Circulating and thymic CD4 CD25 T regulatory cells in myasthenia gravis: effect of immunosuppressive treatment

Abstract

Accumulating evidence indicates an immunosuppressive role of the thymus-derived CD4+ T-cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age-matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non-neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg-cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg-cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg-cell development.

Figure 2

Circulating Treg-cell number (cells/μl) in healthy subjects (HS), not treated (NT), immunosuppressive (IS) therapy and IS therapy/thymectomized (Tx) patients. One patient was tested twice before and after the starting of IS therapy. Open symbols: <45 years old and no-thymoma subjects in healthy subjects (HS) group and IS/Tx group, respectively. In the latter group, all no-thymoma patients are <45 years old. The apparently lower number of Treg cells in IS therapy/Tx patients than in IS therapy patients did not reach significance. Treg cells were assessed as CD4⁺ CD25^high cells as described in Fig. 1. Bars denote mean value. P < 0·05 by one-way anova and Tukey's test.

Figure 1

Dual-colour dot plot displaying the coexpression of CD4 and CD25 in a representative sample. The electronic regions used to measure the CD4⁺ subsets CD25^–, CD25^int and CD25^high are shown.

Figure 3

The influence of thymectomy (Tx) on reducing the extent of increase of circulating Treg-cell level evoked by immunosuppressive (IS) therapy is transient. Each data-point refers to a single patient. x-axis, time from Tx; y-axis, number of circulating Treg cells (cells/μl). Shaded area identifies the normal range assessed in healthy subjects. Circulating Treg-cell level is higher than normal range before Tx because of the concomitant immunosuppressive therapy (see text and Fig. 2). P < 0·05 by paired anova and Tukey's test.

Figure 4

Scatter plots illustrating the coexpression of (a) CD45RO, (b) CD62L and (c) HLA-DR in healthy subjects (HS), not-treated (NT), immunosuppressive (IS) therapy and IS therapy/thymectomized (Tx) patients. Open symbols: <45 years old and no-thymoma subjects in HS group and IS/Tx group, respectively. In the latter group, all no-thymoma patients are <45 years old. Data are expressed as percentage positive cells within circulating Treg cells. Bars denote mean value. P < 0·05 by one-way anova and Tukey's test.

Figure 5

IS therapy and percentages of thymic Treg cells within the CD4SP population and of CD25⁺ cells within the CD8SP population. (a) Representative dual-colour dot plot of the flow cytometric analysis of thymocyte subsets defined by the expression of CD4 and CD8. Shown are the electronic regions used to measure the percentage of Treg and CD25⁺ cells within the CD4SP and CD8SP subsets, respectively. (b,c) dual-colour dot plot exemplifying the enumeration of Treg and CD25⁺ cells within the CD4SP and CD8SP subsets, respectively. (d,e) Scatter plots illustrating the proportion of Treg and CD8SP CD25⁺ cells, respectively. Bars denote mean value. Differences between not treated and IS therapy patients are not significant.