Activation of thyroid antigen-reactive B cells in recent onset autoimmune thyroid disease patients

Abstract

Autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), is the most common autoimmune disorder in the United States, affecting over 20 million people. At the time of diagnosis, both HD and GD are characterized by the accumulation of B and T lymphocytes in the thyroid gland and production of autoantibodies targeting the thyroid, indicating that a breach in tolerance of autoreactive lymphocytes has occurred. However, few studies have sought to understand the underlying pathogenesis of AITD that ultimately leads to production of autoantibodies and loss of thyroid function. In this study, we analyzed the phenotype of thyroid antigen-reactive B cells in the peripheral blood of recent onset and long standing AITD patients. We found that in recent onset patients thyroid antigen-reactive B cells in blood no longer appear anergic, rather they express CD86, a marker of activation. This likely reflects activation of these cells leading to their production of autoantibodies. Hence, this study reports the early loss of anergy in thyroid antigen-reactive B cells, an event that contributes to development of AITD.

Keywords: Anergy; Autoimmune thyroid disease; B lymphocytes; Flow cytometry.

Figure 1. Detection and enrichment of Tg/TPO-binding B cells is specific

A. Diagram of adsorbent used to identify and isolate Tg or TPO-reactive B cells. B. Representative cytograms of Tg/TPO-reactive B cells enriched from the blood of a healthy control when antigen is added or when it is omitted. Cells that bound to the adsorbent and eluted from the magnet are termed enriched, non-binders termed depleted, and un-enriched termed total. Percentages shown are the percent of B cells that are Alexa647 positive. C. Number of Tg and TPO-binding B cells recovered per 10⁷ PBMCS in AITD subjects compared to healthy controls. Each line represents a AITD and healthy control that were analyzed on the same day.

Figure 2. Early onset AITD subjects have a loss of anergic Tg and TPO-bindig B cells in their peripheral blood

A. Representative gating strategy for identification and analysis of Tg/TPO-reactive B cells using Tg binding cells as an example. IgM vs IgD gates are drawn based on staining of T (CD19−) cells, which are IgM-IgD−. B. Percentage of anergic Tg and TPO-reactive B_ND cells in early onset (E/O) AITD, long standing (L/S) AITD, and healthy controls (H/C). C. Percentage of total (Tg/TPO−) anergic B_ND cells in each subject group.

Figure 3. Loss of anergic Tg-reactive B cells is correlated with autoantibody production and activation

A. Anti-Tg and anti-TPO titers for each early onset AITD subject is plotted with their respective percentage of anergic Tg/TPO-reactive B_ND cells to demonstrate correlation between the two. Anti-Tg titers had a significant inverse correlation with % Tg-binding B_ND cells; p=0.015, Spearman’s correlation test). B. Fold increase of geometric mean florescence intensity (gMFI) of CD86 on Tg-binding B cells from AITD E/O and AITD L/S patients over healthy controls. Representative histogram of CD86 expression from Tg-binding B cells from AITD E/O (black line) and H/C (gray line). C. Fold increase of CD86 gMFI on TPO-binding B cells from AITD E/O and AITD L/S patients over healthy controls. D. Fold increase in CD86 gMFI on Tg-reactive B_ND, mature naïve (MN) (CD27−), and memory (CD27+) B cell subpopulations from early onset AITD subjects compared to healthy controls. CD86 gMFI on AITD subjects was normalized to healthy controls in order to account for changes in voltage settings that occurred during the course of the sample collections. Statistical significance determined by paired Student’s t tests.