition the high levels of 5 9 + T cells suggest an increased number of helper T lymphocytes in H.D. We also investigated a possible pathogenetical role of monocytes. The study of the influence of adherent cells of H.D. patients on T-cell cloning efficiency failed to show, with the procedures employed, any abnormal activity of these cells.

Recently it was reported that the peripheral blood and thyroid gland of patients with Hashimoto's thyroiditis contain activated (Ia+ and/or MLR4+) T cells and high levels of 5 9+ (“helper”) T lymphocytes. In normal individuals the 5 9 monoclonal antibody recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5 9+ and 5 9− T lymphocytes were isolated from patients with Hashimoto's disease, and the proliferative response induced by human thyroglobulin was investigated. The results show that the total thyroglobulin-induced lymphocyte DNA synthesis is confined to the 5 9+ T-cell fraction. Further subfractionation of 5 9+ into MLR4+ and MLR4− cells clearly indicates that no substantial differences exist in their proliferative capacities. Whether 5 9 , MLR4, and Ia antigens, all expressed on the thyroglobulin-responsive T-cell subset, are involved in thyroglobulin-induced cell proliferation, was also analyzed. Although both 5 9 and MLR4 monoclonal antibodies had no effect, complete inhibition of antigen-induced blastogenesis was observed upon addition of monoclonal antibodies ( D1 12 and BT2 9 ) directed to common determinants of Ia antigens. This inhibitory effect was also observed when T or non-T fractions were separately incubated with the monoclonal antibodies before culture. These results indicate that in humans, as in animals, the major histocompatibility complex may play a role in autoimmune thyroiditis. The data show that (a) the thyroglobulin-induced proliferative response is confined to a subset ( 5 9+ ) of T lymphocytes and (b) Ia antigens are involved in thyroglobulin-induced lymphocyte DNA synthesis in Hashimoto's disease.

Cyclosporin A (CsA) is an immunosuppressive drug that acts, at least in part, by blocking IL-2 release. Since IL-2-producing human T cells are heterogeneous with respect to their functional capabilities and surface phenotype, we investigated whether differences in sensitivity to CsA existed among different IL-2-producing T-cell clones. Preliminary dose/response experiments showed that 100 ng/ml CsA completely inhibited the PHA- or OKT3-induced IL-2 production by four representative T4+/T8- clones. On the other hand, this drug concentration had virtually no inhibitory effect on the proliferation of CTL-L murine indicator cells to exogeneous IL-2. Clones were derived directly from peripheral blood by applying a microculture system that allows clonal expansion of essentially all T cells: under these experimental conditions growing clones are therefore highly representative of the starting T-cell populations. Among clones so derived, 28 were selected according to their capability to release IL-2 upon PHA stimulation. Six of such clones displayed cytolytic activity in a PHA-dependent assay against P815 murine target cells. CsA (100 ng/ml) abrogated IL-2 production of all clones, including those displaying cytolytic activity and expressing the T4-/T8+ phenotype.

The majority of mature T lymphocytes express CD3-associated antigen receptor molecules (TCR) formed by alpha and beta chains. Recently, a minor subset has been identified that expresses a different CD3-associated heterodimer composed of gamma and delta chains. The TCR gamma/delta+ cell subset differs from conventional T cells for a number of phenotypic and functional characteristics. The simultaneous lack of both CD4 and CD8 antigens allows to greatly enrich TCR gamma/delta+ cells (by monoclonal antibodies and complement). Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which, in most instances, lyse tumor target cells. The formal proof has been provided that TCR gamma/delta+ cells are able to recognize antigens. Indeed they proliferated in response to allogeneic cells in mixed lymphocyte culture (MLC) and MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The use of different monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets which bound BB3 and delta-TCS-1 mAbs, respectively. The BB3-reactive TCR molecules were represented by C gamma 1-encoded disulfide-linked heterodimers, whereas delta-TCS-1 reacted with C gamma 2-encoded nondisulfide-linked molecules. Both BB3 and delta-TCS-1 mAb induced activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca2+ and lymphokine production or cytolytic activity). Analysis of the unfrequent delta-TCS-1+ clones which express surface CD8 molecules revealed that the "heavy" 55 kDa form of (C gamma 2-encoded) gamma chain is selectively expressed by this cell type. Analysis of the distribution of subsets expressing different TCR gamma/delta isotypes showed that the C gamma 1-encoded, BB3-reactive form is prevalent in the peripheral blood, but virtually absent in the thymus. In contrast, cells expressing the C gamma 2-encoded, delta-TCS-1 reactive form are relatively unfrequent in peripheral blood, but represent the majority of TCR gamma/delta+ thymocytes. In addition, upon culture in rIL-2, approximately half of the delta-TCS-1+ thymocytes expressed CD8 antigen, thus providing further evidence that major differences exist in the distribution of TCR gamma/delta+ subsets in thymus and in peripheral blood.

We have investigated at the clonal level the repertoire of intrathyroid and peripheral T lymphocytes in three patients with Graves' disease using a high efficiency cloning technique. Clonal efficiencies ranged from 10 to 31% for intrathyroid, and from 19 to 100% for peripheral T cells. In Graves' disease the phenotypic analysis showed similar percentages of CD3+ CD4+ CD8- and CD3+ CD4- CD8+ clones in thyroid infiltrates and peripheral blood. The functional evaluation showed similar or lower proportions of cytolytic clones in thyroid infiltrates with respect to peripheral blood. Furthermore, the proportions of intrathyroid and peripheral T-cell clones capable of releasing interleukin-2 and/or gamma-interferon in response to mitogen stimulation were similar. Finally, 44% of intrathyroid clones were neither cytolytic nor able to release IL-2 and gamma-interferon. These results are strikingly different from those obtained in Hashimoto's thyroiditis, where the large majority of intrathyroid T-cell clones are cytolytic and the proportions of clones able to release gamma-IFN are remarkably increased in thyroid infiltrates when compared to those obtained from peripheral blood. Taken together, these data suggest a different role for T lymphocytes in the pathogenesis of the two major human autoimmune thyroid diseases.