ÁREA DE ESTUDIO

Identification of the infiltrating cell phenotypes.

The phenotype of the MTg reactive cells has been shown to be Thy-1, Lyt-1 positive by the in vivo administration of appropriate antisera (Simon et al, 1985). Thy-1.2 and Lyt- 1.1 antibodies abolished the proliferative capacity of MTg immunised LNC to MTg and PPD (purified protein derivative); similarly antibodies to H-2A^ were able to block proliferation to MTg and PPD, antibodies to H-2A<^ did not inhibit proliferation. This correlates with the situation seen in the thyroid gland itself in that by day 13 following immunisation, Lyt-1 + cells predominate over Lyt-2+ cells but that by day 21 the level of Lyt-2+ cells gradually increases (Creemers et al, 1984). The kinetic analysis of thyroid infiltration was further characterised by Conaway et a l, (1989); following MTg immunisation in EPS z.v., the thyroid was examined immunohistochemically for the presence of CD4+ (CD4), Lyt-2+ (CD8) k light chain (Ig+ B cell) or F4/80+ (macrophage) cells. The results were consistent with the view that an initial influx of CD4+ cells into the thyroid is responsible for the recruitment of CD8+ cells and macrophages.

the rabbit (Nakamura & Weigle 1967), rat (Jankovic et al, 1969, Twarog et al, 1970) and guinea pig (McMaster & Lemer 1965). Lymph node cells and spleen cells from MTg immunised donors are capable of transferring the disease into syngeneic naive recipients following in vitro activation with the non-specific stimulator. Con A (Okayasu 1985). Cells that were cultured in vitro with medium alone or LPS failed to transfer disease following

Lp. injection of approximately 5x10^ lymph node or spleen cells. Most of the proliferating cells in either the lymph node or spleen cell cultures were shown to be Thy-1.2+ (T-cells) and it was also shown that depletion of Thy-1.2 or Lyt-1.1+(CD5) cells prior to transfer of

in vitro activated cells abrogated transfer of thyroiditis. Therefore the cells responsible for the transfer of EAT by definition are T-cells; that B-cell transfer was not responsible for the transfer was confirmed by the lack of reactivity of the recipient serum to normal thyroids. Okayasu et al, (1985) was also able to show that the development of BAT in recipient mice was very rapid and that thyroid infiltration appeared as early as day 8 following transfer and remained in the gland at least 21 days following transfer.

The relative contribution of cells in the thyroid infiltrate has been assessed by using monoclonal antibodies to the CD4 and CD8 epitopes on T-cells which cause depletion of these cells. Kong et al, (1989b) were able to show that removal of CD4+ T-cells by injection of depleting anti-CD4 on days minus one and six prior to MTg immunisation on days 0 and 7, followed by a single injection on day 13, abrogated development of disease as shown by infiltration and the development of an autoantibody response. In contrast, anti-CD8 had little effect on the induction of disease; all recipients had infiltrated thyroids but the level of infiltration was reduced, antibody titre was unaffected. Anti-CD4 was also inhibited an ongoing response when administered seven days after immunisation whereas anti-CD8 had no effect. On advanced EAT the effect was slightly different; anti CD4 was able reduce infiltration to a greater extent than CD8 alone, but a combination of anti-CD4 and anti-CD8 therapy was able to reduce the degree of infiltration in 50% of animals and clear the infiltration in the remaining animals by day 28 following immunisation. This indicated that the CD8 cells, although not important for the initiation of the disease, may be very important in the maintenance of the infiltration; the fact that depletion of CD8 cells did not enhance thyroiditis or antibody levels suggests that any cells that may be exhibiting a regulatory control in EAT are not CD8+ suppressor cells. The protective effect of these monoclonal antibodies was shown to be long lasting as at 70d post-antigenic challenge and 45d since the last anti-CD4 administration, the level of thyroid infiltration was very low; this effect was greatest when anti-CD4 and anti-CD8 were used in conjunction with each other. Autoantibody expression (anti-Tg) increases over this period, thus despite a continuous antigenic stimulus a chronic inflammation was not able to develop once the initial infiltration was cleared. This lack of infiltration was not due to a depletion of T-cells in the periphery as by day 70 a high enough level of T-cells were detected in the periphery for recruitment into the thyroid, this degree of recovery by day 70 is sufficient for EAT to be induced following peripheral deletion by monoclonal antibody therapy

(Kong et al, 1989a). The composition of the infiltrating cells in control mice was similar to that found by Conaway et al, (1989), from day 21, 30-35% of the infiltrate was comprised of T-cells, <6% B-cells and a small number of polymorphonuclear cells, remaining cells were shown to be macrophages.

This composition is repeated following the transfer of MTg of vitro activated spleen cells (Conway et al, 1990), T-cells accounted for approximately 35% of the infiltrating cells at day 7 following transfer, increasing to 56% by day 10, the ratio of CD4 and CD8 cells also changed in that the proportion of CD8 cells increased with time following transfer. As in the previous work (Conaway et al, 1989) the relative proportions of CD8 cells increased over time, these cells may indeed be responsible for the cytotoxic activity that has been reported in vitro (Creemers et al, 1983) and seen in T-cell clones from Hashimoto’s patients (Bagnasco et al, 1987, Mackenzie & Davies., 1987), once again it is postulated that CD8 cells are involved in the latter stages of the disease.