Eur. J. Immunol. 2012. 42: 195–205 molecule for similar reasons [24, 25], but was subsequently shown to inhibit T-cell activation and thymocyte selection [18]. More recent studies have shown that blocking the CD6–CD166 interaction with soluble recombinant ligands or Abs reduces T-cell activation and proliferation following the interac- tion of T cells with APCs [8–10, 26], supporting a costimulatory role for CD6. In these experiments, however, the blocking Abs and recombinant proteins used signiﬁcantly diminished the Leukocyte signaling 201 number of T cell–APC conjugates that formed, implying that the observed inhibitory effects were not simply due to the blockade of costimulatory processes. Zimmerman et al. [10] observed that a nonblocking CD6 mAb (M-T605) that targets domain 1 of CD6 rather than the ligand-binding domain 3, and therefore ought not to block the CD6/CD166 interaction, is a potent inhibitor of T-cell proliferation. Almost complete inhibition of proliferation was also observed in cultures in which soluble CD166-Fc was used as a blocking agent, whereas the inhibition was much less pronounced when a CD6–Fc fusion protein was used. Similarly, Montero and colleagues showed that in the cultures of T cells interacting with plate-bound recombinant CD166 and CD3 mAb, the inclusion of a nonblocking CD6 domain 1-binding Ab markedly reduced intra- cellular phosphorylation and strongly inhibited T-cell prolifera- tion [27]. Given that only the reagents that interact directly with the CD6 molecule completely abolish T-cell activation, it seems reasonable to propose that in these experiments engaging CD6 delivers a negative signal to the T cells, as is now more exhaus- tively documented in the present experiments. However, it is also possible that CD6 engagement strengthens T-cell activation, albeit indirectly. The interaction of CD6 with CD166 is fairly strong [9], and is likely to increase cellular adhesion and in this way enhance T-cell responses. Thus, a dual function for CD6 could be envisaged, whereby the balance of positive (stronger adhesion) and negative (inhibitory signaling) processes ﬁne-tunes T-cell activation. Hassan et al., while show- ing that the interaction between CD6 and CD166 is important for T-cell activation, have reported that an increase in the level of CD6 expression made T cells less responsive [26], in agreement with our present observations. The overall balance between inhibitory and activatory signals, including the strength of the signals initiated by the TCR, is likely to dictate the outcome of the activation process. The molecular mechanisms through which CD6 constrains signaling are not yet determined, but it is possible that CD6 functions in a similar way of CD5, coupling with inhibitory tyrosine phosphatases, e.g. SHP-1, or modulating the activity of signaling-enhancing tyrosine kinases [28, 29]. Alternatively, given that CD6 associates with several kinases, it might sequester 3 Figure 5. CD6 limits late signaling events. (A) IL-2 production (pg/mL) by hCD6 versus hCD6-expressing (hCD6-FL) E6.1 cells, treated (white bars) or not treated (black bars) with OKT3. Data shown are mean195% conﬁdence intervals from three independent experiments. The prob- ability that the production of IL-2 by hCD6 and hCD6FL E6.1 cells was similar, with and without anti-CD3 mAb stimulation, was assessed using Student’s t-test (for p–values, see graph). (B) CFSE-preincubated human PBLs were allowed to interact for 6 days with sAg-pulsed Raji cells expressing endogenous human CD166, in the presence of blocking (anti-CD166-3A6 and anti-CD6-OX126) or nonblocking (control) Abs. Cell division was determined by the extent of CFSE dilution. (C) Effect of CD6 expression on cell proliferation. An equal number of cells were analyzed by FACS following CFSE labeling. Stacked bars represent the mean number of cells in each generation (G0–G2) from three independent experiments. The probability that the proportion of cells in G0–G2 was the same in hCD6-FL-expressing cells in the presence of control or blocking (anti-CD166-3A6; [po0.0001], anti- CD6-OX126; [po0.0001]) Abs was assessed using the w2-test. po0.0001. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu