60–70 % of IFN-γ−/− NOD.H-2h4 mice given NaI-supplemented water develop a slow onset autoimmune thyroid disease, characterized by thyrocyte epithelial cell hyperplasia/ proliferation (TEC H/P). TEC H/P develops much earlier in CD28−/− mice and nearly 100 % (both sexes) have severe TEC H/P at 4 months of age. Without NaI supplementation, 50% of 5–6 month old CD28−/−IfFN-γ−/− mice develop severe TEC H/P, and 2–3 weeks of NaI is sufficient for optimal development of severe TEC H/P. Mice with severe TEC H/P are hypothyroid and normalization of serum thyroxine (T4) levels does not reduce TEC H/P. Activated CD4+ T cells are sufficient to transfer TEC H/P to SCID recipients. Thyroids of mice with TEC H/P have infiltrating T cells and expanded numbers of proliferating thyrocytes that highly express CD40. CD40 facilitates, but is not required for development of severe TEC H/P, as CD40−/−IFN-γ−/−CD28−/− mice develop severe TEC H/P. Accelerated development of TEC H/P in IFN-γ−/− CD28−/− mice is a result of reduced Treg numbers as CD28−/− mice have significantly fewer Tregs, and transfer of CD28-positive Tregs inhibits TEC H/P. Essentially all female IFN-γ−/− CD28−/−NOD.H-2h4 mice have substantial lymphocytic infiltration of salivary glands and reduced salivary flow by 6 months of age, thereby providing an excellent new model of autoimmune exocrinopathy of the salivary gland. This is one of very few models where autoimmune thyroid disease and hypothyroidism develop in most mice by 4 months of age. This model will be useful for studying the effects of hypothyroidism on multiple organ systems.

CD40 is expressed on cells of the immune system and in some tissues that are targets for autoimmune-mediated damage. It is not known if CD40 expression in target tissues plays a role in the pathology of autoimmune diseases. This study shows that agonistic anti-CD40 induces strong and sustained proliferation of thyroid epithelial cells (TEC) or thyrocytes in IFN-γ−/− autoimmune-prone NOD and NOD.H-2h4 mice. TEC proliferation is accompanied by greatly increased expression of CD40 on TEC, development of fibrosis and hypothyroidism, and increased expression of proinflammatory molecules in thyroids. Bone marrow chimera experiments indicate that TEC expression of CD40 is required for anti-CD40-induced TEC proliferation, but lymphoid cells do not have to express CD40. TEC proliferation is reduced in WT mice given anti-CD40, presumably because they produce IFN-γ, which inhibits TEC proliferation. CD40 also increases on TEC during development of an autoimmune thyroid disease characterized by TEC hyperproliferation that develops spontaneously in IFN-γ−/− NOD.H-2h4 mice (TEC H/P). TEC H/P development is accelerated in mice given agonistic anti-CD40. These studies provide new information regarding the role of target tissue expression of CD40 in development of autoimmunity and suggest that use of agonistic anti-CD40 for tumor therapy could result in autoimmune disease.

IFN-γ−/− NOD.H-2h4 mice develop a spontaneous autoimmune thyroid disease, thyroid epithelial cell hyperplasia and proliferation (TEC H/P) when given NaI in their water for 7+ mo. TEC H/P can be transferred to IFN-γ−/− SCID mice by splenocytes from mice with severe (4–5+) disease, and transfer of TEC H/P is improved when splenocytes are cultured prior to transfer. Older (9+ mo) IFN-γ−/− NOD.H-2h4 mice have elevated numbers of FoxP3+ T reg cells, up to 2 fold greater than younger (2 mo) mice. During culture, the number of T reg decreases and this allows the improved transfer of TEC H/P. Co-culture with IL-2 prior to transfer prevents the decrease of T reg and improves their in vitro suppressive ability resulting in reduced TEC H/P in recipient mice. Therefore, culturing splenocytes improves transfer of TEC H/P by reducing the number of T reg and IL-2 inhibits transfer by preserving T reg number and function.

60-70 % of IFN-γ-/- NOD.H-2h4 mice given NaI in their drinking water for 6-7 mo develop TEC H/P, a CD8-mediated thyroid specific autoimmune disease characterized by uncontrolled TEC proliferation, lymphocytic infiltrates, autoantibody production, and fibrosis, resulting in destruction of TEC and loss of thyroid function. CD28-/- IFN-γ-/- NOD.H-2h4 mice develop very severe TEC H/P at an accelerated rate with nearly 100 % of CD28-/- IFN-γ-/- NOD.H-2h4 mice developing severe TEC H/P 5-8 weeks after exposure to NaI. Thyroids have extensive fibrosis and severe TEC H/P correlates with low serum T4 levels. CD8+ T cell numbers are increased in thyroid, LN, and spleen, and CD8 cells highly express PD-1 and NKG2D. CD28-/- IFN-γ-/- NOD.H-2h4 mice have extensive lymphocyte infiltrates in salivary glands and pancreatic islets, and a few develop diabetes at 4-6 mo of age. CD28-/- IFN-γ-/- NOD.H-2h4 mice with TEC H/P have reduced numbers of peripheral FoxP3+ T reg compared to IFN-γ-/- NOD.H-2h4 mice with TEC H/P. In mice with severe TEC H/P, thyroids from CD28-/- IFN-γ-/- NOD.H-2h4 mice have reduced numbers of FoxP3+ T reg compared to IFN-γ-/- NOD.H-2h4 mice. The accelerated development of TEC H/P in CD28-/- IFN-γ-/- mice may be a result of reduced T reg number or function as preliminary data indicates that development of TEC H/P in CD28-/- IFN-γ-/- NOD.H-2h4 mice is inhibited by transfer of FoxP3+ T reg from CD28 sufficient IFN-γ-/- NOD.H-2h4 mice.

Most IFN-γ-/- NOD.H-2h4 mice given 0.05% NaI in their water develop an autoimmune thyroid disease characterized by infiltrating lymphocytes, expression of TNFα and TGFβ, production of anti-thyroglobulin antibodies, and uncontrolled proliferation of TEC, resulting in destruction of the gland and loss of function. IFN-γ-/- NOD.H-2h4 mice have elevated FoxP3+ CD4+ T reg in cervical LN and spleen (25-40%, and 25-35% of CD4+ T cells) dependent on age, presence of severe disease, and absence of IFN-γ. T reg cells from mice with severe TEC H/P exhibit a suppressive phenotype with expression of high levels of FoxP3, CD25, and TNFαRII (p75), and they suppress anti-CD3 mediated proliferation of T cells in vitro. Splenocytes from mice with severe TEC H/P transfer severe TEC H/P to IFN-γ-/- SCID recipients following 72 hr culture. During culture, percentages of T reg decrease, allowing transfer of severe TEC H/P. However, in recipient mice, percentages of T reg increase, and despite increased numbers of T reg in LN and spleen, the mice develop severe TEC H/P. This data suggests there may be a defect in the ability of these T reg to suppress TEC H/P, either because of an intrinsic defect in the T reg or because effector cells are resistant to suppression. The defect is reversible as addition of IL-2 (100 U/ml) to the culture increases T reg percentages, and inhibits transfer of TEC H/P, restoring the ability of T reg to suppress TEC H/P in recipient SCID mice.

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I would also like to acknowledge the help of Dr. David Brand for his insight and support during my research. I would also like to thank the members of my graduate committee Elizabeth Fitzpatrick, Terrence Geiger, Tony Marion, and Marko Radic for their guidance. Finally, I would like to thank my mentor Dr. Edward Rosloniec for his support, guidance, and understanding throughout the development of my research.