dependent activation of human eosinophils induced a-defensin and ECP release and decreased mycobacteria growth [24]. Furthermore, expulsion of mitochondrial DNA by eosinophils has been shown to contribute to innate immune defences against bacteria [27]. Finally, eosinophil-tumor cell interactions and IL-4- dependent tumoricidal activity of eosinophils have been reported [28,29]. Thus eosinophils appear functionally located at the interface between innate and adaptive immunity. Strikingly, cdT cells are ancestral to other lymphoid populations such as abT cells and B cells, they participate to both innate and adaptive immune responses, have a preferential mucosal localisa- tion and might act as professional antigen-presenting cells [30] recognizing non-peptide antigens found on several pathogens, including mycobacteria, necrotic and tumor cells [31,32]. These surprising similarities between cdT cells and eosinophils prompted us to investigate, whether, in addition to other T cell- associated receptors, human eosinophils expressed a cdT cell Receptor (TCR). We here report that human blood eosinophils express low levels of surface cdTCR/CD3, inducible by mycobacterial ligands. We show that eosinophils release granule proteins and cytokines upon activation by cdTCR agonists, including mycobacteria. Furthermore, we provide evidence that cdTCR contributes to eosinophil cytotoxic potential towards tumors. Results Human eosinophils express CD3 and cdTCR but not abTCR In order to investigate expression by human blood eosinophils of T cell associated receptors, we purified eosinophils by negative selection using magnetic beads. These highly purified eosinophils (Figure S1A) expressed specific granule proteins such as eosinophil peroxidase (EPO) and major basic protein (MBP) but not myeloperoxidase (MPO) associated to neutrophil and monocyte/ macrophage lineages [33] (Figure S1B). Lymphocytes expressed neither of these myeloid markers (Figure S1B). Following permeabilization, binding of anti-CD3 but not anti- CD8 antibodies was detected in eosinophils (Figure 1A). In T cells, CD3 associates with either abTCR or cdTCR. We did not detect abTCR on eosinophils but cdTCR expression was evidenced (Figure 1A). While lymphocytes from PBMC fraction expressed these markers at the expected frequencies (Figure 1B) and unlike a previous report [34], we were unable to detect CD3 or abTCR expression in neutrophils (Figure 1C). Likewise neither CD8 nor cdTCR expression was detected in neutrophils (Figure 1C). Surface expression of CD3 and cdTCR was detected on a fraction of eosinophils following double staining with antibodies against two specific eosinophil granule proteins EPO (Figure 2A) or MBP (Figure 2B). Similarly to intracellular staining, neither CD8 nor abTCR were detected at eosinophil surface. By contrast, another cdT cell marker, NKG2D, was expressed in a lower proportion of cells (Figure 2A–B). As CD3 is required for receptor complex surface expression, CD3+cdTCR+ eosinophils were thus identified following triple staining (Figure 2C). In human blood, cdT cells express either Vc9/Vd2 or Vd1 variable regions. We also evidenced that EPO+ or MBP+ eosinophils expressed Vc9, Vd1 and Vd2 and unexpectedly co-expressed Vd1 and Vd2 at their surface (Figure 2A–C). Expression of cdTCR/CD3 complex was investigated in healthy donors and patients with either reactive eosinophilia (allergies, various skin diseases) or hypereosinophilic syndrome (HES). A wide heterogeneity of surface expression of cdTCR/ CD3 and NKG2D was not only found among the 23 donors but Eosinophil cdTCR Expression also within each group, including healthy donors, however no correlation was observed between receptor expression levels and either eosinophilia, or a specific pathology (Figure 3A–B). Following in vitro activation of cdT cells, CD3, CD8, cdTCR as well as Vc9, Vd2 and Vd1 were detected at the expected frequencies (Figure S2A). Likewise, freshly isolated PBMC from the same eosinophil donors, in which cdT cells represent a very minor population, expressed CD3, abTCR or low levels of cdTCR (Figure S2B). By contrast, monocytes did neither express CD3 nor cdTCR (Figure S2C). Finally, neutrophils neither expressed CD3, cdTCR nor the full abTCR complex at their surface (Figure S2D). Thus, following overnight culture, a fraction of eosinophils, unambigu- ously identified by the presence of specific eosinophil markers, express the cdTCR/CD3 complex at their surface. Besides receptor reexpression at the membrane following a resting period, as demonstrated for NKG2A on cdT cells [35], ligand-dependent induction or upregulation of receptor surface expression has been widely reported, including in the case of FceRI [9]. Thus, we investigated whether a specific cdTCR ligand was able to induce cdTCR/CD3 surface expression on human eosinophils. While CD3 and cdTCR were barely detected at the surface of freshly purified eosinophils (Figure S3A), upon incubation for 2 h with TUBag1, a natural non peptidic ligand from Mycobacterium tuberculosis [36], about 50% of eosinophils expressed CD3 and cdTCR at their surface (Figure S3B). By contrast, neither CD8 nor abTCR expression was induced upon incubation with TUBag (Figure S3B). Finally, besides mature eosinophils present in peripheral blood, eosinophils can be differentiated in vitro from CD34+ cord blood progenitors. In such EPO+ eosinophils, in the absence of possible contamination by cdT cells, cdTCR/CD3 complex was detected at cell surface, after 3 weeks differentiation (Figure S4). As for blood eosinophils, neither CD8 nor abTCR (not shown) could be detected. Eosinophils express all the subunits from cdTCR/CD3 complex To evidence the cellular distribution of cdTCR/CD3 complex and to further exclude that results obtained by flow cytometry could be due to contaminating cdT cells, blood eosinophils were analysed at low magnification by immunofluorescence and at high magnification by confocal microscopy. In full agreement with flow cytometry data, in a preparation without contaminating cells, only a fraction of blood eosinophils, identified by their typical binucleated morphology, expressed the four chains of CD3 and cdTCR. No signal was detected with anti-aTCR, anti-bTCR antibodies or control IgG (Fig 4A). We next characterized the structure of the cdTCR/CD3 complex expressed at the surface of eosinophils, using surface biotinylation, followed by lysis with a mild detergent, co- immunoprecipitation with an anti-cdTCR antibody, SDS-PAGE in reducing conditions and detection of biotinylated proteins. Bands corresponding to TCR, CD3d, e, c and f subunits were detected at the expected molecular weights both on cdT cells and eosinophils (Figure 4B). This result indicates that all the necessary chains are present to allow for surface expression of a complete cdTCR/CD3. However, the amount of material used for eosinophils was 10 times higher than for cdT cells, results consistent with flow cytometry data showing much lower surface expression in eosinophils than in cdT cells (Figure 2 and Figure S2). cdTCR/CD3 is less abundant and more restricted in eosinophils than in cdT cells Transcripts specific for the four chains (e, f, c and d) of CD3 complex, cTCR and dTCR constant regions and CD8 were PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5926