Eosinophil cdTCR Expression Figure 8. Mycobacterium bovis-BCG-induced eosinophil activation. (A and E) Dose-dependent ROS production by BCG- (A) or TUBag (E) activated eosinophils. BCG/eosinophil ratio: 20/1 ($), 10/1 (e), 5/1 (+) and 0/1 (2). (B and F) Dose-dependent EPO release by eosinophils activated by different BCG/eosinophil ratios (n = 3) (B) or TUBag concentrations (F). (C and G) Inhibition of BCG- (ratio 20/1 $) (C) or TUBag- (G) induced ROS production by an anti-cdTCR blocking antibody 5 mg/ml (x) and 10 mg/ml (#) or an isotype control antibody (D). (D and G) Inhibition of BCG- (D) or TUBag- (G) induced EPO production by an anti-cdTCR blocking antibody (5 and 10 mg/ml) or an isotype control antibody. Results are expressed as DEPO cps values (values from medium stimulation are subtracted from values obtained with BCG stimulation for each antibody) (n = 3). (E–F) *, P,0.05; **, P,0.01. doi:10.1371/journal.pone.0005926.g008 organs and tissues would greatly benefit from experiments on animal models. Unfortunately, we were unable to detect cdTCR/ CD3 on mouse blood and tissue eosinophils. This is not extremely surprising since mouse eosinophils are strikingly different from human eosinophils, regarding their granular content [50] and membrane expression of various immune receptors, in particular IgE receptors [51] and IgA receptors [52]. Furthermore, cdT cells also display significant differences between mouse and humans. Indeed, a high proportion of skin lymphocytes express cdTCR in mouse but not in human [53], while only human Vc9Vd2 are responsive to phosphoantigens [54]. Our findings indicate that eosinophils might also contribute, through cdTCR-dependent mechanisms, to immune defences in tissues, where 90% eosinophils are located. One might speculate that cdTCR/CD3 expression is higher in tissue than in blood eosinophils, as previously reported for FceRI in a model of ‘‘humanized’’ mice where human FceRI was quantified and increasingly expressed from blood to peritoneal cavity and spleen eosinophils [9]. Tissue eosinophils are associated to granulomatous diseases such schistosomiasis [55], Crohn’s disease [56] and mycobacterial infections [39]. Eosinophils can damage cell wall and lyse Mycobacterium tuberculosis in vitro[57]. Vc9d2 and Vd1 expression, rapid response to phosphoantigen ligands, as well as BCG-induced activation so far represented hallmarks of cdT lymphocyte-mediated cytotoxicity towards phosphoantigen-ex- pressing mycobacteria [58]. Since tumors produce phosphoanti- gens and are targeted by cdT lymphocytes [58], cdTCR-mediated activation might thus contribute to the anti-tumoral activity of eosinophils. Indeed, while cdT lymphocytes and NK cells display granzyme B/perforin-mediated cytotoxicity, eosinophils addition- ally possess a unique set of cationic proteins with a strong cytotoxic potential [50]. Furthermore, tumor-associated tissue eosinophilia (TATE) is considered by some authors as a marker of good prognosis, including in colon carcinoma [59]. Anti-tumoral activity of eosinophils should be considered in the context of current development of innovative cancer immunotherapies, based on synthetic phosphoantigens [60]. Indeed, due to their high cytotoxic potential and despite their low cdTCR expression in comparison to T cells, eosinophils might thus represent very efficient ‘‘innate’’ effector cells in defence against targets bearing cdTCR ligands, such as tumor cells, in particular when eosinophils surround or infiltrate solid tumors. Our work outlines a previously unexpected link between eosinophils and cdT lymphocytes, which not only share the expression of the cdTCR/CD3 complex but also some major functions in innate immunity. It might also shed light on the Figure 9. cdTCR-dependent tumor-cell killing by human eosinophils. (A–B) Induction of Colo205 killing by eosinophils and inhibition by an anti-cdTCR blocking antibody. Percentage of AnnexinV+ tumor cells in the presence or absence of eosinophils and blocking anti-cdTCR or isotype control antibodies (n = 5–7) at various time-points. Panel A represents the 60 min time-point. (C–D) Potentiation of eosinophil-mediated Colo205 killing by cdTCR ligands. (C) 50 mM SBA (n = 7). (D) 20 mM TUBag (n = 4). *, P,0.05; **, P,0.01. doi:10.1371/journal.pone.0005926.g009 PLoS ONE | www.plosone.org 12 June 2009 | Volume 4 | Issue 6 | e5926