Overall, the rationale of our study is to assess whether GSK-3 inhibition effects are seen at the level of T-cell velocity and interactions with other cells. Our study shows that GSK-3 plays a clear role in regulating the movement of T-cells and interactions with other cells. Indeed, the inhibition of GSK-3 reduced the velocity of T-cells as measured in vitro on plates coated with ICAM1 for adhesion. The net result of the inhibition of T-cell velocity and the reduced distance travelled needed to interact with other cells. However, the actual duration of the interaction or the dwell times was not affected by GSK-3. This is a logical expectation given that movement is needed for T-cells to stochastically encounter other cell types or to respond to chemo-attractants such as chemokines.
The fact that GSK-3 inhibition does not have an effect within minutes of exposure but rather requires longer periods of incubation following activation suggests that its effect on motility may be indirect, possible due to effects on T-cell activation or differentiation. Naive murine T-cells become effector T-cells followed by contraction and the generation of central memory T-cells expressing and effector T-cells (28). We previously showed that GSK-3 regulates this event (22,23,29-31). It is therefore most likely that the targeting effects on GSK-3 is due to effects on the activation or differentiation status of the T-cell. In this instance, the potential disadvantage of reduced motility and interactions with other cells is overridden by the positive intracellular effects on CTL killing of targets (22,23,29,30).
Further, it is important to note that different inhibitors of GSK-3 had the same effect on T-cell motility. L803-mts is structurally unrelated to SB415286 and SB2167763 (25). Further, SB216763 has a preference for the GSK-3alpha isoform, while L803-mts preferentially inhibits the GSK-3beta isoforms. Despite different structures and isoform specificities, the exposure of cells to all drugs overtime resulted in population of cells with reduced motility after long-term exposure. Lastly, the short exposure of CTLs to SB415286 that affects motility did not alter killing suggested that the effects on T-cell motility did not substantially alter the ability of T-cells to kill tumor targets.
The underlying mechanism is not clear. As mentioned, the effects require long-term incubation with T-cells and are therefore most likely related to effects on the activation or differentiation of T-cells. However, effects on more proximal events are also possible since GSK-3 can phosphorylate microtubule-associated protein 2C (MAP2C) which prevents microtubule remodelling (16,17). It is also possible that GSK-3 interfaces with adaptor proteins such as SKAP1 which regulate T-cell motility (32). The N-terminal region of SKAP1 binds to RapL such that a RapL mutation (L224A) abrogates SKAP1 binding and arrests T-cells even in the absence of antigen (17). Lastly, it is possible that GSK-3 may influence cell motility and chemotaxis by regulating PI 3K membrane localization in Dictyostelium (33) or due to effects on phosphatidylinositol-3,4,5-triphosphate (PIP3) metabolism, target of rapamycin complex (TORC) signaling, and remodeling of F-Actin (34,35). Teo et al have reported that gsk3− cells respond to stimuli with a reduced increase of PIP3 and no TORC2 activation (35), decreased adenylyl cyclase, while others have obtained different results (34). Future work will be needed to assess the full range of effects of GSK-3 on aspects of T-cell function linked to motility and migration.
Limitations: Work restricted to non- lymphoid cells