SUMMARYWe have studied the ability of lymphocytes from the blood, thyroid and lymph nodcsof patients with autoimmune thyroid disease (AITD) to produce aiitoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were delectable in plasma from SCiD mice 7 days after transfer of 15-25 x 10'' cells/moiise and ihc highest levels were recorded 2-3 weeks later. In contrast. Tg and/or TPO antibodies were undetcctable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the mosi antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounls of Tg and/or TPO tmtibody detected were in accordance with the ability of Ihyroid and lymph node lymphocytes to secrete these autoanlibodies spontaneously in culture (indicating ihc presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tgand/or TPO antibodies in ctilture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgGl. IgG2and !gG4and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1. 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from paiients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of hutiian antibodies to Tg and TPO-

SUMMARYWe investigated the ability of T cells from patients with Hashimoto's thyroiditis and with Graves' disease as well as control donors to proliferate in response to thyroid peroxidase (TPO) and thyroglobulin using (i) lymphoid cells from different lymphoid organs; (ii) unfractionated or CD8-depleted lymphoid suspensions or T cells + autologous low density cells (LDC); (iii) 200-/jl well cultures and 20-/

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 pg per lo6 cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity ( 2 . 5~1 0~ molar-') and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of '*'I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2'34. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound '*'I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies. with only one type of autoantibody reactive with the same epitope as 2C4.

A panel of human monoclonal thyroglobulin (Tg) autoantibodies (TgAAb) has been used to analyze autoantigenic determinants on human Tg and to investigate the relationship between variable (V) region gene sequences and epitope specificity. Two monoclonal TgAAb bound to the same (or closely related) epitope on Tg, and these were defined as type I TgAAb. Three other monoclonals bound to a different site and were defined as type II TgAAb. Inhibition studies with mixtures of type I and type II monoclonal TgAAb (Fab)2 preparations indicated that a mixture of the (Fab)2s almost completely inhibited (> 75%) labeled Tg binding to intact TgAAb in the sera of apparently healthy blood donors and patients with autoimmune thyroid disease (AITD). Type I TgAAb predominated in apparently healthy blood donors' sera, whereas type II TgAAb predominated in AITD sera. Analysis of V region gene sequences of the TgAAb indicated that a range of light chain and heavy chain genes from different gene families was used. Furthermore, the same germline genes that are used by TgAAb are also well represented in the genes coding for other self- and nonself-reactive antibodies. No homology in terms of light chain and heavy chain gene families, germline gene usage, or complementarity determining region sequences was observed in TgAAb directed to the same or closely related epitopes. Our studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of TgAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different. TgAAb derived from different germline genes and with different complementarity determining region sequences can display similar epitope specificity, and this indicates that AAb directed to the same or a closely related epitope show considerable heterogeneity at the molecular level.

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.