Investigators demonstrate that ectopic expression of PKC-θ V3 domain acts as a decoy and ameliorates inflammatory responses in mouse model.

Scientists say new insights into how an enzyme involved in T-cell activation binds to the co-receptor CD28 indicate that a conserved domain within the enzyme could represent a potential therapeutic target for autoimmune diseases. An international research team led by Amnon Altman, M.D., at La Jolla Institute for Allergy and Immunology, demonstrated that a proline-rich motif in the V3 domain of protein kinase C-θ (PKC-θ) binds with CD28 and that this interaction is essential for PKC-θ-mediated downstream signaling.

Reporting in Nature Immunology the researchers say ectopic expression of the V3 domain was enough to interfere with the functions of endogenous PKC-θ, including TH2 and TH17 differentiation and inflammation but, critically, not TH1 differentia­tion. Their results are described in a paper titled “A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28.”

The induction of an immune response is dependent on communica­tion between antigen-specific T cells and antigen-presenting cells (APCs), the researchers explain. When a T cell expressing a relevant T cell antigen receptor (TCR) meets up with an activated APC, the cells both redistribute receptors and ligands to the interface between the cells, generating a region known as an immunological synapse, which effectively represents the playing field for receptor interaction, signaling, and the recruitment of relevant intracellular proteins to the cell surface.

More specifically, the mature immunological synapse comprises concentric rings with a central core (the central supramolecular activation cluster, or cSMAC) that contains TCRs and co-s­timulatory molecules and a peripheral ring (the peripheral supramolecular activation cluster, or pSMAC) in which adhesion molecules localize.

One of the most prominent proteins to be recruited specifically to the cSMAC after antigen stimulation is the kinase PKC-θ. Studies in mice have demonstrated that the enzyme plays a key role in T-cell activation and survival and that autoim­mune diseases mediated by interleukin 17 (IL-17)-producing TH17 cells require PKC-θ. The enzyme is also needed for allograft rejection and graft-versushost disease but not for the graft-versusleukemia response in mice.

However, the researchers continue, what isn’t yet clear is whether the localization of PKC-θ specifically to the cSMAC is needed for its signaling function in T cells. They first compared the amino acid sequence of PKC-θ and its closest relative PKC-δ, a member of the same  Ca2+-independent PKC family that doesn’t translocate to the immunological synapse after interaction between T cells and APCs.

This comparison highlighted a significant difference between the proteins’ respective V3 (hinge) domains, suggesting the region is involved in targeting PKC-θ to the immunological synapse. “This region has not been linked before, to our knowledge, to the regulation of PKC-θ localization or function other than its general role as a flexible hinge that allows PKC proteins to undergo a conformational change from a resting state into an ‘open’, active conformation,” the authors note.

The researchers then engineered PKC-θ-deficient mice that instead expressed a form of PKC-θ that lacked the V3 domain. In these animals the V3-deficient PKC-θ didn’t translocate to the cSMAC after immune response stimulation but remained in the cytosol.

Equivalent results were demonstrated when the PKC-θ-deficient animals were generated to express a PKC-θ enzyme that incorporated the V3 domain from PKC-δ (PKC-θ+δV3). “These data suggest that the unique V3 domain of PKC-θ is required for its selective localization to the immunological synapse and cSMAC,” the team writes.

To investigate whether PKC-θ+δV3 could activate primary T cells, they then generated bone marrow chimeras in irradiated mice that were defi­cient in recombination-activating gene 1 (Rag1−/− mice) that were reconstituted with Prkcq−/− bone marrow cells that expressed either GFP-tagged wild-type PKC-θ or PKC-θ+δV3.

Eight weeks later, studies confirmed that CD4+ T cells reconstituted with wild-type PKC-θ and co-stimulated with anti-CD3 and anti-CD28 markedly upregulated their expression of both CD69 and CD25, two activation markers regulated by PKC-θ. In contrast, the ability of PKC-θ+δV3 to induce the expression of CD69 or CD25 was about 50% lower. The T cells reconstituted with PKC-θ+δV3 also failed to proliferate and produce IL-2 in response to stimulation with anti-CD3 and anti-CD28.

Previous research has shown that PKC-θ and CD28 co-locate in the immunological synapse and directly interact, and the La Jolla team further confirmed through precipitation studies that co-stimulation with anti-CD3 and anti-CD28 caused PKC-θ to associate with CD28. Deletion of the V3 domain in PKC-θ abolished this interaction, whereas enzyme mutants with N-terminal C2 domain deletion still co-precipitated with CD28.

Even deletion of both the C2 and C1a domains of PKC-θ only reduced interaction with CD28, rather than abolishing it completely. Similar results were again observed with primary Prkcq−/− CD4+ T cells expressing either PKC-θ or PKC-δ: Neither the ΔV3 mutant, the PKC-θ+δV3 mutant, or wild-type PKC-δ immunoprecipitated together with CD28. In contrast, however, the V3 domain was sufficient for interaction with CD28 cells, the team states, as V3 expressed alone in Prkcq−/− primary CD4+ T cells immunoprecipitated together with endogenous CD28, and the V3 domain also localized together with fluorescently tagged CD28 in the immunological synapse of co-transfected T cells.

Further evaluation of the V3 hinge domain identified a proline-rich motif that was conserved in PKC-θ enzymes from a number of species, but was absent in the hinge domains of other PKC enzymes. When this motif was inserted into the V3 domain of PKC-δ (which doesn’t normally translocate to the immunologi­cal synapse) and the resulting PKC-δ+θPR construct expressed in stimulated Prkcq−/− cells, the engineered enzyme did localize to the immunological synapse in a manner similar to that of wild-type PKC-θ.

This indicated that the proline-rich motif in PKC-θ V3 can mediate localization to the immunological synapse and cSMAC, the authors remark. PKC-δ+θPR also immunoprecipitated together with CD28 in transduced Prkcq−/− CD4+ T cells co-stimulated with anti-CD3 and anti-CD28 and was able to stimulate the activity of reporter genes.

Interestingly, switching the two external proline residues in the motif to other amino acids had no effect on the ability of resulting PKC-θ mutants—introduced into relevant cell and bone marrow chimera assays—to translocate to the immunological cSMAC, associate with CD28, activate reporter genes, upregulate activation markers, proliferate, and produce IL-2. In contrast, switching the two internal proline residues for other amino acids blocked or reduced all these activities.

The team’s research had also shown that mouse CD28 similarly contains a proline-rich motif in its cytoplasmic tail and that this was required for CD28–PKC-θ interaction. Independent studies, meanwhile, suggested that the same CD28 motif was required for co-localization of PKC-θ–CD28 to the cSMAC, stabilization of IL-2 mRNA, the reorganization of lipid, and for PKC-θ-dependent TH2- and TH17-mediated inflammatory responses.

Further analysis demonstrated that the interaction between two motifs in PKC-θ and CD28 actually requires an intermediary molecule, in the form of the kinase Lck and in particular the Lck SH3 domain; indeed, interaction between CD28 and V3 was absent in Lck-deficient cells but could be restored after transfection of an expression plasmid for wild-type Lck.

Importantly, Dr. Altman’s team subsequently showed that ectopic expression of V3 by T cells effectively acted as a decoy, causing endogenous PKC-θ to be sequestered from the immunological synapse. Additionally, while cells expressing V3 alone weren’t able to upregulate antigen-induced reported genes, co-expression of the V3 domain together with wild-type PKC-θ resulted in dose-dependent inhibition of the activity of the PKC-θ-dependent reporter genes. This inhibi­tory activity associated with ectopic V3 was eliminated when its critical proline-rich motif was altered or deleted.

The effects of ectopic V3 expression critically encompassed TH2 and TH17 responses. The team preactivated C57BL/6 (B6) CD4+ T cells with retrovirus expressing wild-type or mutant V3 domains and cultured the cells under TH1, TH2, or TH17 differentiation conditions. In support of previous research, they found that differentiation into the TH1 lineage was unaffected by any of the ectopically expressed V3 vectors, while the nonmutant V3 domain inhibited TH17 and TH2 differentiation by about 75%. This inhibition was completely reversed (for TH17) or partially reversed (for TH2) when the protein-rich motif was altered or deleted.

The effects of PKC-θ V3 were finally evaluated in a mouse airway inflammation model using a T-cell adoptive-transfer system. Mice receiving ovalbumin-specific OT-II TCR TH2 cells transduced with an empty vector developed a large inflammatory response. However, introduction of V3 into the trans­ferred TH2 cells ameliorated disease due to a reduction in levels of infiltrating cells and TH2 cytokines to basal amounts. As expected, expression of a V3 domain in which the proline-rich motif was deleted failed to inhibit the inflammatory response.

In contrast, introducing V3 into TH1 effector cells had no effect on the inflammatory response, “consistent with the fact that TH1-mediated lung inflam­mation is relatively independent of PKC-θ,” the team states.

“We have shown that the V3 (hinge) domain of PKC-θ and, specifically, a proline-rich motif in V3, were required for this localization through its physical association with CD28 and, consequently, for PKC-θ-dependent T-cell activation and TH2- or TH17-mediated inflammation,” the authors conclude.

“Given the requirement of PKC-θ in TH2- and TH17-mediated inflammation and graft-versus-host disease but not in TH1 antiviral or graft-versus-leukemia responses, PKC-θ is a likely drug target for a plethora of diseases. Our report of a new approach with which to attenuate the function of PKC-θ by blocking its obligatory interaction with CD28 suggests that such blockade could serve as a basis for the development of new therapeutic agents that would selectively suppress undesired T cell-mediated inflammation while at the same time preserving desired immunity such as antiviral responses.”

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