Impaired in vitro regulatory T cell function associated with Wiskott-Aldrich syndrome

Abstract

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency characterized by the contradictory coexistence of impaired T-cell function and exaggerated T-cell-mediated pathology, including autoimmunity and eczema. WAS protein (WASp)-deficient mice are also immunodeficient and can develop autoimmune disease. Since defects in regulatory T-cells (Treg) are associated with autoimmunity, we examined the presence and function of these cells in WAS patients and WASp-deficient mice. We found that CD4(+)CD25(+)FOXP3(+) Treg cells can develop in the absence of WASp expression. However, Treg cells both from WASp-deficient mice and from four out of five WAS patients studied showed impaired in vitro suppressor function. In WASp-deficient mice, this defect could be partially rescued by pre-activation with IL-2, suggesting that inadequate cell activation may play a role in WASp-deficient Treg dysfunction. These findings may provide insights into the complex pathophysiology and paradoxical phenotypes of WAS and suggest new therapeutic modalities for autoimmunity in these patients.

Figure 1. Impaired suppression function of CD4⁺CD25⁺ Treg cells from WASp-deficient mice

(A) WASp-deficient mice have similar numbers of CD4⁺Foxp3⁺ regulatory T cells, but these cells express lower levels of CD25. Flow cytometry of splenic cells are shown. (B) Freshly isolated WASp-deficient CD4⁺CD25⁺ Treg cells poorly suppress the proliferation of WT CD4⁺CD25^- target cells. WT CD4⁺CD25^- target cells (50,000/well) were plated with accessory cells and co-cultured in triplicate with the indicated numbers of freshly isolated WT or WASp^-/- CD4⁺CD25⁺ Treg cells in the presence of anti-CD3. Treg sample refers to CD4⁺CD25⁺ T cells cultured with accessory cells in the presence of anti-CD3. Cell proliferation was assessed at the end of a 3-day culture period by [³H]-thymidine incorporation and is presented as the mean ± SEM of triplicate cultures. Data is representative of three independent experiments. (C) [³H]-thymidine incorporation by WASp-deficient and WT Treg cells following 3-day pre-activation (stimulation with anti-CD3 + IL-2). (D) Pre-activated WASp-deficient Treg cells show reduced suppression activity against the proliferation of WT CD4⁺CD25^- target cells. Pre-activated CD4⁺CD25⁺ Treg cells were plated and cultured as in Figure 2B. Cell proliferation was assessed by [³H]-thymidine incorporation after 3-days of culture, as above. Data is the mean of triplicate cultures ± SEM and is representative of two independent experiments. Average proliferation of CD4⁺CD25⁺ was less than 220 cpm.

Figure 2. Phenotype of CD4⁺CD25^hi Treg cells in healthy controls and WAS patients

(A) Prevalence of CD4⁺FOXP3⁺ or CD25⁺FOXP3⁺ cells in from healthy control (Ctrl) and WAS CD14^-CD32^- lymphocytes. Quadrant markers were set based on staining with the isotype matched control mAbs and percentage of cells in each quadrant are indicated. Data shown are representative of 5 different experiments. (B) FOXP3 expression in healthy control and WAS CD4⁺CD25^hi Tregs were characterized by intracellular staining with anti-human FOXP3-APC antibody. Open histograms represent isotype controls; solid histograms represent FOXP3-specific staining.

Figure 3. Treg suppression assays after stimulation with anti-CD3 plus anti-CD28 antibodies

(A) Treg suppression activity on autologous target cells. Control (Ctrl) Treg cells and Ctrl target cells were plated alone or co-cultured in a 1:1 ratio and stimulated with anti-CD3 + anti-CD28 in the presence of accessory cells. Similar assays were performed with WAS Treg and WAS target cells. Cell proliferation assessed as incorporation of [³H]-thymidine at the end of 5 days of culture is shown as counts per minute (CPM). Data are the mean ± SD of 5 independent experiments using samples from 5 different healthy controls and the 5 WAS patients described in Supplementary Table 1. (B) Ctrl Treg cells, WAS Treg cells, and Ctrl target cells were plated alone and stimulated with anti-CD3 + anti-CD28 in the presence of accessory cells. Ctrl Treg and WAS Treg were also co-cultured with Ctrl target cells in a 1:1 ratio and stimulated as above. Cell proliferation was assessed as incorporation of [³H]-thymidine at the end of 5 days of culture and is shown as counts per minute (CPM). Data shown are mean values of duplicate cultures.