Agonistic Anti-CD40 Induces Thyrocyte Proliferation and Promotes Thyroid Autoimmunity by Increasing CD40 Expression on Thyroid Epithelial Cells

Abstract

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.

NOD.H-2h4 Mice

“…Agonistic anti-CD40 induces proliferation of thyrocytes in IFN-γ2/2 NOD.H-2h4 mice promotes development of severe TEC H/P As mentioned above, administration of agonistic anti-CD40 to IFN-γ À/ÀCD4 À/À NOD.H-2h4 mice results in increased TEC H/P severity scores and provides a signal that enables their splenocytes to transfer severe TEC H/P to SCID recipients . During the course of these experiments, we unexpectedly observed that SCID mice, whether or not they are given donor splenocytes, have very severe thyrocyte proliferation 7 days after injection of anti-CD40 (Kayes, Fang, et al, 2013). Further studies established that thyroids of NOD and NOD.H-2h4 mice constitutively express CD40 mRNA and very low levels of CD40 protein.…”

“…In contrast to the relatively minimal effects of agonistic anti-CD40 on SAT development, agonistic anti-CD40 profoundly promotes development of TEC H/P and induces sustained thyrocyte proliferation in IFN-γ À/À NOD.H-2h4 mice (Section 3; Kayes, Fang, et al, 2013). NOD.H-2h4 thyrocytes also express CD40L (Kayes, Fang, et al, 2013). The effects of anti-CD40 on thyrocytes of WT and IFN-γ À/À NOD.H-2h4 mice are apparently independent of expression of CD40 ligand in the thyroid, because agonistic anti-CD40 induces the same changes in thyroids of WT and CD154À/À NOD.H-2h4 mice (our unpublished results).…”

“…Further studies established that thyroids of NOD and NOD.H-2h4 mice constitutively express CD40 mRNA and very low levels of CD40 protein. When they are given agonistic anti-CD40, it cross-links CD40 expressed on thyrocytes, and CD40 protein expression markedly increases, resulting in thyrocyte proliferation (Kayes, Fang, et al, 2013). Of particular interest, when agonistic anti-CD40 is administered to WT NOD.H-2h4 or WT NOD mice, CD40 protein expression increases and thyrocytes show proliferative changes that subside 10-14 days later, after which the thyroids appear normal (Kayes, Fang, et al, 2013).…”

“…Because human and mouse serum T4 are cross-reactive, ELISA assays developed for measuring serum T4 in human blood can be used to measure serum T4 in mice (Chen, Wei, Sharp, & Braley-Mullen, 2005). Using kits provided by Biotecx in our early studies (Chen et al, 2005;Yu et al, 2006a) and more recently kits made by Leinco, Inc. (Kayes, Fang, et al, 2013;, values for NOD.H-2h4 normal mouse serum T4 range from 4 to 8 μg/dL of serum, and we consider any values >3 to be normal. WT NOD.H-2h4 mice with SAT almost always have serum T4 values in the normal range, presumably because thyroids with severe SAT (4 +) always have some remaining normal thyroid follicles (Ellis, Hong, et al, 2013).…”

“…Thyroids of NOD.H-2h4 mice express CD40, and CD40 protein is transiently increased on thyrocytes during development of SAT and following administration of agonistic anti-CD40 (Kayes, Fang, et al, 2013). Although agonistic anti-CD40 induces proliferation of thyrocytes in vivo and in vitro and CD40 expression is increased on thyrocytes, these effects are not sustained when IFN-γ is present (Kayes, Fang, et al, 2013), and agonistic anti-CD40 does not promote development of SAT or thyroglobulin antibodies in WT NOD.H-2h4 mice (our unpublished results). In contrast to the relatively minimal effects of agonistic anti-CD40 on SAT development, agonistic anti-CD40 profoundly promotes development of TEC H/P and induces sustained thyrocyte proliferation in IFN-γ À/À NOD.H-2h4 mice (Section 3; Kayes, Fang, et al, 2013).…”