 capture was confirmed by the lack of binding of a CD5 mAb to immobilized CD6 and vice versa; the CD6 mAb did not bind significantly to CD5 immobilized in a similar manner (data not shown).
To test the importance of the C-terminal tyrosine motif in native CD6 for SLP-76 binding, wild-type human CD6 (CD6) and human CD6 with 662Y mutated to phenylalanine (662F) were expressed in a mouse T-cell hybridoma (see Fig. 5A). The human and mouse CD6 cytoplasmic regions are well conserved, including the SLP-76 binding tyrosine motif at the C terminus; thus, it was expected that human CD6 would engage functionally with the murine intracellular signaling machinery and be phosphorylated. Lysates were prepared from cells treated or not with pervanadate, and similar amounts of phosphorylated CD6 and 662F and unphosphorylated CD6 were immobilized. Unphosphorylated CD6 was used a negative control, as the SLP-76 SH2 domain did not bind to the unphosphorylated C-terminal CD6 peptide. Examples of the data for SLP-76 SH2 domain binding at 37°C are shown in Fig. 3A. Phosphorylation-dependent binding to CD6 was determined by subtracting equilibrium values for unphosphorylated CD6. Analyses of equilibrium binding data are shown in Fig. 3B. The SLP-76 SH2 domain bound CD6 with an affinity comparable to that found with the synthetic peptide.
CD6 costimulation of antigen-specific responses is dependent on 662Y. To address the functional consequences of mutating the C-terminal region of CD6, which is involved in phosphorylation-dependent recruitment of SLP-76, we tested 2B4 murine T-cell hybridoma cells transduced with human CD6 and mutants for differences in responses. The biochemical data above had shown that the C-terminal 662Y was the main site of binding for SLP-76. There was evidence for a weaker phosphorylation-dependent interaction with another region of the cytoplasmic tail. To test whether mutation of 489Y, which could bind weakly to SLP-76, had the same effect as 662Y, we produced cells expressing a 489F mutant and a double mutant in which both 662Y and 489Y were mutated to F, 489F/662F.
In this system, human CD6 would be engaged by mouse CD166 on APC. It has been shown that human CD6 binds mouse CD166 (43). To establish how effective this cross-species binding would be in the antigen-presentation experiments, we measured the affinity of human CD6 for mouse CD166 by surface plasmon resonance in the same way as we measured the interaction between human CD6 and human CD166 previously (19). A series of concentrations of human CD6 were passed over immobilized mouse CD166 (Fig. 4A). Calculation of the equilibrium-binding dissociation constants of human CD6 binding mouse and human CD166 at 37°C were the same (Fig. 4B and data not shown). 2B4 cells express mouse CD6, so the consequences of engaging endogenous mouse CD6 could also be investigated in this model. Mouse CD6 was tested for binding to immobilized mouse CD166 (Fig. 4C) and vice versa (Fig. 4D). The mouse CD6/CD166 interaction had an affinity similar to that of the human CD6/CD166 interaction, a KD of
1 µM at 37°C (19).
Compared with the wild-type human CD6, cells expressing 662F showed a reduced response to antigen (Fig. 5B and C), consistent with CD6-dependent costimulation being compromised by mutation of 662Y to F, which would prevent phosphorylation-dependent recruitment of SLP-76 to the C-terminal region of CD6. Mutations of both 489Y and 662Y to F resulted in decreased IL-2 production, whereas the single mutation of 489Y to F did not (Fig. 5B and C), consistent with 489Y not recruiting a positive regulator such as SLP-76. The behavior of 662F, the double mutant 489F/662F, and 489F is consistent with the interaction between CD6 and SLP-76 being critical for costimulation by CD6. The double mutant consistently showed less of a decrease than 662Y, suggesting that 489Y may be important for a negative effect of CD6.
The effect of increased levels of CD6 in the hybridoma cells is comparable with the effect of upregulation of accessory T-cell proteins in T-cell development. Once thymocytes are positively selected and become mature T cells, the requirement for tight regulation of their proliferative capacity is increased. This correlates with increased expression of regulatory accessory proteins CD5 and CD2 (40). To test whether mouse CD6 is also upregulated in a similar manner, as has been shown for human CD6 (36), we compared expression of CD6, CD5, and CD2 on mouse thymocytes expressing low and high levels of TCR. As expected, increased expression of all three receptors correlated with TCR levels (Fig. 5D).
CD6 costimulation is dependent on recruitment by CD166. Experiments with the hybridoma cells expressing human CD6 and mutants revealed a role for the cytoplasmic region of CD6 in regulating responses. Thus, costimulation by CD6 depends on both extracellular and intracellular engagement. To test the dependence of the response in this model on CD6/CD166 engagement, we compared responses when CD6 was available or not to bind CD166. As expected, antigen-specific IL-2 production by untransduced 2B4 hybridoma cells was sensitive to inhibition by soluble CD6 (Fig. 6A). The response of cells expressing the additional human CD6 was also sensitive to inhibition by soluble CD6, but to a lesser extent, reflecting the higher levels of CD6 at the cell surface. The reduced response by cells expressing 662F was not sensitive to inhibition by soluble CD6, showing that the decreased response is due to loss of signaling effects in 662F cells rather than extracellular engagement, which would be the same as for cells expressing wild-type human CD6.
CD6 costimulation is dependent on recruitment of SLP-76. The SLP-76/CD6 interaction requires CD6 to be phosphorylated. We had shown that human CD6 and the mutant 662F could be phosphorylated in the murine hybridoma cells in response to pervanadate treatment (Fig. 3). If the recruitment of SLP-76 to the C-terminal 662Y is critical for costimulation by CD6, which is not lost in 489F, it was important to know that 489F could be phosphorylated. We analyzed the phosphorylation status of CD6 and mutants in the hybridoma cells in response to pervanadate treatment. Phosphorylated human CD6 was immunoprecipitated specifically from NP40 lysates of pervanadate-treated cells expressing human CD6 and mutants but not from untransduced cells (Fig. 6B, upper panel). This experiment showed that CD6 can be phosphorylated on tyrosine residues other than 662Y and 489Y, suggesting that additional activation-dependent interactions of the CD6 cytoplasmic region can occur. A separate nonreduced gel showed that CD6 was expressed at similar levels in all the cell lines (Fig. 6B, lower panel), corresponding to the cell surface analysis (Fig. 5A).
We tested the SLP-76 mAb for recognition of murine SLP-76. The SLP-76 mAb precipitated a phosphorylated band from cells corresponding to the molecular weight expected for CD6,
130 kDa (Fig. 6C). Blotting with the SLP-76 mAb confirmed the presence of murine SLP-76 in the immunoprecipitate (Fig. 6D). As in human cells (Fig. 3E), SLP-76 immunoprecipitates from Brij-96 lysates of pervanadate-treated cells expressing human CD6 contained a band comigrating with CD6. By comparing SLP-76 and CD6 precipitates from cells expressing human CD6 and mutants, it was clear that a band comigrating with CD6 was coprecipitated by the SLP-76 mAb specifically from CD6 and 489F cells, precisely the forms of CD6 predicted to be able to bind SLP-76 (Fig. 6C). Blotting with SLP-76 confirmed the presence of SLP-76 in all the SLP-76 immunoprecipitates and that it was coprecipitated with CD6 and 489F but not 662F or 489F/662F.
It has been previously reported that truncation of CD6 at amino acid 661 resulted in reduced phosphorylation of CD6 in response to CD3 mAb cross-linking (22). We compared the effect of CD3 mAb cross-linking on human CD6 and 662F phosphorylation (Fig. 6E). Receptor-mediated phosphorylation of CD6 was reduced by the single-point mutation 662F. This confirms the importance of phosphorylation of 662Y in CD6 in response to CD3 stimulation.
The discovery of a positive regulator binding to the cytoplasmic region of CD6 is in keeping with CD6 recruitment being essential for an optimal immune response (15, 19). The interaction between CD6 and SLP-76 is the first direct activation-dependent interaction to be described for the large cytoplasmic tail of CD6 (Fig. 1) and provides a mechanism for linking CD6 engagement by CD166 or other ligands to T-cell regulation through the well-characterized adaptor SLP-76. SLP-76 was the only protein isolated in pulldown experiments from cell lysates with the peptide containing the DDYDDI motif at the extreme C terminus of CD6. This sequence fits with the motifs in ADAP and HPK1, which are the other proteins which bind the SH2 domain of