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Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.

Foxp3(+)CD4(+)CD25(high) regulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4(+) T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.

The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment.

Ligation of the B cell antigen receptor (BCR) complex initiates tyrosine phosphorylation of the receptor's transducer components, Ig-alpha and Ig-beta and tyrosine kinase-dependent accumulation of GTP-bound, activated p21ras. The mechanism of receptor coupling to p21ras activation and the roles of Ig-alpha and Ig-beta are unknown. The results reported here indicate that the resting, nonphosphorylated BCR associates with the Grb-2/Sos-linker SHC via the Ig-alpha immunoreceptor-based tyrosine activation motif (ITAM). Ig-alpha specificity of this interaction is determined by the sequence DCSM found in Ig-alpha, but not Ig-beta. Tyrosine phosphorylation of Ig-alpha and Ig-beta ITAM allows recruitment of SHC, which now binds directly to both Ig-alpha and Ig-beta via a phosphotyrosine/SH2 interaction. In confirmation of recent studies by Saxton et al. (J. Immunol. 1994. 153: 623) receptor ligation leads to tyrosine phosphorylation of SHC and to the formation of a phospho-SHC/Grb2/Sos complex. In view of previous studies which demonstrated p21ras co-capping with ligated BCR, the data presented here suggest that Ig-alpha/beta- and SHC tyrosine phosphorylation-dependent recruitment of the Grb2/Sos complex to the receptor can occur and may provide a mechanism by which the nucleotide exchange activity of Sos could mediate activation of BCR-localized p21ras.