Follicular B Cells in Thyroids of Mice with Spontaneous Autoimmune Thyroiditis Contribute to Disease Pathogenesis and Are Targets of Anti-CD20 Antibody Therapy

Abstract

B cells are required for development of spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice where they function as important antigen presenting cells (APCs) for activation of CD4+ T cells. Depletion of B cells using anti-CD20 effectively inhibits SAT development. The goals of this study were to characterize the B cells that migrate to thyroids in SAT, and determine if anti-CD20 effectively targets those B cells in mice with established SAT. The results showed that most thyroid infiltrating B cells in mice with SAT are follicular (FO) B cells. Expression of CD80, CD86 and CD40 was significantly increased on follicular (FO), but not marginal zone (MZ) splenic B cells after SAT development. Thyroid-infiltrating and peripheral blood B cells had lower expresion of CD20 and CD24 compared to splenic and LN FO B cells. Despite reduced CD20 expression, anti-CD20 depleted most B cells in thyroids of mice with established SAT within 3 days. B cell depletion in thyroids of mice given anti-CD20 was more complete and longer lasting than in spleen and LN and was comparable to that in blood. Circulation of B cells was required for effective and rapid removal of B cells in thyroids since preventing lymphocyte egress by administration of FTY20 abrogated the effects of anti-CD20 on thyroid B cells. Therefore the FO subset of B cells preferentially contributes to SAT development and persistence, and anti-CD20 targeting of FO B cells effectively eliminates B cells in the target organ even though thyroid B cells have decreased CD20 expression.

NOD.H-2h4 Mice

“…These results suggest that Th1 and Th17 cells, or CD4+ T cells that produce both Th1 and Th17 cytokines, may both have a pathogenic role in SAT. In our colony of NOD.H-2h4 mice, IFN-γ is absolutely essential for SAT development, and CD4+ T cells expressing IFN-γ and TNF-α are much more prevalent in thyroid infiltrates of mice with SAT than are IL-17-producing CD4 + T cells (Hong & Braley-Mullen, 2014;our unpublished results). Others also showed that IFN-γ-and IL-12-producing cells are present in thyroids of mice with SAT and are increased in areas where there are accumulations of inflammatory cells (Bonita et al, 2003).…”

“…For example, when SAT develops following Treg depletion in NP-Tg NOD.H-2h4 mice, there are many B cells in the thyroid infiltrates, but all B cells are NP-specific and do not secrete Ig (Yu, Maiti, et al, 2006), suggesting that their function in the thyroid is to present antigen and not to secrete autoantibody. Splenic B cells in NOD.H-2h4 mice include marginal zone (MZ), follicular (FO), and T2 subsets based on differential expression of CD21 and CD23 (Hong & Braley-Mullen, 2014;Yu, Dunn, et al, 2008), and essentially all B cells that traffic to the thyroid are FO B cells (Hong & Braley-Mullen, 2014). We believe that FO B cells are the primary effector and antigen-presenting cells in SAT, based on their upregulation of MHC class II during disease development, their presence in thyroid infiltrates and the fact that anti-CD20, which has relatively little effect on MZ B cells in adult NOD.H-2h4 mice, but depletes most FO B cells (Yu, Dunn, et al, 2008;Yu et al, 2012), can be administered to mice with established SAT, depleting B cells in the thyroid and greatly reducing SAT severity scores (Hong & Braley-Mullen, 2014).…”

“…The NOD.H-2h4 mouse provides an especially useful model for studying the role of B cells in development of spontaneous autoimmune diseases, since both SAT and SS (see below) require B cells for their development and the target organs of both of these spontaneous autoimmune diseases become infiltrated by B cells that form clusters and tertiary lymphoid organ-like structures when autoimmunity develops (Hong & Braley-Mullen, 2014;Karnell, Mahmoud, Herbst, & Ettinger, 2014;Yu, Dunn, et al, 2008;Yu et al, 2001). Another feature of this mouse model that is useful for mechanistic studies of the role of B cells in autoimmunity is that SAT is a chronic disease that is maintained for the life of the animal with essentially little change after lesions develop.…”

“…CD4 + T cells are the first lymphocytes to migrate to the thyroid, and can be found in thyroid infiltrates as early as 2 weeks after mice are given NaI in their drinking water (Bonita et al, 2003;Yu et al, 2001). As SAT severity increases, thyroid infiltrates have characteristic clusters of CD4 + T cells and B cells, which often progress to form germinal center-like structures, e.g., tertiary lymphoid organs (Hong & Braley-Mullen, 2014;Yu et al, 2001). CD4 + T cells in thyroids of mice with SAT produce proinflammatory cytokines such as IFN-γ and TNF-α (see below).…”

“…CD8+ T cells are less numerous and are scattered throughout the thyroid. Germinal center-like structures, characteristic of tertiary lymphoid organs, are also common in thyroids of mice with SAT (Hong & Braley-Mullen, 2014;Yu et al, 2001).…”