Here we demonstrate that S1P2 has a dual role in GC B cells, regulating survival and promoting clustering at the follicle center. We provide evidence that S1P2 acts through a signaling module involving G12–G13, p115RhoGEF, and most likely Rho and ROCK, to dampen Akt activation in GC B cells. When S1P2 is lacking, elevated Akt activity can lead to increased phosphorylation of 4E-BP1, a modification that releases this inhibitor from eIF4E, allowing for small increases in cap-dependent translation of a range of transripts37
. 4E-BP1 regulates translation of transcripts with complex 5’UTRs, including those for a number of pro-survival molecules38–40
. Our data do not exclude the possibility that Akt promotes GC B cell survival through additional pathways, such as regulation of GSK3 or Glut141
. S1P2 can signal via Akt-independent pathways 31,32
and it is possible that their reduction also contributes to the growth advantage observed in S1P2-deficient cells.
Although the ex vivo
analysis suggests a strong prosurvival effect of S1P2-deficiency, the in vivo
advantage only becomes evident over periods of weeks in chronically stimulated GCs. This suggests that S1P2’s contribution is relatively small during acute antigen-driven GC responses, while it is more strongly revealed under the conditions associated with cell isolation. This may be a consequence of GC B cell exposure to the higher amounts of S1P present outside the GC during tissue preparation as well as the stresses of in vitro culture. We suggest that S1P2’s contribution in vivo
may become most evident under conditions of elevated genotoxic stress arising due to chronic GC stimulation at mucosal sites. The survival advantage may increase the likelihood of cells acquiring and surviving secondary oncogenic hits, thereby setting the stage for progression to DLBCL. Gq negatively regulates Akt activation in naïve B cells42
, indicating that multiple GPCRs exert a controlling influence over Akt activation in B cells. B cells with elevated Akt activity due to PTEN deficiency or Akt over-expression were reported to have reduced isotype switching as a result of decreased activation induced cytidine-deaminase (AID) function43,44
. We did not observe defects in isotype switching or affinity maturation in S1P2-deficient Hy10 B cells, suggesting AID function is intact. These differences may reflect a lesser increase in Akt activity in S1P2-deficient compared to PTEN-deficient B cells, or they may indicate differences in the way distinct pools of activated Akt are integrated into downstream signaling networks45
S1P has a well-established role in promoting lymphocyte egress from lymphoid tissues9,12
. The present findings establish that S1P also has a role in regulating cell behavior within lymphoid tissue. Follicular S1P is derived from radiation resistant cells other than FDCs and is rapidly degraded by B cells. S1P has a half-life in plasma shorter than 15 minutes10
. Given that the most frequent cells in blood (red blood cells) do not express S1P degrading enzymes9
whereas B cells do, it seems likely that the S1P half-life in densely packed B cell follicles will be at least this short.
We propose a model where S1P concentrations are relatively high in the outer follicle and decay to a low point over the FDC network at the follicle center. We suggest that S1P2 induction in GC-precursor cells, by inhibiting their propensity to migrate towards CXCL13 and other attractants in the S1Phigh
outer follicle, helps to focus the cells to the follicle center. S1P2 may also act by promoting chemorepulsion46
, though we have not found GC B cells to move away from S1P in transwell migration assays. EBI2 down-regulation is also important in allowing GC cells to move away from the outer follicle7
. Once a GC forms, S1P within this microenvironment may be kept at low levels by local degradation and by minimal carriage of S1P into the structure by newly arriving cells. When an S1P2-expressing GC B cell reaches the GC perimeter it likely encounters higher amounts of S1P, causing activation of Rho at the leading edge, prompting retraction of cellular processes47
and cell turning. Because S1P2 signaling also causes Akt inhibition, this growth-regulatory effect may be strongest in GC B cells migrating near the GC perimeter. Thus, through dual growth regulatory and migration inhibitory activities, S1P2 may act as one of multiple factors that helps link GC size to the volume of the supportive niche at the follicle center. In future studies it will be important to develop techniques to measure interstitial S1P concentrations in order to have a better understanding of how S1P2 could be controlling both processes. Nevertheless, we feel it reasonable to suggest, based on the existing data, that a general property of G12–G13-coupled receptors may be to help coordinate niche confinement and cell survival.