Eosinophil cdTCR Expression Figure 5. cdTCR/CD3 complex expression and rearrangements in human peripheral blood eosinophils. (A) Real time PCR analysis of expression of individual chains from cdTCR/CD3 complex in eosinophils, cd T cells and Colo-205 cells. Average expression in Colo-205 cells is chosen as reference (samples were analysed in triplicate using primers listed in Table S1). (B) TCRc and d rearrangements in peripheral blood eosinophils analysed by RT-PCR using primers listed in Table S2. CD8 amplification is performed to exclude lymphocyte contamination. Colo-205 are chosen as negative control (C) Spectratyping analysis of CDR3 length profiles for Vc9-Jc1/2 ; Vc9-JcP and Vd2-Cd families in eosinophils and in cd T cells from the same donor. (D) Expression of rearranged c and dTCR at the single cell level in peripheral blood eosinophils and cdT cells detected by in situ hybridization using anti-sense and control sense probes specific for Vd2-Cd, VcI-JcP and for Vd1-Jd4 used as negative control. doi:10.1371/journal.pone.0005926.g005 in eosinophils. These rearrangements were all found in cdT lymphocytes but neither in abT lymphocytes nor Colo-205 cells (Figure 5B). Spectratyping analysis of CDR3 length profiles for Vc9-Jc1/2, Vc9-JcP and Vd2-Cd evidenced a limited Vd2-Cd diversity (2 peaks) on eosinophils compared to cdT cells (Figure 5C), while no signal was detected on eosinophils for the 2 other rearrangements, in contrast to cdT cells (data not shown). Furthermore, to unambiguously demonstrate that TCR rear- rangements occur in eosinophils, at single cell level, we performed in situ hybridization using sequences from rearranged TCR genes as RNA probes. Hybridization with anti-sense probes correspond- ing to Vd2-Cd and VcI-JcP rearrangements, that were detected by RT-PCR, gave a positive signal on nuclei from eosinophils at times clearly bilobed. In keeping with RT-PCR results, a significantly stronger signal was detected on cd T lymphocytes, while corresponding sense probes only gave background amplification on both cell types (Fig. 5D). Vd1-Jd4 anti-sense probe gave positive signal on cd T lymphocytes but not on eosinophils in which this rearrangement was not detected by RT-PCR. Finally, DNA sequencing of Vd1Jd3 Vd2-Cd regions from eosinophils and cdT cells show that V and J regions were virtually identical to the IMGT sequence, while significant differences between eosinophils and 2 different cloned sequences of cdT cells from the same donor were found in D regions and in junctional regions (Figure 6). This further confirms that cdTCR/CD3 complex expressed by eosinophils is less expressed and diverse than the corresponding receptor on cd T cells. cdTCR/CD3-mediated eosinophil activation induces ROS production, degranulation and cytokine release Upon activation, eosinophils very rapidly produce Reactive Oxygen Species (ROS) and release, by selective degranulation, eosinophil-specific granule proteins, including highly cytotoxic cationic proteins such as EPO and eosinophil cationic protein (ECP) [6]. In purified blood eosinophils, cdTCR/CD3 complex activation by immobilized antibodies to CD3, cdTCR or Vd1 led to a similar kinetics of ROS production (Figure 7A) and to a significant EPO and eosinophil-derived neurotoxin (EDN) release (Figure 7D and G). Stimulation with a soluble ligand, bromohy- drin pyrophosphate (BrHPP), a phosphoantigen agonist selective for human c9d2+ T lymphocytes [37], induced dose-dependent ROS production as well as EPO release (Figure 7B and E). Furthermore, eosinophil activation by sec-butylamine (SBA) [38], an inducer of endogenous phosphoantigens, yielded to a sustained dose-dependent ROS and EPO production (Figure 7C and F), comparable to values obtained following activation with IgA/anti- IgA immune complexes, one potent physiological eosinophil activator. Of note, plate-bound antibodies induced stronger ROS release but weaker EPO release than soluble cdTCR activators. Since eosinophils also release several immuno-regula- tory and proinflammatory cytokines [10], we further show that activation with anti-CD3, -cdTCR or -Vd1 antibodies efficiently induced IFN-c and TNF-a production by eosinophils (Figure 7H and I). Thus, cdTCR/CD3-expressing eosinophils respond to selective agonists similarly to human cdT lymphocytes and produce both specific eosinophil granule proteins, myeloid cell mediators as well as cytokines. Mycobacterium bovis induce cdTCR/CD3-dependent eosinophil activation Human cdT cells respond to non-peptide antigens including mycobacterial and tumor-derived ligands. Patients with mycobac- terial infections may exhibit blood and tissue eosinophilia and mycobacteria-induced eosinophil-associated experimental acute inflammation [39] as well as rapid eosinophil chemotaxis in vivo. Mycobacteria- eosinophil interactions might thus reflect the reactivity conferred by cdTCR/CD3 complex. Accordingly, incubation of blood eosinophils with increasing ratios of Mycobacterium bovis-BCG induced dose-dependent ROS production and EPO release (Figure 8A–B), that could be inhibited, in a dose- dependent manner, by an anti-cdTCR antibody [40] (Figure 8C– D). Similar eosinophil activation, and inhibition by an anti- cdTCR antibody, was also obtained following incubation with TUBag, a natural mycobacterial component (Figure 8E–H). Thus mycobacteria appear capable of activating eosinophils, at least partly, in a cdTCR-dependent manner. cdTCR/CD3-dependent eosinophil cytotoxicity toward tumor cells Eosinophils have been associated with many tumors in vivo. Furthermore, both cd T cells [41] and eosinophils [19] exert potent cytotoxic activity towards many tumor cells. Therefore, we investigated cdTCR-mediated eosinophil cytotoxicity towards Colo-205 carcinoma cell line. Indeed, eosinophils induced time- dependent apoptosis of Colo-205 tumor cells in vitro, which was significantly inhibited following addition of neutralizing anti- cdTCR Ab (Figure 9A). This effect was more prominent at earlier time-points suggesting that cdTCR-mediated eosinophil-tumor interactions are important for the initiation of the cytotoxic reaction (Figure 9B). Finally preincubation of eosinophils with cdTCR ligands, SBA and TUBag, potentiated eosinophil cytotoxicity towards tumor cells (Figure 9C–D). Altogether, these data confirm the potential role of eosinophils in anti-tumor cytotoxicity and strongly suggest the involvement of cdTCR/CD3 complex in this mechanism. Discussion We here provide evidence that highly purified human blood eosinophils, as well as eosinophils differentiated in vitro from CD34+ cord blood progenitors, gated on the basis of presence of specific eosinophil granule proteins, express a functional cdTCR/ CD3 complex, so far almost exclusively associated to T lymphocytes, but lack abTCR. As previously observed for other receptors such as CD25 [42], CD4 [18], FceRI, CD89 and CD28 [10], cdTCR/CD3 surface expression on eosinophils is highly heterogeneous between individual donors. Such an heterogeneity in cdTCR/CD3 expression has also been observed for human blood cdT lymphocytes [35]. As it is the case for several receptors, including FceRI [9], surface expression of cdTCR/CD3 can be PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5926