Circulating mononuclear cells from euthyroid patients with thyroid-associated ophthalmopathy exhibit characteristic phenotypes

Abstract

Thyroid-associated ophthalmopathy (TAO) is a common yet poorly understood component of Graves' disease involving inflammation, congestion and soft tissue remodelling of the orbit. Unlike most autoimmune disorders, TAO has variable severity but follows a predictable course and is usually self-limited. The objective of this study was to investigate the phenotypic profile of peripheral blood mononuclear cells in euthyroid patients with TAO. The study was a prospective, consecutive analysis of the peripheral blood mononuclear cell phenotype in patients with TAO and normal controls. We demonstrate that the fraction of T cells expressing CD69, CD25 or CXCR4 is significantly greater in patients with TAO compared to control donors. In addition, the fraction of CD19(+) CD25(+) B cells is significantly greater. We did not find differences between the two groups of subjects in monocytes expressing these markers. There is a phenotypic shift in peripheral blood lymphocytes associated with TAO that appears durable and persists beyond the hyperthyroid phase of Graves' disease. These changes may support the immune reaction provoking orbital disease development.

Fig. 1

A larger fraction of T cells from patients with thyroid-associated ophthalmopathy (TAO) display CD69 than do those from control donors. Representative dot plots demonstrate expression of CD69 using multi-parameter flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with fluorochrome-labelled antibodies against CD3, CD19, CD14 and CD69. Analysis of CD3 positive cells is demonstrated using a live cell gate. Isotype matched control antibodies were used to establish gating parameters such that positive cells constituted less than 0·5% of the quadrant gate for the isotype. The fraction of T cells expressing CD69 is smaller in control donors (7%) compared to TAO (41%).

Fig. 2

Increased fraction of T cells from patients with thyroid-associated ophthalmopathy (TAO) express CD69. Display of CD69 by CD3⁺ T cells and CD14⁺ monocytes was examined by multi-parameter flow cytometry. 30 ± 7% CD3⁺ T cells from patients with TAO (n = 20, dark bar) express CD69 compared to 14 ± 3% from control donors (n = 10, shaded bar: P < 0·02). A similar fraction of monocytes from TAO patients (81 ± 5%, n = 22) and from control donors (69 ± 5%, n = 12) express CD69.

Fig. 3

A larger fraction of T cells from patients with thyroid-associated ophthalmopathy (TAO) display CD25 expression. Representative histograms demonstrate expression of CD25 using multiparameter flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with fluorochrome-labelled anti-CD3, CD19, CD14 and CD69 antibodies. A live cell gate was performed and analysis of CD3⁺ cells is demonstrated. Isotype matched labelled control is shown as an open tracing while CD25 expression is represented by the solid histogram. The fraction of T cells expressing CD25 in TAO T cells (39%) is greater than that from control donors (12%). The fraction of control B cells expressing CD25 is smaller (8%), compared to those from patients with TAO (38%).

Fig. 4

(a) Increased fraction of T cells from patients with thyroid-associated ophthalmopathy (TAO) express CD25. Expression of CD25 by CD3⁺ T cells, CD19⁺ B cells and CD14⁺ monocytes from control donors and those with TAO was examined by multi-parameter flow cytometry. With regard to T cells, 38 ± 4% from donors with TAO (dark bar, n = 19) express CD25 compared to 19 ± 4% of those from controls (shaded bar, n = 10: P < 0·001). In the case of B cells, 31 ± 6% from TAO (dark bar, n = 11) express CD25 compared to 11 ± 4% of those from controls (shaded bar, n = 8: P < 0·02). Similar fractions of monocytes from patients with TAO (10 ± 1%) and from controls (9 ± 2%) express CD25. (b) An increased fraction of CD3⁺ CD4⁺ T cells from patients with TAO express the antigen, but this population does not express forkhead box P3 (Foxp3). CD3⁺ CD4⁺ and CD25⁺ cells are gated as shown (density plot) and minimally express Foxp3 (solid histogram, < 5%).

Fig. 5

A greater fraction of T cells from patients with thyroid-associated ophthalmopathy (TAO) display CXCR4 expression. A live cell gate was performed and analysis of CD3⁺ cells is demonstrated. Isotype-matched labelled control is shown as an open histogram while CXCR4 expression is represented by the solid histogram. CXCR4 is greater in TAO (86%) versus controls (25%).

Fig. 6

Increased fraction of T cells from patients with TAO express CXCR4. Expression of CXCR4 by CD3⁺ T cells was determined by multi-parameter flow cytometry. TAO, 86 ± 6% (dark bar, n = 15) versus control, 55 ± 10% (shaded bar, n = 9: P < 0·02).