Patients with Systemic Lupus Erythematosus Show Increased Levels and Defective Function of CD69⁺ T Regulatory Cells

Figure 1

Flow cytometry strategy for the analysis of peripheral blood CD69⁺ Treg lymphocytes. Mononuclear cells were isolated from blood samples and labeled with mAbs against CD4, CD25, CD69, and LAP (TGF-β). Then, cells were fixed, permeabilized, and stained for IL-10 and Foxp3. (a) Dot plots of FITC and APC isotype antibody controls. (b) Strategy for the analysis of CD69⁺ Treg cells. Lymphocytes were gated on the basis of their size and complexity (not shown) and then analyzed for the expression of CD4 and CD25, CD69, LAP and IL-10, and finally for Foxp3. Results were expressed as the percent of CD4⁺CD25^varCD69⁺LAP⁺IL-10⁺Foxp3⁻ cells, referred to mononuclear cells. (c) Strategy for the analysis of CD69⁺NKG2D⁺ Treg cells. Lymphocytes were gated on the basis of their size and complexity (not shown) and then analyzed for the expression of CD4 and NKG2D, CD69, LAP and IL-10, and finally for Foxp3. Results were expressed as the percent of CD4⁺NKG2D⁺CD69⁺LAP⁺IL-10⁺Foxp3⁻ cells, referred to total lymphocytes. Numbers correspond to the percent of cells in the highlighted gate, referred to the total of events in the previous gate. Data correspond to a blood sample from a representative patient with SLE. In all cases, at least 1 × 10⁶ events were analyzed, and gates were set up by using fluorescence minus one controls.

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