CD40/CD40L interaction regulates CD4+CD25+ T reg homeostasis through dendritic cell-produced IL-2.

CD4+CD25+ regulatory T cells (T reg) development and homeostasis require IL-2 and costimulation through same TNF-receptor family members. CD40KO mice have reduced number of T reg in peripheral blood, thymus and spleen. Herein we show that naive T reg express low basal level of CD40L that is upregulated upon TCR-triggered mediated activation. Treatment of wt mice with Ab blocking CD40/CD40L interaction results in a fast decrease in T reg number that rapidly recovers upon Ab withdrawal. CFSE-labeled T reg from wt mice injected into CD40KO, but not wild-type (wt) mice, showed reduced survival and proliferation in homeostatic setting. In vitro, dendritic cells from CD40KO mice but not wt mice produce diminished amount of IL-2 upon T reg encounter and are impaired in expanding T reg, a defect corrected by the addition of rIL-2. Accordingly, four daily IL-2 administrations to CD40KO mice normalize T reg number by promoting both their survival and homeostatic proliferation. Such IL-2 effect is transient since T reg number returns to the low constitutive level described in CD40KO mice within 5 days upon IL-2 withdrawal thus suggesting that IL-2 is persistently needed to assure T reg homeostasis.

Triggering of OX40 (CD134) on CD4(+)CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR.

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor family that is transiently expressed on T cells after T-cell receptor (TCR) ligation. Both naive and activated CD4(+)CD25+ regulatory T cells (T reg's) express OX40 but its functional role has not been determined. Since glucocorticoid-induced tumor necrosis factor receptor (GITR), a related TNF receptor family member, influences T reg function, we tested whether OX40 might have similar effect. Triggering either GITR or OX40 on T reg's using agonist antibodies inhibited their capacity to suppress and restored effector T-cell proliferation, interleukin-2 (IL-2) gene transcription and cytokine production. OX40 abrogation of T reg suppression was confirmed in vivo in a model of graft-versus-host disease (GVHD). In a fully allogeneic C57BL/6>BALB/c bone marrow transplantation, GVHD was lethal unless T reg's were cotransferred with the bone marrow and effector T cells. Strikingly, T reg suppression of GVHD was abrogated either by intraperitoneal injection of anti-OX40 or anti-GITR monoclonal antibodies (mAbs) immediately after transfer, or by in vitro pretreatment of T reg's with the same mAbs before transfer. Cumulatively, the results suggest that in addition to controlling memory T-cell numbers, OX40 directly controls T reg-mediated suppression.

Defects in cytokinesis, actin reorganization and the contractile vacuole in cells deficient in RhoGDI.

Rho GDP-dissociation inhibitors (RhoGDIs) modulate the cycling of Rho GTPases between active GTP-bound and inactive GDP-bound states. We identified two RhoGDI homologues in DICTYOSTELIUM: GDI1 shares 51-58% similarity to RhoGDIs from diverse species. GDI2 is more divergent (40-44% similarity) and lacks the N-terminal regulatory arm characteristic for RhoGDI proteins. Both are cytosolic proteins and do not relocalize upon reorganization of the actin cytoskeleton. Using a two-hybrid approach, we identified Rac1a/1b/1c, RacB, RacC and RacE as interacting partners for GDI1. Cells lacking GDI1 are multinucleate, grow slowly and display a moderate pinocytosis defect, but rates of phagocytosis are unaffected. Mutant cells present prominent actin-rich protrusions, and large vacuoles that are continuous with the contractile vacuole system. The actin polymerization response upon stimulation with cAMP was reduced, but the motile behavior toward the chemoattractant was unaffected. Our results indicate that GDI1 plays a central role in the regulation of signal transduction cascades mediated by Rho GTPases.