The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was −/− mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was −/− mice as compared with wild-type controls. Moreover analysis of was −/− iNKT cell maturation revealed a complete arrest at the CD44 + NK1.1 − intermediate stage. Notably, generation of BM chimeras demonstrated a was −/− iNKT cell-autonomous developmental defect. was −/− iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon γ upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology.

A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4 + CD25 + FOXP3 + natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS −/− mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS −/− nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS −/− nTreg cells failed to proliferate and to produce transforming growth factor β upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS −/− nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS −/− nTreg cells showed reduced in vitro suppressor activity on both WT and WAS −/− effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4 + effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.

The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4 + CD45RO/RB high T cells but not in CD8 + T cells. In addition, CD4 + T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-γ production by TT-specific effector T cells by an interleukin-10–dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8 + T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-γ production by MP.58-66–specific effector CD8 + T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4 + and CD8 + regulatory T cells.

T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4 + T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β. The relationship between CD25 + CD4 + T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25 + CD4 + T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25 − CD4 + T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25 + CD4 + T cells. Cloned human CD25 + CD4 + T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25 + CD4 + T cell clones with suppressive function produce IL-10, but all produce TGF-β. Suppression mediated by CD25 + CD4 + T cell clones is partially dependent on TGF-β, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25 + CD4 + T cells are distinct from IL-10–producing Tr1 cells.

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4 + Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25 + CD4 + Tr cells in humans has not been reported. Here we show that human CD25 + CD4 + Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25 + CD4 + Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25 + CD4 + Tr cells strongly inhibited the proliferative responses of both naive and memory CD4 + T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25 + CD4 + Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25 + CD4 + Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice ( xid ). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-x L . Ectopic expression of Bcl-x L in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-x L .