In this report, we present evidence that, like CD5, CD6 is capable of generating inhibitory signals in T lymphocytes. Our main findings are (i) that transfected rCD6 and hCD6 reduce Ca2+ responses in human T cells and a T-cell line, respectively, and that the suppression of hCD6 expression increases these responses; (ii) that the ligand-dependent localization of CD6 to the synapse is neither required for these effects nor, surprisingly, further enhances them; (iii) that the cytoplasmic domains of human and rCD6 are responsible for these inhibitory effects; and (iv) that, in addition to receptor proximal signaling, CD6 expression suppresses longer term events such as cytokine secretion and T-cell proliferation. We conclude from these experiments that CD6 has an important role in establishing thresholds for T-cell activation.
CD6 was previously regarded to be a costimulatory receptor however, this assumption was largely built on reports by ourselves and others in which CD6 was cross-linked together with the TCR using Abs 6, 7, 13
. An explanation for these observations is that CD6 associates with protein tyrosine kinases such as Lck, Fyn, and ZAP-70 13
, and it could be expected that mAb-induced coaggregation of CD6 with the TCR might promote the Lck/Fyn-mediated phosphorylation of CD3 ITAMs, leading to signaling. CD5 was originally proposed to be a costimulatory 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 interaction 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 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 intracellular phosphorylation and strongly inhibited T-cell proliferation 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 exhaustively 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 showing 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 these kinases away from the TCR apparatus or it could function analogously to the IgSF glycoprotein CD2, which transduces mitogenic signals via its association with lipid rafts and the tyrosine kinases Lck and Fyn 30, 31
, and inhibitory signals via its association with CD5 32–34
. Since CD5 and CD6 also associate at the surface of T cells and one major effect of CD6 stimulation is the phosphorylation of CD5 13
, it is feasible that CD5 can integrate or transform the signals generated by the triggering of CD6.
A striking finding of the present study is that CD6 appears to be capable of strong inhibitory signaling in the absence of ligand. Our findings suggest that CD6 is a general attenuator of T-cell activation, whose level of expression at the plasma membrane, rather than its interactions with ligand or localization at the immunological synapse, determines the amplitude of the signal. At present, we are unable to offer an explanation for why the localization of CD6 to the synapse does not induce stronger inhibitory signaling. A correlation between the levels of CD6 expression and the heightened inhibition of cell responsiveness has been proposed previously 9
. However, given that very few T cells do not express CD6, finer-grain regulation may be achieved by the generation of CD6 intracellular isoforms produced at distinct stages of activation or differentiation 12, 35, 36
. Since, as we show, the cytoplasmic domain is crucial for the inhibitory role of CD6, variation in its composition may translate into different associations with effector molecules that could very conceivably fine-tune activation thresholds in T cells.
Nevertheless, the full-length isoform seems to be the most abundant, and hence at least at some stages of immune responses, CD6 will display some capacity for downmodulating activation signals as was shown for the full-length constructs used in the present study. As the cytoplasmic part of CD6 is highly tyrosine phosphorylated after T-cell activation, the example of CD5 could be followed 16, 17
, with a systematic and careful analysis of the various tyrosine residues present within the domain of CD6 undertaken. The observation that, like CD6, CD5 also attenuates signaling in T and B cells stimulated via their physiological receptors in a ligand independent manner, suggests that CD6 is not alone in its capacity to establish activation thresholds via its expression per se, and that this could be a general feature of inhibitory signaling proteins.