Y. M. Kong

T-cell regulation in autoimmune thyroiditis.

The mechanisms of induction and maintenance of self-tolerance are still problematical. Several basically different strategies have been proposed to explain the bodys apparent refusal to respond immunologically to its own constituents. 1. Self-reactive lymphocytes may be eliminated by clonal deletion during fetal life, by later clonal abortion or anergy, by receptor blockade or by other antigen-dependent methods of inducing and maintaining unresponsiveness. 2. Active immunoregulatory procedures, similar to those that normally limit immunological responses, may prevent autoimmunity by suppression. The leading candidates as active regulatory mechanisms are the generation of a distinct subpopulation of suppressor T lymphocytes, the production of specific anti-idiotypic antibodies or a combination thereof; that is, induction of antiiodiotypic cytotoxic T lymphocytes (Fig. 1). 3. Both clonal deletion and active suppression may be involved, providing double insurance against self-destruction by immunological means. The decisive question for distinguishing the several mechanisms of selfrecognition is whether lymphocytes that recognize unaltered self-antigens persist in normal animals. Clonal deletion theories in all of their various forms predict that they do not, whereas the concept of active regulation implies the persistence of functional, self-reactive lymhocytes in normal as well as disease states. To study this question we have been analyzing two different models of autoimmune thyroiditis. The first is experimental autoimmune thyroiditis

TOLERANCE TO THYROGLOBULIN BY ACTIVATING SUPPRESSOR MECHANISMS

The studies of Vladutiu and Rose demonstrated that the induction of autoimmune thyroiditis with emulsified mouse thyroglobulin ( MTg) in good responder mice is linked to the H-2 complex and requires T cells.l! Subsequent studies in H-2 congenic mice with the same B10 background,& confirmed the H-2 linkage of the gene controlling the immune response to thyroglobulin (the Zr-Tg gene). Similar segregation of good and poor responder strains on Balb/c and C3H backgrounds according :o the H-2 haplotype further solidified the major role of the MHC in autoimmune thyroiditis. Both the H-2 linkage and the need for T cells arc found despite the use with MTg of different adjuvantswhich have included complete Freunds adjuvant (CFA) ,l, * lipopolysaccharide (LPS) ,3 and polyadenylic-polyuridylic acid complex (poly A:U) .4 LPS and poly A:U are both soluble adjuvants and have enabled investigators to study autoimmune responses induced by aqueous, unmodified MTg. Self tolerance or the lack of autoimmunity in genetically susceptible, unimmunized mice cannot be attributed to the absence of antigen, inasmuch as thyroglobulin circulates in ng amounts in the natural Nor can it be attributed to the lack of autoreactive T cells, since we have recently shown that autoreactive T cells exist in good responder mice.fi The presence of MTgreactive T cells was revealed by repeated injections (16 times in four weeks) of syngeneic MTg in the absence of adjuvant. IgG production reached high levels in all animals and 50% showed infiltration of the thyroid by mono-

Graves’ hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA

Familial and twin studies in Caucasians have established that the MHC class II allele HLA‐DRB1*0301 (DR3) is a strong susceptibility gene in Graves’ hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II‐knockout NOD mice (H2Ag7–). DR3+g7– mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL‐4 or GM‐CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid‐stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid‐stimulating blocking Abs and/or TSH‐binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimotos thyroiditis model induced with human thyroglobulin.

REGULATION OF EXPERIMENTAL AUTOIMMUNE THYROIDITIS: INFLUENCE OF NON-H-2 GENES

Experiments were conducted to investigate the non‐H‐2 genetic effects on experimental autoimmune thyroiditis (EAT). Strains having C3H or BALB backgroud in general produced higher autoimmune responses to mouse thyroglobulin (MTg) than the B10 or A strains. Comparisons of C3H and B10 congenic strains carrying similar H‐2 haplotypes demonstrated that the C3H congenics had significantly higher MTg antibody titres and more severe thyroid damage, even when the strains carry the low responder H‐2 haplotypes. These observations show that non‐H‐2 gene(s) influences EAT, in addition to genes in the MHC.

Depletion of CD4+CD25+ regulatory T cells exacerbates sodium iodide‐induced experimental autoimmune thyroiditis in human leucocyte antigen DR3 (DRB1*0301) transgenic class II‐knock‐out non‐obese diabetic mice

Both genetic and environmental factors contribute to autoimmune disease development. Previously, we evaluated genetic factors in a humanized mouse model of Hashimotos thyroiditis (HT) by immunizing human leucocyte antigen DR3 (HLA‐DR3) and HLA‐DQ8 transgenic class II‐knock‐out non‐obese diabetic (NOD) mice. DR3+ mice were susceptible to experimental autoimmune thyroiditis (EAT) induction by both mouse thyroglobulin (mTg) and human (h) Tg, while DQ8+ mice were weakly susceptible only to hTg. As one environmental factor associated with HT and tested in non‐transgenic models is increased sodium iodide (NaI) intake, we examined the susceptibility of DR3+ and/or DQ8+ mice to NaI‐induced disease. Mice were treated for 8u2003weeks with NaI in the drinking water. At 0·05% NaI, 23% of DR3+, 0% of DQ8+ and 20% of DR3+DQ8+ mice had thyroid destruction. No spleen cell proliferation to mTg was observed. Most mice had undetectable anti‐mTg antibodies, but those with low antibody levels usually had thyroiditis. At 0·3% NaI, a higher percentage of DR3+ and DR3+DQ8+ mice developed destructive thyroiditis, but it was not statistically significant. However, when DR3+ mice had been depleted of CD4+CD25+ regulatory T cells prior to NaI treatment, destructive thyroiditis (68%) and serum anti‐mTg antibodies were exacerbated further. The presence of DQ8 molecules does not alter the susceptibility of DR3+DQ8+ mice to NaI‐induced thyroiditis, similar to earlier findings with mTg‐induced EAT. Susceptibility of DR3+ mice to NaI‐induced EAT, in both the presence and absence of regulatory T cells, demonstrates the usefulness of HLA class II transgenic mice in evaluating the roles of environmental factors and immune dysregulation in autoimmune thyroid disease.

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ‐specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6u2003×u2003CBA)F1 mice, recently shown to be susceptible to mouse TPO‐induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL‐12 or GM‐CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPOu2003+u2003GM‐CSF immunized group. However, after TPO DNA immunization of HLA‐DR3 transgenic class II‐deficient NOD mice, thyroiditis was present in 23% of mice injected with TPOu2003+u2003IL‐12 or GM‐CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti‐TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti‐TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA‐DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA‐DR4 or HLA‐DQ8, with differential susceptibility to Tg‐induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3+ mice, similar to our reports on Tg‐induced thyroiditis and thyrotropin receptor DNA‐induced Graves’ hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

The Essential Role of Circulating Thyroglobulin in Maintaining Dominance of Natural Regulatory T Cell Function to Prevent Autoimmune Thyroiditis

Several key findings from the late 1960s to mid-1970s regarding thyroid hormone metabolism and circulating thyroglobulin composition converged with studies pertaining to the role of T lymphocytes in autoimmune thyroiditis. These studies cemented the foundation for subsequent investigations into the existence and antigenic specificity of thymus-derived natural regulatory T cells (nTregs). These nTregs prevented the development of autoimmune thyroiditis, despite the ever-present genetic predisposition, autoantigen (thyroglobulin), and thyroglobulin-reactive T cells. Guided by the hypothalamus-pituitary-thyroid axis as a fixed set-point regulator in thyroid hormone metabolism, we used a murine model and compared at key junctures the capacity of circulating thyroglobulin level (raised by thyroid-stimulating hormone or exogenous thyroglobulin administration) to strengthen self-tolerance and resist autoimmune thyroiditis. The findings clearly demonstrated an essential role for raised circulating thyroglobulin levels in maintaining the dominance of nTreg function and inhibiting thyroid autoimmunity. Subsequent identification of thyroglobulin-specific nTregs as CD4(+)CD25(+)Foxp3(+) in the early 2000s enabled the examination of probable mechanisms of nTreg function. We observed that whenever nTreg function was perturbed by immunotherapeutic measures, opportunistic autoimmune disorders invariably surfaced. This review highlights the step-wise progression of applying insights from endocrinologic and immunologic studies to advance our understanding of the clonal balance between natural regulatory and autoreactive T cells. Moreover, we focus on how tilting the balance in favor of maintaining peripheral tolerance could be achieved. Thus, murine autoimmune thyroiditis has served as a unique model capable of closely simulating natural physiologic conditions.

Immunogenetic aspects of human thyroglobulin-reactive T cell lines and hybridomas

The in vitro proliferative response of T cells primed with human thyroglobulin (Tg) was compared in 11 independent haplotypes on B10 background. B10.K and B10.S mice were the most responsive, whereas, with the exception of B10.PL (H‐2u, all other B10 congenics were intermediate responders. The two best responders to in vitro challenge with human Tg, of the k and s haplotype, are the same as those showing H‐2‐linked susceptibility to induction of experimental autoimmune thyroiditis (EAT) with mouse Tg. Since shared epitopes on human and mouse Tgs have been shown to be thyroiditogenic by adoptive transfer studies in CBA (H‐2k) mice, the findings indicate that shared epitopes may be studied in appropriate (i.e. EAT‐susceptible) strains of mice. Therefore, we proceeded to develop methods to produce T‐cell lines and hybridomas to human Tg in B10.K and B10.S mice, test their cross‐reactivity to heterologous Tgs and their Ia restriction patterns. By using antigen‐presenting cells from recombinant strains, we identified restriction elements encoded by the I‐A subregion alone and a combinatorial molecule from the I‐AI/I‐E subregions.

Experimental Autoimmune Thyroiditis in the Mouse and Rat

Publisher Summary This chapter presents the condition of experimental autoimmune thyroiditis (EAT) in the mouse and rat. Murine EAT became a firmly established model for Hashimotos thyroiditis (HT), when susceptible and resistant strains were segregated according to the major histocompatibility complex, the mouse H-2, and particular attention was paid to preparing ingredients in complete Freunds adjuvant for emulsion with the thyroid antigen. There is no spontaneous model in murine AT except for the coexistence of mononuclear cell infiltration in endocrine organs, such as the salivary and thyroid glands, in the nonobese diabetic mouse, a recent model for insulin-dependent, Type I diabetes. Both an induced and a spontaneous model have been used to study rat EAT. For both induced mouse and rat EAT, the in vitro proliferative response to Tg correlates well with thyroiditis development but not severity. The measurements of serum thyroxine and thyroid stimulating hormone levels, on the other hand, are not reliable indicators of thyroid dysfunction.